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Edema

GlossarySuccess Chemistry Staff

Edema

Edema refers to the increased accumulation of fluid (water, lymph) in the tissue of the body, outside of cells and blood vessels. Normally, about two-thirds of body water is in cells, while the remaining third is made up of tissues (25 percent) and vessels (8 percent). In edema, this ratio shifts in favor of the tissue.

The Short version:

  • The causes of edema may vary.

  • Edema, depending on its cause, can be divided into different forms.

  • Symptoms of water retention in the tissues are swelling.

  • The treatment of edema depends on the underlying disease.

Edema can occur in a specific location, for example, only on a lower leg or the whole body. From a temporal perspective, the fluid can accumulate very suddenly (acute), repeatedly (relapsing) or permanently (chronically)

How does edema develop?

The causes of edema can be very different and still lead to the same appearance. Edema may be due to the following factors:

  • an increase in pressure in the capillaries (the smallest blood vessels in the body)

  • a reduced concentration of albumin (a protein produced by the liver that, among other things, binds water)

  • a combination of these two mechanisms

  • damage to the vessel shell

The following diseases and medicines can lead to such changes:

  • Heart failure (right heart failure, left heart failure)

  • Renal dysfunction, renal insufficiency

  • cirrhosis

  • Venous insufficiency (chronic venous insufficiency, CVI) on the leg

  • Malnutrition or digestive disorders (inflammatory bowel disease, celiac disease )

  • allergies

  • A deposit

  • Hypothyroidism

  • Disorders of the lymph

  • Hormonal disorders/changes (hyperaldosteronism, Cushing's syndrome, pregnancy, before menstruation)

  • Medicines (non-steroidal anti-inflammatory drugs, antihypertensives, estrogens)

Sometimes no identifiable cause can be determined. After excluding all other causes, one then speaks of idiopathic edema.

Special forms of edema

There are special forms of edema, which are usually differentiated by the cause:

brain edema

Increase in the volume and pressure in the brain. The causes of this increase may be different (including tumors, bleeding, meningitis complications)

eyelid edema

Swelling of the eyelids. Can be caused by insect bites, eye inflammation, and allergies.

lymphedema

Edema due to weak lymphatic vessels. Most severe swelling, hardly noticeable in late stages. It is differentiated between the primary (innate) and secondary (acquired, among other things by tumors, inflammations, operations) lymphedema.

Angio-edema or Quincke's edema

Is caused by an allergy. Swelling of the facial area (lips, eyelids) and gastrointestinal tract is typical. May also occur in inheritable form.

Reinke's edema

Fluid accumulation in the vocal folds by long-term stimuli such as smoking, particulate matter or overstressing the vocal cords.

Schwangerschaftsödem

Pregnancy can lead to water retention. These may be physiological ("normal"), but also due to kidney disease or pregnancy-related hypertension.

myxedema

Water retention often under the skin due to hypothyroidism, typically on the tibia.

hunger edema

Edema caused by a diet-related lack of protein (eg fasting, low-protein diet, zero diet), which manifests itself above all as ascites (edema in the abdomen).

pulmonary edema

Water accumulation in the lungs, which is often caused by left heart failure. May also be caused by other causes, such as infections, medications, toxins, ingestion of fluids into the respiratory tract.

macular edema

Water retention in the retina in the area of the macula. The macula, also called a yellow spot, is the area of the sharpest vision.

glottis

Water retention in the mucous membrane of the larynx, which can also affect the vocal folds. Medications, infections, allergies, and injuries can cause glottic edema. Generalized edema of the unborn child, which may be due to various diseases (maternal and child blood group incompatibility, heart defects, infections).

nuchal translucency

From the 11th to the 14th week of pregnancy the unborn child has edema in the neck area that is completely normal up to a certain size. An enlargement is associated with an increased likelihood of chromosome disorders or malformations.

lipoedema

Atypical symmetric accumulation of adipose tissue laterally on the hips and thighs and upper arms, later also on the lower legs, forearms, and neck; colloquially called "saddle pants syndrome". Occurs mainly in women. The primary cause is damage to the lymphatic system.

What symptoms cause edema?

Edema is swellings that are either confined to one area or can occur all over the body.

Depending on the cause of the edema, there are different types of swelling and additional symptoms:

right heart failure

Bilateral swelling of the feet, ankles and later on the shins. Water retention in the abdomen (ascites) possible. In bedridden patients, fluid accumulation may also occur around the sacrum in accordance with gravity.

left ventricular failure

Often the cause of pulmonary edema. Shortness of breath, rapid pulse, coughing up of foamy mucus, partly with bloody fluid. The skin may be pale and bluish due to the lack of oxygen while rattling breath sounds.

cirrhosis

Typical for liver cirrhosis is in addition to generalized swelling water retention in the stomach (ascites).

Venous insufficiency, thrombosis (blood clots)

Unilateral swelling of the lower leg and/or ankle/foot.

Renal dysfunction

Often swelling in the face and around the eyes. If the disease progresses, fluid in the abdomen (ascites) or in the chest (pulmonary edema) may accumulate.

Lymphstörungen

Swelling usually on the ankles, ankle, and legs. They can harden, which is often associated with a feeling of tension and pain. The toes are swollen ("box toe"). In secondary lymphoedema (due to tumors or infections) spread from the trunk towards the extremities.

Allergy (angiogenic or quincke edema)

Common in the facial area (lips, eyelids) and gastrointestinal tract. Tension and itching on the affected areas.

How do doctors make a diagnosis?

Edema is usually recognized by a good "clinical look". Together with a thorough anamnesis and a manual examination of the swelling, it is then usually possible to clarify the causes of the edema.

The doctor examines where the edema occurs, what the skin looks like and what the swelling is like: is the edema compressible, painful, does it form dents?

Of particular importance is the questions about medications taken and long-standing illnesses of internal organs and injuries.

In case of ambiguity, laboratory examinations (blood, urine) and examinations with equipment (eg ultrasound, X-ray, magnetic resonance) can be added.

How is edema treated?

The treatment of edema depends on the underlying disease. Apart from angioedema and acute pulmonary edema - two life-threatening forms of edema that require immediate treatment - fluid retention should not be too rapid, leaving enough room for treatment.

Generally, generalized edema is usually treated with a diuretic and a low-fluid and low-salt diet. Weight, kidney values, urinary excretion and electrolytes should be kept in mind. If the cause is known, the treatment depends on the underlying disease:

underlying diseasetreatment methodsright heart failureDiuretic, possibly nitrates (preload reduction)left ventricular failureDiuretic, ACE inhibitors, possibly aldosterone antagonists, beta blockerscirrhosisdiureticThrombosis (blood clots)Compression stockings, elevation, dissolution (lysis) or surgical removal of the clot venous insufficiencyVariceal surgeryAllergy (angiogenic or quincke edema)Antihistamines & SteroidsRenal dysfunctionDiuretic (loop diuretics)

Treatment for lymphatic disorders

  • Complex physical decongestive therapy (KPE): lymphatic drainage , compression therapy  with compression stockings or replacement bandages, decongestant movement and respiratory therapy, skin care

  • Physiotherapy, apparatus intermittent compression, thermotherapy

  • Medications are used to treat the complications: antibiotics, antihistamines, cortisone, diuretics under certain circumstances.

  • In the operative treatment, an interrupted lymphatic system can be restored, the lymph can be derived by other means (establishing a connection between the lymph and veins) and diseased tissue can be removed.

  • Clinical-psychological treatment with possibility of continuation outside of hospitalization

Prognosis and disease course of edema

The prognosis depends very much on the underlying disease. If the underlying disease can be treated well, usually the edema disappears again.

Oregano

Glossary, HerbsSuccess Chemistry Staff
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Oregano

is a close relative of the marjoram and applies as this also as a medicinal plant. With its mild-spicy taste, oregano plays an important role in the Mediterranean cuisine. Classically, this herb is found on pizza and in tomato sauce. But it also has a lot of other uses. Oregano blends well with fruity tomatoes, cucumbers, and salads. Also with egg dishes, sauces, potato, and tomato soup, this aromatic herb fits.

Oregano as a medicinal herb

Although oregano is usually known only as a kitchen herb, it finds in the folk medicine relatively much attention. Oregano can be used for a variety of ailments due to its containing ingredients.

Oregano was already known in antiquity and the Middle Ages as a herb with great healing power. Oregano was used to treat painful hemorrhoids. In gynecology, the medicinal herb was used to initiate the birth. Furthermore, oregano was in the Middle Ages as a tried and tested means to shield themselves from demons, witches and the devil. From oregano lentils in the house and incense with oregano promised the same effect as garlic used against vampires.

In old herbal books, oregano, as well as some oregano species commonly referred to as Wohlgemuth (Wolgemut) or Dosten. In the herbal book by Petri Andreae Matthioli, the herb was used for both internal and external applications. Oregano was recommended among other things with itching (application: Oreganobad), with tumors of the almonds (presumably almond inflammation) or with the Wundbehandlung. Oregano has also been used to treat gastrointestinal complaints, coughing or tracheal diseases. The herb was usually mixed with red wine or drunk as pressed juice.

Today, however, oregano is used for problems in the gastrointestinal tract. The tannins and bitter substances in the oregano have an anticonvulsant effect, thus helping with gastric and/or intestinal cramps as well as flatulence and casually stimulate the appetite.

The plant parts of the oregano, especially the leaves and the herb contain a variety of different active ingredients. The containing essential oils, tannins, and bitter substances have the following healing effect:

  • antibacterial

    1. carminative

    2. appetizing

    3. cough expectorant

    4. antifungal

    5. antioxidant

To relieve stomach and intestinal complaints or chronic bronchitis, oregano is usually taken in the form of tea. One teaspoon of dried cabbage or two teaspoons of fresh oregano is poured over with 250 to 300 ml of boiling water. The infusion is left covered for about 10 minutes and then drunk. Even with a bacterial cough and infestation of Candida fungi, Oreganotee can be drunk supportive.

externally, oregano is used in inflammatory skin problems, as the ingredients in the essential oil of oregano (especially phenols and carvacrol) are classified as antibacterial and anti-inflammatory. Oily ginger tinctures are used to treat greasy, blemished skin, which is used to dab the affected areas of the skin.

Oregano - Medicinal herb of the Mediterranean

Oregano is an indispensable part of the Mediterranean cuisine. In the West, it is almost exclusively associated with delicious dishes from Italian cuisine.

Oregano is not only used as a medicinal but also as a spice plant.

But also the people of the eastern Mediterranean know and love oregano. For example, anyone traveling to Turkey will come across this tasty herb again and again; as well as in Greece and Spain. Incidentally, in the Middle Ages, it was often used there for dyeing, for example, wool. But back to the present and to Italy, where Oregano has also been known for centuries and is prized for its unmistakable flavor. Pasta sauces are mainly prepared here. But also with lamb dishes and everything that has anything to do with tomatoes, oregano is often used for seasoning. In addition, it is indispensable for a real Italian pizza.

Where does oregano come from?

Oregano is not only widespread in the Mediterranean, but the herb also has its original home here. It belongs botanically to the family Lamiaceae. In Germany, oregano is also known by names such as "wild marjoram", "Dost", "Dorst" and "Wohlgemuth". The latter points to the healing properties associated with the condiment. It helps the digestive tract to the legs and relieves coughing. For a soothing tea, add one teaspoon of dried oregano to a one-quarter liter of boiling water and infuse for five minutes.

How is oregano used?

Herbs are very important in Italian cuisine. Without basil, parsley, sage, and oregano, Italian dishes are almost unthinkable. They are mainly used fresh in salads, in sauces for pasta dishes, for egg dishes and in the context of tomatoes. A classic example is "Insalata Caprese"; an appetizer consisting of tomatoes, mozzarella, and fresh basil. Oregano is often used in a dried form for meat dishes. Classically, the Italian mom will use it in lamb dishes; Oregano also goes well with pork and beef.

When is oregano available and what should you pay attention to when buying?

Oregano is grown today almost everywhere in the world and is therefore available all year round fresh in the supermarket, in the vegetable trade and at weekly markets. It is usually offered in pots. In addition, the herbs are also dried and rubbed. It then has a slightly changed, woody and intense taste. In fresh plants, make sure that the leaves do not hang limp and have no brown edge.

Grow oregano yourself

As long as you have a sunny spot in the garden, this spice herb can easily be grown by yourself. You can sow just as well as-as of May - buy also bought young plants. Those who prefer seeds should only lightly press the earth over them. Oregano is a so-called "light germ," so the seeds should not be buried too deep in the ground. Moderate, but regularly poured, there will soon be a rich harvest. Partly dry part of it for the winter months.

 

 

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Sage

Herbs, GlossarySuccess Chemistry Staff

Sage medicinal benefits 

With over 60 ingredients, sage combines the healing properties of eucalyptus, rosemary, wormwood & tea tree oil.. It is a popular Mediterranean herb and is known for its use in saltimbocca or entrecote. Sage is ideal for refining meat dishes of all kinds and pasta. If the aroma is too intense, you can fall back on the much milder young leaves. The healing effects of sage can be used, for example, in homemade cough syrup, sage cough sweets or tooth-care throat drops.

The true sage - Latin name Salvia officinalis - is considered with its disinfecting and astringent effect as one of the most important medicinal herbs and played a major role in ancient and Celtic mythology. The Egyptians also appreciated him very much. They gave their women sage juice to promote fertility. Perhaps the following advice comes from this: women should eat a raw sage leaf every time they walk the garden. Sage leaves are also an integral part of ancient medicine, the ancient Ayurveda. In the Middle Ages, the herb was considered a panacea and was mainly grown in monastery gardens.

Pharmacists and doctors at that time appreciated him especially as a wound treatment for wound cleansing and hemostasis. Already in the 6th-century monks brought the medicinal plant across the Alps in northern regions. Therefore, it is not surprising that at early mention the plant appears in the monastery plan of St. Gallen, the monk Strabo from the monastery Reichenau and Hildegard von Bingen. The herbal boom in the 1980s made sure that the forgotten herb gardens were recreated in many monasteries and opened to the public. Even in cottage gardens, Salvia officinalis is a traditional plant that must not be missed. Today, more and more people trust in herbal medicine, and so the herb finds application in many complaints. As a spice herb,

Origin of sage

The home of sage is the Mediterranean. But now you can find this herb and spice herb in many gardens, where it prefers a heat-loving plant full sun and light soil. He does not love waterlogging and too much fertilizer, on the other hand, he withstands drying times very well. As a heat-loving plant, however, it needs a winter shelter in rough locations, for example, brushwood. Wild it still grows in Italy, mainly on the Adriatic coast in sandy and calcareous areas. In addition to Salvia officinalis, there are numerous other species of sage, which are cultivated partly in the garden as ornamental plants. All sorts of sage belong to the family of the mint family and bloom in different colors. The true shows from May to July its approximately two inches long blue to purple flowers, which make an excellent bee pasture. The perennial, bushy plant grows up to 80 cm high and lightens easily if you do not prune it back after flowering. Striking are the elongated silvery-green and slightly felty leaves. They emanate an aromatic fragrance through the contained essential oils. The leaves also contain the manifold healing powers.

Special features of sage

Sage leaves have a sharp, dry and strong, slightly bitter taste. In the kitchen, they are therefore particularly suitable for high-fat meals. However, mainly sage is used as a medicinal herb. Its known active ingredients are essential oils that include thujone, borneol, cineole, and camphene among other things, the leaves are rich in tannins and bitter substances, terpenes and numerous flavonoids. For the bitter substances, it is above all the typical salvin. The amount and composition of these substances are highly dependent on climatic conditions and the harvest time, which is most ideal before flowering. Together, the ingredients have antibacterial, antiviral, fungicidal, astringent and antiseptic properties. Thujone is poisonous, therefore you should not consume sage tea or drops for a long time. The name Salvia comes from Latin and means salvation or healing. This says a lot about the broad effect of the plant. The medicinal herb is used for tea, tincture, drops, sage oil, sage wine, and powder; in the industry for throat sweets and pastilles. For the applications mainly the dried sage leaves are taken. To do this, cut off some stems before blooming, hang them up in tufts or lay them out to dry in layers not too thick in the shade. In the oven or in the Dehydrator you can also dry at a maximum of 40 degrees. After drying keep dark and cool. You can also buy dried sage leaves. Pay attention to the quality.

Sage as a medicinal herb - earlier and today

In natural medicine, the use of medicinal herbs has a long tradition. Even Hippocrates, Hildegard von Bingen, and Paracelsus - to name just the best known - recommended the use of sage in fever, colic, urinary tract disorders, loss of appetite, colds, dental disease and red dysentery. During the various plague epidemics, the herb also played a significant role. Thieves rubbed their bodies with a mixture of sage leaves, lavender, rosemary, and thyme to protect them against the pest and were able to plunder the houses of the dead without becoming infected. The medicinal herb was also used for cramps, itching, pneumonia, somnolence, digestive problems, and body aches. Old recipes recommend sage powder or fresh leaves for teeth and gums for cleansing and strengthening. As early as the 10th century, Arab doctors, scholars, and philosophers used sage tea with honey to enhance their mental abilities. As an essential sage oil, it has a disinfecting and antispasmodic effect. In addition, the oil is suitable for colds for inhalation. Due to the disinfecting effect sage leaves were burned for a long time as fumigants in the room of the seriously ill.

Even today, many of these recommendations are valid and medical research can largely confirm this ancient knowledge. The active ingredients actually provide relief from everyday problems. Naturally, the herb is used today in:

  • Gingival and oral mucosal inflammations

  • Colds and sore throat

  • Tonsillitis, pharynx, and laryngitis

  • heavy sweating

  • for wound healing

  • mild indigestion

  • bronchitis

  • Panting and smoker cough

  • rheumatism

  • headaches

  • Nervousness and weak nerves

  • for breastfeeding during lactation

  • thin, graying hair

  • skin problems

The best-known uses of sage are certainly inflammation in the mouth and throat and various dental diseases. For this, rinsing or gargling with sage tea or mouthwash is recommended. Even special ready-made salvia preparations can help with these complaints. Because sage has anti-inflammatory and antispasmodic properties, it is also often included in toothpaste and cough sweets. Known is the effect of sage tea in case of excessive sweating, especially during menopause. Sole tea is also helpful for girls during puberty and for women in menstrual disorders. Responsible for this are probably the contained tannins and terpenes.

Regular drinking of cold tea - 2-3 cups a day, but not more than four weeks, then take a break - inhibits sweat production. The tea can bring relief for indigestion. These include the irritable stomach syndrome, which can cause flatulence, cramps, pain, and loss of appetite. It is even believed, according to recent studies, that sage tea has an effect on the lowering of blood sugar levels. This would be ideal for diabetics. The antibacterial and anti-inflammatory effect of sage is ideal for wound healing and for bad and oily skin. Salvia bites from a strong decoction are recommended here. They have a calming, astringent and anti-inflammatory effect. In lactating women, sage tea prevents milk flow and should therefore not be consumed during breastfeeding. In the case of weaning, however, it can be helpful. Thin and graying hair regularly flushed with a strong broth, makes the hair stronger and gives it a nice dark color.

Even with greasy hair helps a flush with the broth. Seating baths with salvia or the daily intake of sage wine to support the nervous system, such as stress and excitement. The herb calms and brings serenity, at the same time it also stimulates and strengthens the circulation. In studies, even an anticarcinogenic effect of sage was found. The contained diterpenes are said to trigger the cell death of the tumor cells. Leukemia and lymph node cancers are being discussed. Like the Egyptians, the Englishman John Gerard found in the Middle Ages: "Sage is uniquely good for the head and brain and accelerates the nerves and the memory". A team from the Newcastle up Tyne Medical Plant Research Center did a study on this and the result confirmed this statement by Gerard.

The reason is an enzyme of sage, which inhibits the degradation of the messenger acetylcholine. In Alzheimer's disease, this messenger substance is broken down in the brain. It is now being researched which ingredients of sage are responsible for this and whether a drug can be developed from them. Studies have also observed the inhibitory effect on the herpes virus. Ritual fumigation with the purifying effect of medicinal herbs has always existed. Today, these fumigations are again increasingly carried out, for example, for cleaning rooms at home, after illnesses, significant changes in life or in agriculture, the stables.

How can you apply sage 

Applied internally, tea is the simplest remedy. It strengthens the resistance of the organism and the nervous system. Ideal for menopausal symptoms with palpitations, blood rush in the head and sweating. Add 2 teaspoons of dried sage leaves and bake with 1/4 l of boiling water. Let it rest for 10 minutes, strain. Depending on your taste you can add some honey or a splash of lemon juice. In case of sweating and indigestion drink cold.

A weekly sitz bath can contribute to mental health. Women also help a sitz bath in case of discharge or soreness in the genital area. For this, let 4 handfuls of dried sage leaves in about three liters of cold water overnight. Heat to boiling the next day, strain and place in the bath water (38-40 degrees). Bathing in it for 20 minutes. Sage wine can also strengthen the nerves. Add 80 g of dried sage leaves and strain for 10 days in 1 liter of sweet wine. Then drain through a cloth. Take 1 tablespoon after each meal.

The sage tincture is like tea against perspiration, diarrhea, and stomach upset. For this, 50 g of dried sage leaves are mixed with 1/2 liter of 50% alcohol in a bottle. Close and allow to shake for 10 days, shaking frequently. Then drain through a cloth and fill in a brown pharmacy bottle - possibly with a pipette. Take 30 drops of tincture 3 times a day for 1 tablespoon of water.

Sage envelopes can help with spotty skin. The herb regulates sebum production and fights bacteria. The envelopes can also help with wound healing. Add 2 tablespoons of dried sage leaves to 150 ml of boiling water, infuse for 20 minutes, strain. Soak a clean cotton cloth in the warm infusion, gently wring it out and place it on the wound. Envelopes can also be applied to the face for blemished facial skin or cotton swabs placed in the appropriate places. Leave for 20 minutes, then rinse with lukewarm water.

Ointment water helps with bleeding gums, it heals and strengthens the gums. To chop 6 fresh sage leaves small. Bring 1/2 liter of water and a pinch of salt to a boil and infuse the sage leaves. Allow to cool, pour through a fine sieve and squeeze the leaves well. After brushing, use as gargle water.

For gingivitis, chewing fresh sage leaves or rubbing in and rubbing with leafy pulp is recommended. Fresh sage leaves are also said to help with bad breath. For pressure points of dentures or braces repeated rubbing with fresh leaves can help.

The homemade sage syrup also helps with colds and is also taken by older children. To do this, boil 1 kg of sugar in 1 liter of water. Then add 4 handfuls of fresh sage leaves and 2 sliced bigotries. Run for two days in a cool place, strain and boil briefly again. Bottling hot and sealing well. For cold and sore throat, drink 1 tablespoon of syrup 3 times a day on 1 glass of warm water.

Beware of side effects!

In principle, internal applications with sage tea, tincture or oil should only be carried out for the duration of the symptoms. The active ingredient thujone can cause symptoms of intoxication if it is taken longer and in larger quantities. Signs of toxic effects include accelerated heartbeat, convulsions, nausea, dizziness and heat. External applications and use as spice are generally harmless. For pregnant women, infants and nursing mothers, the medicinal herb is in no form. Suckling tea, on the other hand, is a popular remedy for reducing milk flow. Allergic reactions occur sporadically.

lavender

Glossary, HerbsSuccess Chemistry Staff

lavender 

Lavender (Lavandula angustifolia) - Characteristics, cultivation, use, and healing properties

The purple flowers of lavender give the herb bed an unmistakable splash of color and its wonderful scent enchants everyone's senses. Lavender is an integral part of French cuisine and can be found in every herb-of-Provence mixture. Because of its particularly intense aroma, it is advisable to dose lavender very sparingly. The herb is good for flavoring meat dishes, but also desserts, jams, and drinks get a unique touch with a touch of lavender. The flowers can also be used for salads and dips.

Origin and occurrence of lavender

The real lavender is a classic Mediterranean plant, which occurs wild mainly in the countries of the Mediterranean, including Greece, southern France, and Italy. It can be found there especially in dry, barren and sun-drenched altitudes in regions with calcareous soils. A region of lavender that is very attractive to tourists is French Provence, where lavender fields are very common.

Due to its great popularity, lavender has been introduced and cultivated in many countries. Significant cultural occurrences of lavender can be found next to France in England, Morocco and the United States.

Real lavender

The true lavender (Lavandula angustifolia, sometimes also Lavandula officinalis) is a member of the labiate (Lamiaceae). It is also part of the botanical subfamily Nepetoidaea, which includes many other well-known herbs such as rosemary, sage, savory or peppermint .

The genus of lavender plants (Lavandula) includes more than 30 different types of lavender . In addition to the real lavender, lavender lavender (Lavandula stoechas), broadleaf lavender (Lavandula latifolia) and French lavender (Lavandula dentata) are well-known representatives of this genus.

Characteristics of lavender

In the botanical sense, lavender is a perennial subshrub that can reach heights of growth of up to 140 cm. Most of the plants in this country, however, achieve much lower stature heights of 40 to 70 cm. Lavender forms elastic, stable and relatively short roots.

Lavender leaves are quite distinctive and easy to recognize. They usually have a light green to greyish green color. The lanceolate, ganzrandigen and elongated leaves are up to 5 cm long and are on both sides of the leaves with a white felt (leaf hair) occupied. The leaves of the lavender sit on the ascending and upright branches on which they are arranged opposite. The branches are usually heavily branched. As you grow older, the leaves turn greener.

The real lavender usually flowers between late May to mid-September . There it forms striking violet, rarely white flowers, which are arranged in so-called Scheinquirlen. These in turn form false notes, in which the lip-shaped flowers sit. Each flower consists of four stamens, a two-part upper and three-part lower lip and a four-part ovary. After the flowering period, at the time of fruit ripeness, small brownish nut fruits are formed.

Real lavender - sowing and care

Although it is usually easier to transplant finished lavender plants from the hardware store or garden center in the garden or on the balcony, it can sometimes be more sustainable to grow the lavender from your own seeds. Many purchased plants are sometimes overfertilized, so they survive only one season.

Who wants to sow lavender, should know how the natural habitat of the plant is. It thrives magnificently on sun-exposed slopes, with calcareous, well-drained and nutrient-poor soils. It is therefore recommended not to cultivate the lavender pure in commercially available potting soil or in clayey or clayey garden soils. If no optimal substrates are available, the soil or the soil should be mixed with aggregates such as pumice, lava or zeolite.

The sowing succeeds best in Vorkultur on the window sill or in a room greenhouse. Since germ temperatures around 20 ° C are required, the lavender seeds should also have a correspondingly warm environment. Ideally, the pre-culture should take place between the end of February and the end of March, in order to transplant the young plants into the field or on the balcony. Germination requires some patience. Germination can take up to four weeks. It may be advantageous to use a mineral growing substrate for sowing. A 50:50 mixture of vermiculite and perlite appears optimal. Always keep the seed soil moist, but not too humid.

sowing in the field is also possible, though a bit more difficult. Here it should be ensured that no more night frosts occur. The seeds can be easily pressed into the ground at a distance of about 30 x 30 cm. Since the lavender is a light germ, the grains should be incorporated only about 0.5 cm deep into the soil. The best time for sowing outdoors are the months of April and until the end of May.

Fertilization: Lavender is used to nutrient-poor locations and therefore does not need a lush supply of fertilizers . It is usually sufficient to provide the plants once a year with compost or light NPK fertilizer. If you have plants in tubs or smaller pots, you may need to fertilize a little more often.

Water supply: The herb needs only a little water. Therefore, should only be poured when the soil or the soil is almost dried out. The plant will cope without any problems for a while without water. Significantly more harmful is an oversupply of water. Too much water can promote root rot and other diseases.

Overwinter: Lavender is considered frost tolerant, ie the plant can survive short periods of freezing. If you want to let the plant hibernate outdoors, you should take some measures. The cover appears optimally with very coarse mulch material, straw as well as twigs and foliage. The latter protects against dehydration. Without cover, the water freezes in the near-surface soil layers, so that the lavender can no longer absorb water. It is important that the lavender branches should be cut back already in late summer, otherwise frostbite may occur. Lavender plants that are in pots can be stored in the garage or in the basement in unheated but frost-free areas (see Making herbs winter proof ).

Lavender and its uses

The lavender is not only a popular ornamental plant, it is also a very versatile herb. As a fragrance, it is used in numerous cosmetic products. As a culinary herb lavender lends a spicy touch to many Mediterranean dishes and as a medicinal herb it can sometimes cause minor miracles.

Lavender in the kitchen

Lavender is considered an excellent culinary herb that can be used for numerous recipes. The flowers, the leaves as well as entire branches are used. Care should always be taken to use the real lavender (Lavandula angustifolia) and not the shaggy lavender. The real lavender has a distinctive spicy and camphor-like flavor, which is sometimes flowery and slightly bitter.

Fresh lavender branches, for example, flavor meat dishes of all kinds . Chicken, lamb and fish dishes have an excellent taste when lavender is combined with thyme or rosemary.

Chased or chopped lavender leaves provide a refined taste in many cheeses such as goat cheese, soft cheese, gorgonzola or raclette cheese with lavender. The aroma of lavender is also suitable for hearty creamy soups. In principle, the dosage should always be handled very sparingly, since the leaves are usually very aromatic and produce an intense taste.

Lavender flowers are often used for sweets. For example, fruit juices that receive berry fruit can develop a very excellent aroma when mixed with a few lavender flowers. Lavender is also very suitable for cakes (eg Guglhupf), truffles and ice cream.

A special feature is lavender honey , which tastes lovely and has a fine floral aroma. For high-quality honeys the lavender taste is fine. Lavender honey is also considered very healthy due to its many minerals.

Gerebelter lavender leaves are occasionally contained in herbal mixtures such as herbs of Provence. However, the herb is not an ingredient in the original French herbal blend . Although lavender is listed as an ingredient in many sources, this seems to be more of a German peculiarity, mixing herbs from Provence with lavender.

Lavender as a medicinal herb

The true lavender looks as a medicinal herb on a long and impressive history. The Romans, Egyptians and Greeks considered the lavender to be an almost sacred herb. In the Roman military, the plant was used as a wound healing and stimulant, among other things. Both Greeks and Romans used lavender as an ingredient for their bathwater. The Roman physician Dioskurides has already reported in detail in his book Materia Medica (1st century AD) about the lavender and its applications.

The herb and its healing properties were also known in European countries, so that it was cultivated in many monastery gardens and was also used in many diseases and complaints. Hildegard von Bingen described the lavender as a warm and dry herb, which was administered by her among other things in lung and liver diseases as well as in psychological conditions. Paracelsus already knew its calming effect and used the herb as a sedative and for the treatment of heart and digestive complaints.

The medicinal herb is mentioned in almost all medieval herbal books . In the herbal book by Pietro Andrea Matthioli (late 16th century) numerous internal and external applications have been described. For example, lavender has been used to treat paralysis, cramps, stomach disorders as well as liver and spleen disorders. There it was recommended to boil the lavender in wine or water (tea) and to drink it or to spread it on the arteries as schnapps (distilled water).

In addition, the lavender was recommended for various dental diseases, language problems and body aches. He was also given to pregnant women who have contractions. In the latter, pure lavender branches were placed on the stomach.

 

Today, lavender is mainly used for nervous restlessness, mild depression, insomnia and as a mild sedative. The sleep-inducing, sedative and muscle-relaxing effects of lavender have been confirmed in many scientific studies. Responsible for the calming, sedative effects are certain essential oils in lavender, vader fabric linalool .

In addition to the sedative effect of the ingredients of the lavender also have antibacterial (including Shigella, Staphylococcus aureus, Escherichia coli), antifungal, chiral and anticonvulsant [1]. For the antibacterial and antifungal properties, especially the essential oils are responsible. Depending on the variety and growing area of more than 40 essential oils may occur. The cholagogue properties are mainly caused by the containing tannins.

Lavender is used in natural medicine today as it was then as a tea or as a bath additive. Other dosage forms include aromatherapy with essential oils or the use of lavender pillows.

Preparation of lavender tea

For the preparation of 250 ml of lavender tea, cover a spoonful of lavender leaves or flowers with 250 ml of boiling water. The tea should last at least 7 minutes, but not longer than 10 minutes. The tea should not be sweetened if possible.

Lavender tea can be drunk in nervous restlessness, insomnia and minor stomach and intestinal complaints such as bloating or diarrhea.

To prepare a herbal bath with lavender, pour about 40 to 50 grams of lavender flowers with one liter of boiling water in a container and leave for about 15 minutes. The container can then be poured into the prefilled tub. Even if the lavender bath is quite relaxing and soothing, the bath should be stopped after 25 minutes at the latest.

Some ingredients of lavender may also help in the treatment of so-called Hodgin lymphoma. Under laboratory conditions, lymphoma cells could be inhibited in their growth (proliferation).

Another medical research approach is the use of lavender extracts in Alzheimer's disease. For example, learning deficits in Alzheimer's patients could be reduced, or the enzyme acetylcholinesterase, which plays a role in the onset and progression of the disease, can be sustainably inhibited.

Buying lavender - here is what to pay attention to

Lavender is available in all imaginable shapes today. Fresh lavender plants can usually be bought in early summer in supermarkets, hardware stores and garden centers. If you want to buy the lavender as a useful plant, you should pay attention to the botanical name (Lavendula angustifolia). Sometimes also the Schopflavendel comes into the trade, which looks very similar to the real lavender.

It should also be ensured that the leaves do not hang, the soil is not too wet and that the ratio plant height to pot size fits. A lavender plant that is three times as high as the pot should not be purchased if possible.

Dried lavender leaves and flowers, which are used for teas, recipes and herbal baths, are available from many herbalists, sometimes in larger supermarkets or in numerous online shops. Again, the botanical name should be checked. The herbs should be packed aroma-tight and spread when opening a strong lavender-scented aroma. Lavender flowers are usually much more expensive than lavender leaves.

For aromatherapy, scented candles, lavender soaps or lavender bath also finished lavender oil can be purchased. If you want to use lavender oil for aromatheurotherapeutic purposes, you should be careful to get real lavender oil. There are many synthetic products available on the market, but they are not suitable for this purpose.

Aloe Vera

GlossarySuccess Chemistry Staff

INTRODUCTION

Aloe vera is one of the oldest known medicinal herbs with

a history of use that spans thousands of years. Today, aloe

vera is cultivated and used in a large variety of commercial

preparations. It is an economic driver in the food, dietary

supplement, and personal care industries worldwide. The

two main commercial materials derived fromaloe vera are

aloe vera juice and aloe latex. Aloe vera juice is used for

various dietary, cosmetic, and medical purposes such as

burn treatment, wound healing, and skincare. It is available

in several forms including liquid juice, juice powder,

and concentrates. Aloe latex was formerly recognized as

an over-the-counter (OTC) laxative drug in the United

States. It has seen limited use in dietary supplements as

a laxative and in the personal care industry as a skin

lightener.

Confusion among consumers, researchers, and regulatory

bodies has arisen from the fact that products from

aloe latex are often referred to as simply “aloe” or “aloe

juice” (including in pharmacopoeias and other official

documents around the world), which is physically, chemically,

and biologically distinct from products made from

the charcoal filtered whole leaf or inner leaf aloe vera juice.

These latex-free juice products represent the vast majority

of aloe products on the market. Regardless, the prominence

of, interest in, and use of aloe vera products for

centuries attests to the plant’s myriad value and benefits.

BACKGROUND

Aloe vera (L.) Burm. f. is one of more than 400 known Aloe

species in the Asphodelaceae family, though it is sometimes

classified in Aloaceae. Because most aloe species

are indigenous to Africa, it is most likely that aloe vera

also originated from that continent. However, because of

its now worldwide cultivation, its origin is difficult to establish.

Linnaeus classified aloe vera as the “true aloe”

hence the name “vera,” meaning true in Latin. Although

it has also been known as Aloe barbadensis, Aloe chinensis,

Aloe indica, Aloe vulgaris, and others, A. vera (L.) Burm.

f. has precedence (1). Its standardized common name is

“aloe vera” though it has also been called Barbados aloe,

Curac¸ao aloe, true aloe, West Indian aloe, Ghrita kumari,

or simply aloe (2).

The plant is cactus-like in appearance with succulent

leaves that grow in a spiral form from a basal rosette

(Fig. 1). An inflorescence is produced annually (typically

December through March) with yellow flowers in a trident

configuration from a single central stalk with many

flowers in each of the three branches. Aloe vera does not

normally reproduce from seeds but from offshoots often

called “pups” that grow out from the mother plant. When

the green outer rind of the leaves is cut or damaged, a

bitter yellow exudate from pericyclic tubules located between

the outer rind and the inner leaf is released. This

sap is commonly referred to as “aloe latex” (3) and contains

several anthraquinone glycosides that have powerful

stimulant laxative properties.

When the rind is completely removed, a semitransparent,

semicrystalline gel-like layer composed of large

thin-walled parenchyma cells is revealed. This inner leaf

material is often called “aloe gel,” or “inner leaf fillet,”

because of its similarity in shape to a fish fillet. When

crushed, it produces a very viscous fluid usually containing

approximately 98.5% water. The solids are composed

mainly of polysaccharides and other carbohydrates,

pectin, and organic acids.

As mentioned earlier, aloe latex–derived products

are used as a laxative agent and the processed leaf or

inner leaf is often employed topically for the treatment

of burns and injury. More recent applications range from

skin-moisturizing agents to the management of cancers in

animals to impregnation in articles of clothing and mattresses

for its softening and moisturizing properties. Aloe

vera juice is also orally ingested to manage digestive ailments

and for its immune-modulating activities and is

sold worldwide in beverage form as a food-based drink

product available in various flavors. Aloe vera is also used

widely in Ayurvedic medicine (4).

CULTIVATION

Aloe vera is cultivated in subtropical regions around the

globe for commercial use and is widely grown by indoor

and outdoor plant enthusiasts as an ornamental plant because

of its hardiness and beauty. The species is resistant

to most insect pests and needs very little maintenance

or care to flourish, given appropriate temperature

conditions (5).

Because of its very low inner leaf solid content of

0.5% to 1.5%, aloe vera plants are highly susceptible to

freezing, which causes extensive damage, even killing

them when the temperature falls below 32◦F. For this reason,

commercial cultivations are typically carried out in

warm weather areas (USDA zones 8–11). Aloe vera is the

most cultivated species of the various Aloe species because

it produces the largest, thickest leaves and therefore yields

the greatest amount of juice. It is cultivated extensively in

removing most of the outer, lower, older leaves. Typically one to

four leaves are removed at a time per plant per harvest.

This way it is possible to obtain three to six harvests in a

year depending on how many leaves are collected from

each plant at harvest.

Cultivation practices for the industrial production

of aloe extracts made from the yellow latex sap are radically

different from those used to grow aloe vera for juice.

In the case of sap production, plants are not irrigated

and are grown in arid regions. The leaves turn brown

and thin under these conditions but when cut produce

the maximum amount of an anthraquinone glycoside–

rich latex, the principal constituents of which are the

compounds known as “aloins A and B.” The sap exudates

are collected and further processed to produce

two main products, aloe latex concentrate also known as

“aloin paste” and a product commercially known as “aloin

spray dried.”

DESCRIPTION

The leaf of aloe vera is normally described as consisting of

three major parts that are used in commercial products: the

outer mesophyll (rind or cuticle), the interior parenchyma

(inner leaf, gel or gel fillet, inner gel, inner leaf gel fillet),

and the aloe latex (sap, bitter element, yellow sap, yellow

latex). Researchers, raw material manufacturers, and finished

goods manufacturers have utilized all three plant

parts separately or in combination for aloe vera research

and in the formulation of consumer products.

Outer Mesophyll (Rind)

Aloe vera rind or cuticle is the site of photosynthesis and

primarily consists of cellulose, monosaccharides, water

soluble and insoluble carbohydrates, chlorophyll, amino

acids, proteins, and lipids.

Interior Parenchyma (Inner Leaf)

Aloe vera inner leaf is the colorless, mucilaginous

parenchyma of the aloe vera plant leaf consisting of

water, monosaccharides, water-soluble carbohydrates,

water-soluble polysaccharides, and water-insoluble fibrous

pulp. The compound -(1–4)-acetylated mannan,

a polysaccharide also known as “acemannan” or “acetylated

polymannose,” is widely considered to be the biologically

most important component of the inner leaf. After

removal of fibrous pulp from the inner leaf, the resulting

juice contains about 0.5% to 1.5% solids.

Histological examination of aloe vera inner leaf pulp

has shown it to be composed of large cells made up of 16%

cell walls, about 1% microparticles, and 83% of a viscous

gel on a dried weight basis. The carbohydrate portion

of each of these components was distinct, with the cell

walls composed of 34% galacturonic acid (an unusually

high level), the microparticles composed of galactose-rich

polysaccharides, and the liquid gel contained mannan (6).

These findings showed that different pulp structures are

associated with different polysaccharides and may therefore

confer different biological activities.

 

Aloe Vera

Harvest of the

leaf or the

whole plant—

root not used

Cleaning and

sanitation of

the leaves

Reduction of

the leaves to a

puree

consistency

Removal of

the insoluble

pulp and rind

Enzymatic treatment to

reduce viscosity

(not all manufacturers)

Charcoal filtration step to

remove phenolic compounds

(not all manufacturers)

Reduction of

microbial load by

pasteurization

Preservation and packaging

Reduction of water content to

increase the percent juice solids

or to produce juice powder

Flowchart 1 Aloe vera whole leaf processing.

Aloe Latex (Aloe Sap, Aloe Bitters)

Aloe latex is a yellow-green bitter exudate that contains

the anthraquinone glycosides aloins A and B, formerly

known as “barbaloin” and “isobarbaloin,” respectively.

The aloin content of aloe latex changes with the season

and the age of the leaf but usually makes up 10% to 25%

of the dried latex by weight. Products made fromaloe latex

have been used historically as a laxative. The source plant

is most commonly Aloe ferox from Africa or Argentina.

COMMERCIAL RAW MATERIAL PROCESSING

Aloe Vera Juice

Aloe vera juice can be manufactured from raw leaves in

two ways-–from the entire leaf or from only the inner leaf

material. In both cases, the leaves are first processed to

remove the side thorns and tips. For aloe vera juice made

from the entire leaf, the leaves are macerated in a grinder

into what is commonly called “guacamole” and then further

processed by enzymatic treatment (usually with cellulase)

to break up cell walls and then charcoal filtered to

remove anthraquinones and other phenolic constituents.

The resulting aloe vera juice is commonly referred to as

“filtered aloe vera juice” or “purified whole leaf aloe vera

juice.” See flowchart 1 for more detail.

When creating juice from only the inner leaf material,

the inner leaf is separated from the outer rind either

manually with a knife or by machine and then washed to

rinse away any aloe latex present. The remaining material

is crushed and further processed to produce the aloe vera

juice. See flowchart 2 for more detail.

At this stage, regardless of starting material, the

now-processed aloe vera juice is typically called “single strength.”

The juice from the leaf or inner leaf can also be

further processed to produce concentrates and powders

and are often spray or freeze dried. Some heat is usually

applied in the industrial production of aloe vera juice to

deactivate enzymes that would break down the mannans

into oligosaccharides and simple sugars. Heating also

serves to control the normal microbial load present on the

plant. Enzymatic treatment can be used to further break

down cell walls, with filtration removing any remaining

insoluble fiber. The resulting filtered juice contains all the

major groups of components from the original aloe vera

inner leaf.

Aloin-Rich Materials

The commercial production of aloin-rich materials starts

with the specialized cultivation practices mentioned earlier.

In contrast to aloe juice production, the leaves are

  • Leaf harvest

  • Cleaning and sanitation of the leaves

  • Removal of most of the rind

  • eduction of fillet to puree

  • Removal of the insoluble pulp and rind Enzymatic treatment to

  • reduce viscosity

  • Reduction of microbial load by pasteurization

  • Preservation and packaging

  • Reduction of water content to increase the percent juice solids or to produce juice powder

  • Dicing/slicing of fillet typically for use in beverage or food, the pulp is not removed

  • Reduction of microbial load by pasteurization

  • Preservation and packaging

Aloe vera inner leaf processing.

Acemannan

Mannan is a generic name for polysaccharides that are

polymers of the sugar mannose. In aloe vera juice, the

mannose moieties are connected by -(1–4) linkages,

which are partially substituted with acetate units and with

galactose-rich side chains on the mannose backbone. This

-(1,4)-acetylated-polymannose material is also known by

other names such as “aloverose” and “acemannan”; the

latter is also a name given to a proprietary substance

covered by many patents (7) and has been assigned as a

generic name by the United States Adopted Names Council

(8). It is based on the chemical name as it refers to the

acetylated mannan found in all aloe vera inner leaf fillets

(Fig. 3).

Acemannan is not sold as a pure material; however,

many commercial products contain varying amounts of it

depending on the processing of the aloe vera leaf as mentioned

earlier. The therapeutic properties of aloe vera juice

have been largely attributed to its polysaccharide component

and acemannan in particular. This high-molecularweight

material is perhaps the most studied component

of the aloe vera plant aside from the anthraquinone glycosides.

Many industrial methods have been developed

to stabilize the aloe vera juice and prevent polysaccharide

degradation. Drying the juice at temperatures over 60◦C

has been shown to cause deleterious changes in acemannan

and also pectin from the cell walls (9).

ANALYSIS OF COMMERCIAL PRODUCTS

Analysis of 32 commercial products showed wide variations

in polysaccharide content when compared by molecular

weight (10), which was attributed to different manufacturing

procedures. A second study of nine commercial

powders used a method that hydrolyzed the mannan into

mannose as a rapid way to measure the total polysaccharide

content in the powder. One sample was found to have

an abnormally high concentration of free glucose, four

showed signs of spoilage, and all but three were found to

have low levels of polymannose present (11). Both studies

found all samples to have a significantly lower amount

of aloins than the raw unwashed inner leaf fillet, with a

high of 16 ppm of aloin A found in one sample in one of

the studies. Because of the widespread use of aloe vera

juice in personal care products, a voluntary industry limit

of 50ppm aloin content for use in cosmetics as a topical

agent has been established (12).

PRECLINICAL STUDIES

General

Manyof the biological properties of aloe vera have been attributed

to acemannan. This compound has been studied

precipitate

fraction demonstrated wound healing activity (17),

suggesting that more than one aloe compound could be

useful in treating both inflammation and wounds.

 

Skin Moisturizing

Aloe vera applied topically has a moisturizing effect on

skin (18) and has been used for this purpose and as a

conditioning agent.

Antidiabetic Activity

Rodent studies have shown blood glucose regulating activity

of an aloe vera alcoholic extract (19) and processed

aloe vera inner leaf juice (20), suggesting its utility in treating

non–insulin-dependent diabetes.

Antitumor Activity

Acemannan has shown significant antitumor activity via

immune system activation. In a mouse model, IP injection

of acemannan at the time of implantation of sarcoma cells

resulted in a 40% survival rate in the treated animals versus

0% of the controls, most likely because of the production

of monokines from macrophage peritoneal stimulation.

The data suggested that this acemannan-stimulated

synthesis “resulted in the initiation of immune attack,

necrosis, and regression of implanted sarcomas in mice”

(21). A study involving acemannan treatment as an adjunct

to surgery and radiation in confirmed fibrosarcoma

in dogs and cats showed tumor shrinkage in one-third

of the animals after four to seven weeks of treatment administered

by intraperitoneal and intralesional injections

(22). An earlier study by the same group showed similar

results (23).

Studies on Aloin-Rich Materials

Aloin-rich extracts derived from aloe latex belong to the

stimulant laxatives drug class. Aloins are inactive until

deglycosylated by intestinal flora to form aloe-emodin, the

putative active compound (24). Their mechanism of action

is believed to involve increasing peristalsis and water accumulation

in the colon (25). Aloe latex has been subjected

to a human clinical trial as a laxative in combination with

other ingredients (26).

The potential toxicities of aloin and its metabolites

are not well established though studies have shown it

does not promote colon cancer in a mouse model (27) and

induces cell changes that could be a sign of anticancer

activity (28). Selective activity against certain cancers has

also been demonstrated by aloe-emodin, a metabolite of

aloins A and B (29,30).

 

CLINICAL STUDIES

Wound Healing

Although one study showed a delay in healing wound

complications after cesarean delivery or gynecological

surgery following treatment with “aloe vera dermal

wound gel” (31), another recorded a 6-day statistically

significant reduction (from 18 to 12 days) in the healing

time of partial thickness burns. A systematic review of

the literature for the use of topical aloe vera in treating

burn wounds included four controlled clinical trials involving

371 patients. A meta-analysis based on the time

for healing showed almost nine fewer days required for

the aloe vera–treated group over the controls (32). No specific

conclusions could be drawn because of the difference

in preparations and outcome measures. Further studies

with well-characterized materials were called for by the

authors as cumulative evidence tended to support the use

of aloe vera for the treatment of first- and second-degree

burns. A bioadhesive patch of an aloe vera preparation

was evaluated in an open uncontrolled trial for the management

of mouth ulcers in children with apparent good

results (33).

 

Ulcerative Colitis and Irritable Bowel Syndrome (IBS)

Ulcerative colitis is caused by a dysfunction of the immune

system (34). A 2004 clinical trial involving 44 patients

with mild or moderately active ulcerative colitis compared

100 mL twice-daily oral aloe vera juice treatment

with placebo for four weeks. The aloe vera–consuming

patients showed positive clinical responses more often

than placebo. Clinical remission was seen in 30% of the

active group, clinical improvement in 37%, and a clinical

response in 47% of patients compared with 7%, 7%,

and 14% in the placebo group, respectively. Histological

scores and the Simple Clinical Colitis Activity Index did

not change in the placebo group but decreased significantly

for those who consumed aloe vera. No significant

differences were seen between the two groups with regard

to laboratory values or sigmoidoscopic scores (35).

Ahuman clinical study using aloe vera for treatment

of irritable bowel in refractory secondary care patients

failed to show a benefit, though the authors could not rule

out that diarrhea-predominant patients were helped (36).

Antidiabetic Activity

Aloe dried sap has demonstrated hypoglycemic activity

in a study involving five patients with non–insulindependent

diabetes (37).

Antitumor Activity

A preliminary clinical trial on the use of orally administered

“aloe vera tincture” for untreatable metastatic solid

tumor patients with and without melatonin treatment

showed a significantly higher percentage of nonprogressing

patients in the group that received the aloe treatment

(50% vs. 27%, P < 0.05) (38). Another human trial on 240

patients treated with Aloe aborescens (used because of purported

immunostimulating activity from this plant owing

to its acemannan component) suggested that oral aloe

therapy may be a successful adjunct to chemotherapy in

patients with metastatic solid tumors. Tumor regression

rate and survival time were improved in this study (39).

No conclusions can be drawn from this study because

details on characterization of the test material were not

provided.

SAFETY

A four-week subacute oral toxicity study in mice administered

a freeze-dried aloe juice product reported no remarkable

subacute toxic effects but did note a decrease

in male kidney weights. The report also provided a review

of several adverse reaction case studies associated

with aloe vera (40). They ranged from skin irritation from

topical use to one report of acute hepatitis in a 73-yearold

female taking oral aloe vera capsules for constipation.

A second case of acute hepatitis involving a 26-year-old

man who had been drinking “aloe vera tea” has also been

reported (41).

The National Toxicology Program of the U.S. government

nominated “aloe vera gel” for study in 1998 (42).

No long-term carcinogenicity studies of aloe vera gel in

animals were identified at that time. NTP subsequently

chose to conduct a two-year carcinogenicity study on mice

and rats with a “whole leaf extract” (43) that includes a

considerable amount of latex aloins. The majority of aloe

vera juice products intended for long-term internal use

are either charcoal filtered whole leaf preparation or are

made from washed inner leaf juice with aloin concentrations

typically under 10 parts per million. The NTP

report was still in progress at the time of publication of

this chapter.

REGULATORY STATUS

United States

Aloe and aloin are present in the first approved food additives

list published by the U.S. Food and Drug Administration

in 1959. Although initially approved in the United

States in 1975 as an OTC drug to treat chronic constipation,

aloe latex is no longer approved for such use in the

United States as of May 9, 2002 (44). Standard quality tests

for aloe latex have been described in detail in many official

pharmacopeias including the United States Pharmacopeia,

Japanese Pharmacopoeia, and the European Pharmacopoeia,

though, as mentioned in the introduction, these texts typically

define aloe latex as simply “aloe” or incorrectly as

“aloe juice.”

Aloe vera juice products can be labeled and marketed

as dietary supplements. Aloe latex may also be used

in dietary supplements in the United States with laxative

or constipation claims as long as such claims are not for

the treatment of chronic constipation.

Australia

Aloe vera inner leaf (called “aloe barbadensis”) is eligible

for use as an active or excipient ingredient in Australia

in “Listed” medicines in the Australian Register of Therapeutic

Goods. Acemannan is approved as a component.

Components are not approved as substances for use in

their own right and can only be used in conjunction with

an approved source.

Some aloe vera juice and juice concentrate beverages

are viewed as “nontraditional foods” and not as “novel

foods” and there are some listed medicines described as

“aloe vera drinking gel” or as “aloe vera juice.”

Canada

Aloe vera inner leaf, when included as a Natural Health

Product (NHP) active ingredient, requires premarket authorization

and a product license number for OTC human

use. Such products must comply with the minimum specifications

outlined in the current NHPD Compendium of

Monographs (45).

European Community

Aloe vera inner leaf was listed as “currently not on the

priority list” in the inventory of herbal substances for assessment

by the European Medicines Agency as of March

2009. There is an EU regulatory limit established for aloin

content of 0.1 ppm in orally ingested products based on

a flavoring regulation in which the aloin is defined as an

added ingredient as opposed to naturally occurring. The

International Aloe Science Council (IASC) (a trade association)

has taken a position that these regulations are not

applicable to aloe vera juice products.

Japan

Aloe vera juice is regulated as a food beverage product in

Japan and is not to contain more than 0.60 mg/kg of benzoic

acid. Various forms of aloe vera and extracts thereof

are used as components of functional food products or in

Foods for Specified Health Use such as in fortified waters

and fermented yogurt drinks.

South Korea

Aloe products, known as “edible aloe concentrate” and

“edible aloe gel,” are regulated as food products by the

Korean Food and Drug Administration. Juice or concentrate

from the inner leaf or dried and powdered inner

leaf material containing not-less-than 30 mg/g of total

aloe polysaccharides is able to carry the health claim

of “smoothing the evacuation” on the basis of 20 to

30 mg delivered as aloe polysaccharides. Processed aloe

vera leaf or concentrates thereof, after removal of the

inedible parts, and containing 2.0 to 50 mg/g of anthraquinones

(as anhydrous barbaloin), is permitted to

make the same health claim at the specified daily intake.

Aloe vera is also one of the four botanical ingredients allowed

to make immune system enhancement claims in

South Korea.

CONCLUSION

Of the 400 known species of aloe, Aloe vera is the most

commonly used in commerce and is cultivated in many

different areas of the world. The plant yields two raw

materials for use in various consumer products including

foods, dietary supplements, cosmetics, and drugs, namely

aloe vera juice and aloe latex. Aloe vera juice can be made

from processing either the entire leaf or only the inner leaf

material. Aloe vera juice is often further processed into a

powder or concentrate.

Preliminary scientific evidence suggests that aloe

vera has therapeutic benefits; however, more studies need

to be conducted to definitively demonstrate efficacy. Consumers

should be aware and informed when buying aloe

vera products; although there are many quality products

on the market, there are also many products that may

bring little or no benefit to the user. The IASC maintains a

certification program using validated analytical methods

to determine and ensure products displaying the IASC

program seal contain aloe vera of a particular quality. It

is recommended that consumers verify that products displaying

the IASC seal are current participants in the IASC

certification program.

 

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Accessed January 15, 2010.

Adenosylmethionine

GlossarySuccess Chemistry Staff

ABBREVIATIONS:

  • CSF, cerebrospinal fluid; GNMT, glycine N-methyltransferase;

  • GSH, glutathione; HCC, hepatocellular carcinoma;

  • Hcy, homocysteine; MAT, methionine adenosyltransferase;

  • MTA, 5-deoxy-5-methylthioadenosine; MTHFR,

  • 5,10-methylenetetrahydrofolate reductase; NASH, nonalcoholic

  • steatohepatitis; SAH, (S)-adenosylhomocysteine;

  • SAMe, (S)-adenosylmethionine.
     

INTRODUCTION

Common and Scientific Name

S-Adenosyl-L-methionine, also known as 5-[(3-Amino-3-

carboxypropyl) methylsulfonium]-5-deoxyadenosine; (S)-

(5-deoxyadenosine-5-yl) methionine; [C15H23N6O5S]+, is

abbreviated in the scientific literature as AdoMet, SAM,

or SAMe. In the early literature, before the identification

of its structure, SAMe was known as “active methionine.”

General Description

SAMe was discovered in 1953 and since then has been

shown to regulate key cellular functions such as differentiation,

growth, and apoptosis. Abnormal SAMe content

has been linked to the development of experimental and

human liver disease, and this led to the examination of the

effect of SAMe supplementation in various animal models

of liver disease and in patients with liver disease. Both

serum and cerebrospinal fluid (CSF) levels of SAMe have

been reported to be low in depressed patients, which has

led to the examination of the effect of SAMe treatment

in this condition. The effect of SAMe in the treatment of

other diseases, such as osteoarthritis, has also been investigated.

This chapter reviews (i) the biochemistry and

functions of SAMe; (ii) altered SAMe metabolism in liver

disease; (iii) SAMe deficiency in depression; and (iv) the

effect of SAMe treatment in liver disease, depression, and

osteoarthritis.

BIOCHEMISTRY AND FUNCTIONS

SAMe Discovery

Although SAMe was discovered by Giulio Cantoni in

1953, the story of this molecule begins in 1890 with

Wilhelm His when he fed pyridine to dogs and

isolated N-methylpyridine from the urine and emphasized

the need to demonstrate both the origin of the

methyl group as well as the mechanism for its addition

to the pyridine (1). Both questions were addressed

by Vincent du Vigneaud who, during the late 1930s,

demonstrated that the sulfur atom of methionine was

converted to cysteine through the “transsulfuration”

pathway and discovered the “transmethylation” pathway,

that is, the exchange of methyl groups between

methionine, choline, betaine, and creatine. In 1951, Cantoni

demonstrated that a liver homogenate supplemented

with ATP and methionine converted nicotinamide to N Methylnicotinamide.

Two years later, he established that

methionine and ATP reacted to form a product, that he

originally called “Active Methionine,” capable of transferring

its methyl group to nicotinamide, or guanidinoacetic

acid, to form N-methylmethionine, or creatine in the

absence of ATP, which, after determination of its structure,

he called “AdoMet” (Fig. 1). Subsequently, Cantoni

and his colleagues discovered the enzyme that synthesizes

SAMe, methionine adenosyltransferase (MAT);

(S)-adenosylhomocysteine (SAH), the product of transmethylation

reactions; and SAH hydrolase, the enzyme

that converts SAH into adenosine and homocysteine

(Hcy). At about the same time, Bennett discovered that

folate and vitamin B12 could replace choline as a source of

methyl groups in rats maintained on diets containing Hcy

in place of methionine, a finding that led to the discovery

of methionine synthase (MS). In 1961, Tabor demonstrated

that the propylamino moiety of SAMe is

converted via a series of enzymatic steps to spermidine

and spermine. In the biosynthesis of polyamines,

5-deoxy-5-methylthioadenosine (MTA) was identified

as an end product. Thus, by the beginning of the 1960s,

Laster’s group could finally provide an integrated view,

similar to that depicted in Figure 2, combining the

transmethylation and transsulfuration pathways with

polyamine synthesis.

Since then, SAMe has been shown to donate (i) its

methyl group to a large variety of acceptor molecules

including DNA, RNA, phospholipids, and proteins;

(ii) its sulfur atom, via a series of reactions, to cysteine and

glutathione (GSH), a major cellular antioxidant; (iii) its

propylamino group to polyamines, which are required

for cell growth; and (iv) its MTA moiety, via a complex

set of enzymatic reactions known as the “methionine salvage

pathway,” to the resynthesis of this amino acid. All

these reactions can affect a wide spectrum of biological

processes ranging from metal detoxification and catecholamine

metabolism to membrane fluidity, gene expression,

cell growth, differentiation, and apoptosis (2), to

establish what Cantoni called the “AdoMet Empire.”

SAMe Synthesis and Metabolism

MAT is an enzyme extremely well conserved through evolution

with 59% sequence homology between the human

and Escherichia coli isoenzymes. In mammals, there are:

  • 1

  • 2 Mato and Lu

  • N O

  • N

  • N

  • O O

  • S+

  • N

  • N N

  • O

  • CH3

  • S--Adenosylmethionine

AdoMeit,, SAM,, SAMee

Figure 1 Structure of SAMe. (S)-adenosylmethionine (SAMe) has been

shown to donate: (i) its methyl group to a large variety of acceptor molecules

including DNA, RNA, phospholipids, and proteins; (ii) its sulfur atom, via a

series of reactions, to cysteine and glutathione, a major cellular antioxidant;

(iii) its propylamino group to polyamines, which are required for cell growth;

and (iv) its MTA moiety, via a complex set of enzymatic reactions known as

the “methionine salvation pathway,” to the resynthesis of this amino acid.

  • MS

  • MTA

  • Putrescine Spermidine

  • Spermine

  • MTA

  • Met

  • SAMe

  • SAH

  • Hcy

  • Cys

  • CBS

  • BHMT GNMT

  • MAT

  • MTs

  • Cystathionine

  • THF

  • 5,10-MTHF

  • 5-MTHF

  • XX-

  • CH3

  • Ser

  • α-Ketobutyrate

  • Betaine

  • N,N-Dimethyl-Gly

  • Serine

  • Glycine

  • GSH

into homocysteine (Hcy) via (S)-adenosylmethionine (SAMe) and (S)-

adenosylhomocysteine (SAH). The conversion of Met into SAMe is catalyzed

by methionine adenosyltransferase (MAT). After decarboxylation, SAMe can

donate the remaining propylamino moiety attached to its sulfonium ion to

putrescine to form spermidine and methylthioadenosine (MTA) and to spermidine

to form spermine and a second molecule of MTA. SAMe donates

its methyl group in a large variety of reactions catalyzed by dozens of

methyltransferases (MTs), the most abundant in the liver being glycine-N Methyltransferase

(GNMT). The SAH thus generated is hydrolyzed to form

Hcy and adenosine through a reversible reaction catalyzed by SAH hydrolase.

Hcy can be methylated to form methionine by two enzymes: methionine

synthase (MS) and betaine homocysteine methyltransferase (BHMT). In the

liver, Hcy can also undergo the transsulfuration pathway to form cysteine via

a two-step enzymatic process. In the presence of serine, Hcy is converted

to cystathionine in a reaction catalyzed by cystathionine -synthase (CBS).

Cystathionine is then hydrolyzed by cystathionase to form cysteine, a precursor

of the synthesis of glutathione (GSH). In tissues other than the liver,

kidney, and pancreas, cystathionine is not significantly converted to GSH due

to the lack of expression of one or more enzymes of the transsulfuration

pathway. The expression of BHMT is also limited to the liver. All mammalian

tissues convert Met into Hcy, via SAMe and SAH, and remethylate Hcy into

Met via the MS pathway. Abbreviations: THF, tetrahydrofolate; 5,10-MTHF,

methylenetetrahydrofolate; 5-MTHF, methyltetrahydrofolate; Ser, serine; Gly,

glycine; X, methyl acceptor molecule; X-CH3, methylated molecule.

three isoforms of MAT (MATI, MATII, and MATIII) that

are encoded by two genes (MAT1A and MAT2A). MATI

andMATIII are tetrameric and dimeric forms, respectively,

of the same subunit (1) encoded by MAT1A, whereas the

MATII isoform is a tetramer of a different subunit (2) encoded

by MAT2A. A third gene, MAT2β encodes for a

subunit that regulates the activity of MATII (lowering the

Km and Ki for methionine and SAMe, respectively) but not

ofMATI orMATIII (2). Adult differentiated liver expresses

MAT1A, whereas extrahepatic tissues and fetal liver express

MAT2A. MAT1A expression is silenced in HCC. It

is an intriguing question why there are three different

MAT isoforms in the liver. The predominant liver form,

MATIII, has lower affinity for its substrates, a hysteretic

response to methionine (a hysteretic behavior, defined as

a slow response to changes in substrate binding, has been

described for many important enzymes in metabolic regulation),

and higher Vmax, contrasting with the other two

enzymes. On the basis of the differential properties of hepatic

MAT isoforms, it has been postulated that MATIII is

the truly liver-specific isoform. Under normal conditions,

MATI would, as MATII outside the liver, synthesize most

of the SAMe required by the hepatic cells. However, after

an increase in methionine concentration, that is, after

a protein-rich meal, conversion to the high-activity

MATIII would occur and methionine excess will be eliminated

(Fig. 2). This will lead to accumulation of SAMe

and activation of glycine N-methyltransferase (GNMT),

the main enzyme involved in hepatic SAMe catabolism.

Consequently, the excess of SAMe will be eliminated and

converted to homocysteine via SAH. Once formed, the

excess of homocysteine will be used for the synthesis of

cysteine and -ketobutyrate as a result of its transsulfuration.

This pathway involves two enzymes: cystathionine

-synthase (CBS), that is activated by SAMe, and

cystathionase. Cysteine is then utilized for the synthesis

of GSH as well as other sulfur-containing molecules

such as taurine, while -ketobutyrate penetrates the mitochondria

where it is decarboxylated to carbon dioxide

and propionyl CoA. Because SAMe is an inhibitor of 5,10-

methylenetetrahydrofolate-reductase (MTHFR), this will

prevent the regeneration of methionine after a load of this

amino acid. At the mRNA level, SAMe maintains MAT1A

and GNMT expression while inhibiting MAT2A expression.

This modulation by SAMe of both the flux of methionine

into the transsulfuration pathway and the regeneration

of methionine maximizes the production of cysteine

and -ketobutyrate, and consequently of ATP, after a methionine

load minimizing the regeneration of this amino

acid (oxidative methionine metabolism).

ALTERED SAMe METABOLISM AND DISEASE

Altered SAMe Metabolism in Liver Disease

Accumulating evidence supports the importance of maintaining

normal SAMe level in mammalian liver, as both

chronic deficiency and excess lead to liver injury, steatosis,

and development of hepatocellular carcinoma (HCC)

(2,3). Majority of the patients with cirrhosis have impaired

SAMe biosynthesis because of lower MAT1A mRNA levels

and inactivation of MATI/III (4,5). However, patients

with GNMT mutations have been identified and they also

S-Adenosylmethionine 3

have evidence of liver injury (6). In mice, loss of GNMT

results in supraphysiological levels of hepatic SAMe and

aberrant methylation (7). The molecular mechanisms responsible

for injury and HCC formation are different in

MAT1A and GNMT knockout mice but these findings illustrate

the importance of keeping SAMe level within a

certain range within the cell.

In contrast to normal non proliferating (differentiated)

hepatocytes, which rely primarily on MATI/III to

generate SAMe and maintain methionine homeostasis,

embryonic and proliferating adult hepatocytes as well

as liver cancer cells instead rely on MATII to synthesize

SAMe (2). Liver cancer cells often have very low

levels of GNMT and CBS expression and increased expression

of MAT2β, which, as mentioned earlier, lowers

the Km for methionine and the Ki for SAMe of MATII.

Consequently, proliferating hepatocytes and hepatoma

cells tend to utilize methionine into protein synthesis regardless

of whether methionine is present in high or low

amounts and to divert most homocysteine away from the

transsulfuration pathway by regenerating methionine and

tetrahydrofolate (THF) (aerobic methionine metabolism).

MAT2A/MAT2β-expressing hepatoma cells have lower

SAMe levels than cells expressing MAT1A, which also favors

the regeneration of methionine and THF. From these

results, it becomes evident that proliferating hepatocytes

and hepatoma cells do not tolerate well high SAMe levels

for converting methionine via the transsulfuration pathway

to cysteine and -ketobutyrate.

The finding that MAT1A, GNMT, MTHFR, and CBS

knockout mice spontaneously develop fatty liver (steatosis)

and, in the case of MAT1A- and GNMT-deficient animals,

HCC also (3) demonstrates the synchronization of

methionine metabolism with lipid metabolism and hepatocyte

growth.

The medical implications of these observations are

obvious, since the majority of cirrhotic patients, independent

of the etiology of their disease, have impaired

metabolism of methionine and reduced hepatic SAMe

synthesis and are predisposed to develop HCC (4,5); and

individuals with GNMT mutations that lead to abnormal

SAMe catabolism develop liver injury (6). Moreover, the

observation that genetic polymorphisms that associate

with reduced MTHFR activity and increased thymidylate

synthase activity, both of which are essential in minimizing

uracyl misincorporation into DNA, may protect

against the development of HCC in humans (8) further

supports that this synchronization may be an adaptive

mechanism that is programmed to fit the specific needs of

hepatocytes, and that alterations in the appropriate balance

between methionine metabolism and proliferation

may be at the origin of the association of cancer with fatty

liver disease.

An explanation for these observations connecting

methionine metabolism with the development of fatty

liver and HCC has remained elusive because the association

of SAMe with lipid metabolism and hepatocyte

proliferation is, at first glance, not intuitive. During

the past years, a signaling pathway that senses cellular

SAMe content and that involves AMP-activated protein

kinase (AMPK) has been identified to operate in hepatocytes

(9,10). AMPK is a serine/threonine protein kinase

that plays a crucial role in the regulation of energy homeostasis

and cell proliferation. AMPK is activated by stress

conditions leading to an increase in the AMP/ATP ratio,

such as during liver regeneration. Once activated, AMPK

shuts down anabolic pathways that mediate the synthesis

of proteins, fatty acids, lipids, cholesterol, and glycogen

and stimulates catabolic pathways such as lipid oxidation

and glucose uptake restoring ATP levels and keeping

the cellular energy balance. The finding that in the

liver AMPK activity is tightly regulated by SAMe (9,10)

has provided a first link between methionine metabolism,

lipid metabolism, and cell proliferation. Moreover, excess

SAMe can induce aberrant methylation of DNA and

histones, resulting in epigenetic modulation of critical

carcinogenic pathways (7). Finally, there is evidence indicating

that SAMe regulates proteolysis, widening its

spectrum of action. In hepatocytes, the protein levels of

prohibitin 1 (PHB1) (11), the apurinic/apyrimidininc endonuclease

(APEX1) (12), and the dual specificity MAPK

phosphatase (DUSP1) (13) are stabilized by SAMethrough

a process that may involve proteasome inactivation. PHB1

is a chaperone-like protein involved in mitochondrial

function, APEX1 is a key protein involved in DNA repair

and genome stability, and DUSP1 is a member of a family

of mitogen-activated protein kinases (MAPKs) phosphatases,

which simultaneously dephosphorylates both

serine/threonine and tyrosine residues.

SAMe Deficiency in Depression

Major depression has been associated with a deficiency

in methyl groups (folate, vitamin B12, and SAMe) (14,15).

Thus, depressed patients often have low plasma folate and

vitamin B12 and reduced SAMe content in the CSF. Moreover,

patients with low plasma folate appear to respond

less well to antidepressants. The mechanism by which low

SAMe concentrations may contribute to the appearance

and evolution of depression is, however, not well known.

SAMe-dependent methylation reactions are involved in

the synthesis and inactivation of neurotransmitters, such

as noradrenaline, adrenaline, dopamine, serotonin, and

histamine; and the administration of drugs that stimulate

dopamine synthesis, such as L-dihydroxyphenylalanine,

cause a marked decrease in SAMe concentration in rat

brain and in plasma and CSF in humans. Moreover, various

drugs that interfere with monoaminergic neurotransmission,

such as imipramine and desipramine, reduce

brain SAMe content in mice (14,15). As in the liver, abnormal

SAMe levels may contribute to depression through

perturbation of multiple metabolic pathways in the brain.

Interestingly, alterations in methionine metabolism that

lead to a decrease in the brain SAMe/SAH ratio associate

with reduced leucine carboxyl methyltransferase-1

(LCMT-1) and phosphoprotein phosphatase 2AB (PP2AB)

subunit expression, and accumulation of unmethylated

PP2A (16). PP2A enzymes exist as heterotrimeric complexes

consisting of catalytic (PP2AC), structural (PP2AA),

and regulatory (PP2AB) subunits (17). Different PP2AB

subunits have been described that determine the substrate

specificity of the enzyme. PP2AC subunit is methylated

by SAMe-dependent LCMT-1 and demethylated by a specific

phosphoprotein phosphatase methylesterase (PME1).

PP2AC methylation has no effect on PP2A activity but has

a crucial role in the recruitment of specific PP2AB subunits

4 Mato and Lu

to the PP2AA,B complex and therefore PP2A substrate

specificity. Downregulation of LCMT-1 and PP2AB and

accumulation of unmethylated PP2A are associated with

enhanced Tau phosphorylation and neuronal cell death

(16).

INDICATIONS AND USAGE

SAMe Treatment in Animal Models of Liver Disease

The importance of the metabolism of methyl groups in

general, and SAMe in particular, to normal hepatic physiology,

coupled with the convincing body of evidence

linking abnormal SAMe content with the developmental

of experimental and human liver disease, led to the

examination of the effect of SAMe supplementation in

various animal models of liver disease. SAMe administration

to alcohol-fed rats and baboons reduced GSH depletion

and liver damage (2,18). SAMe improved survival

in animal models of galactosamine-, acetaminophen- and

thioacetamide-induced hepatotoxicity, and in ischemia reperfusion–

induced liver injury (18). SAMe treatment

also diminished liver fibrosis in rats treated with carbon

tetrachloride (18) and reduced neoplastic hepatic nodules

in animal models of HCC (19,20). Similar to the liver,

SAMe can block mitogen-induced growth and induce

apoptosis in human colon cancer cells (21,22).

SAMe Treatment in Human Diseases

SAMe has been used in humans for the past 20 years for the

treatment of osteoarthritis, depression, and liver disease.

In 2002, the Agency for Healthcare Research and Quality

(AHRQ) reviewed 102 individual clinical trials of SAMe

(23). Of these 102 studies, 47 focused on depression, 14

focused on osteoarthritis, and 41 focused on liver disease.

Of the 41 studies in liver disease, 9 were for cholestasis of

pregnancy, 12 were for other causes of cholestasis, 7 were

for cirrhosis, 8 were for chronic hepatitis, and 4 were for

various other chronic liver diseases.

Pharmacokinetics of SAMe

Orally administered SAMe has low bioavailability, presumably

because of a significant first-pass effect (degradation

in the gastrointestinal tract) and rapid hepatic

metabolism. Peak plasma concentrations obtained with

an enteric-coated tablet formulation are dose related, with

peak plasma concentrations of 0.5 to 1 mg/L achieved

three to five hours after single doses ranging from 400 to

1000 mg (23). Peak levels decline to baseline within 24

hours. One study showed a significant gender difference

in bioavailability, with women showing three- to sixfold

greater peak plasma values than men (23). Plasma-protein

binding of SAMe is no more than 5%. SAMe crosses the

blood–brain barrier, with slow accumulation in the CSF.

Unmetabolized SAMe is excreted in urine and feces.

Parenterally administered SAMe has much higher

bioavailability. However, this form is currently not approved

for use in the United States.

SAMe Treatment in Liver Diseases

Out of the 41 studies in liver disease analyzed by AHRQ,

8 studies were included in a meta-analysis of the efficacy

of SAMe to relieve pruritus and decrease elevated

serum bilirubin levels associated with cholestasis of pregnancy

(23). Compared with placebo, treatment with SAMe

was associated with a significant decrease in pruritus and

serum bilirubin levels. Similar results were obtained when

six studies were included in a meta-analysis of the efficacy

of SAMe to relieve pruritus and decrease bilirubin levels

associated with cholestasis caused by various liver diseases

other than pregnancy.

In 2001, the Cochrane Hepato-Biliary Group analyzed

eight clinical trials of SAMe treatment of alcoholic

liver disease including 330 patients (24). This meta analysis

found SAMe decreased total mortality [odds

ratio (OR) 0.53, 95% confidence interval (CI): 0.22 to 1.29]

and liver-related mortality (OR 0.63, 95% CI: 0.25 to 1.58).

However, because many of the studies were small and

the quality of the studies varied greatly, the Cochrane

Group concluded, “SAMe should not be used for alcoholic

liver disease outside randomized clinical trials” (24). The

AHRQ reached a similar conclusion, “For liver conditions

other than cholestasis additional smaller trials should be

conducted to ascertain which patient populations would

benefit more from SAMe, and what interventions (dose

and route of administration) are most effective” (23). The

Cochrane Hepato-Biliary Group also concluded that only

one trial including 123 patients with alcoholic cirrhosis

used adequate methodology and reported clearly on mortality

and liver transplantation. In this study (25), mortality

decreased from 30% in the placebo group to 16% in

the SAMe group (P = 0.077). When patients with more

advanced cirrhosis (Child score C) were excluded from

the analysis (eight patients), the mortality was significantly

less in the SAMe group (12%) as compared with the

placebo group (25%, P=0.025). In this study, 1200 mg/day

was administered orally. Unfortunately, new controlled

prospective double-blind multicenter studies on the benefits

of SAMe for liver diseases are lacking.

 

SAMe Treatment in Depression

Out of the 39 studies in depression analyzed by the AHRQ,

28 studies were included in a meta-analysis of the efficacy

of SAMe to decrease symptoms of depression (23). Compared

with placebo, treatment with SAMe was associated

with an improvement of approximately six points in the

score of the Hamilton Rating Scale for Depression measured

at three weeks (95% CI: 2.2 to 9.0). This degree of

improvement was statistically as well as clinically significant.

However, compared with the treatment with conventional

antidepressant pharmacology, treatment with

SAMe was not associated with a statistically significant

difference in outcomes. With respect to depression, the

AHRQ report concluded, “Good dose-escalation studies

have not been performed using the oral formulation of

SAMe for depression” (23). The AHRQ report also concluded,

that “Additional smaller clinical trials of an exploratory

nature should be conducted to investigate uses

of SAMe to decrease the latency of effectiveness of conventional

antidepressants and to treat of postpartum depression”

(23). Unfortunately, these clinical studies are still

lacking.

SAMe Treatment in Osteoarthritis

Out of the 13 studies in osteoarthritis analyzed by the

AHRQ, 10 studies were included in a meta-analysis of

S-Adenosylmethionine 5

the efficacy of SAMe to decrease pain of osteoarthritis

(23). Compared with placebo, one large randomized clinical

trial showed a decrease in the pain of osteoarthritis

with SAMe treatment. Compared with the treatment

with nonsteroidal anti-inflammatory medications, treatment

with oral SAMe was associated with fewer adverse

effects while comparable in reducing pain and improving

functional limitation. In 2009, the Cochrane Osteoarthritis

Group analyzed 4 clinical trials including 656 patients, all

comparing SAMe with placebo (26). The Cochrane Group

concluded, “The effects of SAMe on both pain and function

may be potentially clinically relevant and, although

effects are expected to be small, deserve further clinical

evaluation in adequately sized randomized, parallel group

trials in patients with knee or hip osteoarthritis.

Meanwhile, routine use of SAMe should not be

advised” (26).

Adverse Effects

The risks of SAMe are minimal. SAMe has been used in

Europe for more than 20 years and is available under prescription

in Italy, Germany, United Kingdom, and Canada,

and over the counter as a dietary supplement in the United

States, China, Russia, and India. The most common side

effects of SAMe are nausea and gastrointestinal disturbance,

which occurs in less than 15% of treated subjects.

Recently, SAMe administration to mice treated with cisplatin

has been found to increase renal dysfunction (27).

Whether SAMe increases cisplatin renal toxicity in humans

is not known.

Interactions with Herbs, Supplements, and Drugs

Theoretically,SAMe might increase the effects and adverse

effects of products that increase serotonin levels, which

include herbs and supplements such as Hawaiian Baby

Woodrose, St. John’s wort, and L-tryptophan, as well as

drugs that have serotonergic effects. These drugs include

tramadol (Ultram), pentazocine (Talwin), clomipramine

(Anafranil), fluoxetine (Prozac), paroxetine (Paxil), sertraline

(Zoloft), amitriptyline (Elavil), and many others. It is

also recommended that SAMe should be avoided in patients

taking monoamine oxidase inhibitors or within two

weeks of discontinuing such a medication.

CONCLUSIONS

Although evidence linking abnormal SAMe content with

the development of experimental and human liver disease

is very convincing, the results of clinical trials of

SAMe treatment of liver disease are not conclusive. Consequently,

SAMe should not be used outside clinical trials

for the treatment of liver conditions other than cholestasis.

A new clinical study enrolling a larger number of patients

should be carried out to confirm that SAMe decreases

mortality in alcoholic liver cirrhosis. This is important because

if SAMe improves survival, SAMe will become the

only available treatment for patients with alcoholic liver

cirrhosis.

Although depression has been associated with a deficiency

in SAMe, it is not yet clear whether this is a consequence

or the cause of depression. To clarify this point,

more basic research and the development of new experimental

models are needed. Clinical trials indicate that

SAMe treatment is associated with an improvement of

depression. Dose studies using oral SAMe should be performed

to determine the best dose to be used in depression.

New studies should also be carried out where the

efficacy of SAMe is compared with that of conventional

antidepressants.

With respect to osteoarthritis, at present there is no

evidence associating a deficiency in SAMe with the appearance

of the disease. Moreover, the efficacy of SAMe

in the treatment of osteoarthritis is also not convincing at present.

 

ACKNOWLEDGMENTS

This work was supported by grants from NIH DK51719

(to S. C. L.) and AT-1576 (to S. C. L. and J. M. M.) and

SAF 2008-04800 (to J. M. M.). CIBERehd is funded by the

Instituto de Salud Carlos III.

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4. Duce AM, Ortiz P, CabreroS, et al. S-Adenosyl-L-methionine

synthetase and phospholipid methyltransferase are inhibited

in human cirrhosis. Hepatology 1988; 8(1):65–68.

5. Avila MA, Berasain C, Torres L, et al. Reduced mRNA

abundance of the main enzymes involved in methionine

metabolism in human liver cirrhosis and hepatocellular carcinoma.

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6. Mudd SH, Cerone R, Schiaffino MC, et al. Glycine N Methyltransferase

deficiency: A novel inborn error causing

persistent isolated hypermethioninemia. J Inherit Metab Dis

2001; 24;448–464.

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Loss of the glycine N-methyltransferase gene leads to steatosis

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8. Yuan J-M, Lu SC, Van den Berg D, et al. Genetic polymorphisms

in the methylenetetrahydrofolate reductase and

thymidylate synthase genes and risk of hepatocellular carcinoma.

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9. Mart´ınez-Chantar ML, Vazquez-Chantada M, Garnacho M,

et al. S-Adenosylmethionine regulates cytoplasmic HuR

via AMP-activated kinase. Gastroenterology 2006; 131;

223–232.

10. V´azquez-Chantada M, Ariz U, Varela-Rey M, et al. Evidence

for LKB1/AMP-activated protein kinase/endothelial nitric

oxide synthase cascade regulated by hepatocyte growth factor,

S-adenosylmethionine, and nitric oxide in hepatocyte

proliferation. Hepatology 2009; 49:608–617.

11. Santamar´ıa E, Avila MA, Latasa MU, et al. Functional proteomics

of non-alcoholic steatohepatitis: Mitochondrial proteins

as targets of S-adenosylmethionine. Proc Nat Acad Sci

U S A 2003; 100(6):3065–3070.

12. Tomasi ML, Iglesias-Ara A, Yang H, et al. S Adenosylmethionine

regulates apurinic/apyrimidinic

endonuclease 1 stability: Implication in hepatocarcinogenesis.

Gastroenterology 2009; 136(3):1025–1036.

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regulates dual-specificity mitogen activated

protein kinase phosphatase expression in mouse

and human hepatocytes. Hepatology 2010, in press.

14. Bottiglieri T. S-Adenosyl-L-methionine (SAMe): From the

bench to the bedside—Molecular basis of a pleiotrophic

molecule. Am J Clin Nutr 2002; 76(5):1151S–1157S.

15. Miller AL. The methylation, neurotransmitter, and antioxidant

connections between folate and depression. Altern Med

Rev 2008; 13;216–226.

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deficiency induces in vitro and mouse brain region-specific

downregulation of leucine carboxyl methyltransferase-1 and

protein phosphatase 2A B subunit expression that correlate

with enhanced Tau phosphorylation. J Neurosci 2008;

28(45):11477–11487.

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2A and S-adenosylmethionine in mitogen induced

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Androstenedione

Glossary, sports nutritionSuccess Chemistry Staff

INTRODUCTION

Androstenedione (chemical name: 4-androstene-3,17-

dione) is a steroid hormone produced primarily in the reproductive

system and adrenal glands in men and women.

It circulates in the bloodstream and is the immediate precursor

to the potent anabolic/androgenic hormone testosterone

in the steroid synthesis pathway. Despite this well known

physiological classification, as well as a growing

body of evidence demonstrating that orally administered

androstenedione is converted to more potent steroid hormones,

the United States Food and Drug Administration

originally classified the hormone as a “dietary supplement.”

As such, it was available to the general public

without a prescription and for nearly a decade could be

easily purchased in health clubs, nutrition stores, and

over the Internet. This over-the-counter availability of

androstenedione came to an end when Food and Drug

Administration banned its sale in early 2004. The ban

was then codified with the passing of the 2004 Anabolic

Steroid Control Act. This law reclassified androstenedione

as an anabolic steroid and hence a controlled

substance.

GENERAL DESCRIPTION

The original and seemingly contradictory classification of

androstenedione as a dietary supplement was based on

the definition set forth in the 1994 Dietary Supplement

Health and Education Act (DSHEA). According to the

DSHEA, a substance was defined as a dietary supplement

if it was a “product (other than tobacco) intended

to supplement the diet that bears or contains one or more

of the following dietary ingredients: a vitamin, mineral,

amino acid, herb or other botanical. . . or a concentrate,

metabolite, constituent, extract, or combination of any ingredient

described above.” Hence, because androstenedione

could be synthesized from plant products, it fell

under that umbrella. Furthermore, the DSHEA specified

that the Department of Justice could not bring action to

remove a product unless it was proven to pose “a significant

or unreasonable risk of illness or injury” when

used as directed. Not surprisingly, after the passing of the

DSHEA, the use of dietary supplements increased dramatically.

In fact, by 1999, the dietary supplement industry

in the United States was generating annual sales of $12

billion (1).

Initially, androstenedione use was primarily confined

to athletes in strength and endurance-related sports,

an interest that seems to have sprung from reports of

its use in the official East German Olympic athlete doping

program. The event that most dramatically sparked

widespread curiosity in androstenedione, however, was

the media report that the St. Louis Cardinals baseball

player Mark McGwire had used androstenedione in the

1999 season (during which he broke the record for most

home runs in a season). The publicity that surrounded

this supplement also prompted an increased interest in

related “prohormones,” such as norandrostenedione and

androstenediol. This then led to a proliferation of claims

concerning the potential benefits of androstenedione use.

Manufacturers credited these products not only with

promoting muscle growth and improving athletic performance

but also with increasing energy, libido, sexual

performance, and general quality of life. Additionally,

androstenedione was often packaged in combination

with other substances as part of an intensive nutritional

approach to performance enhancement. An example

of such a combination is shown in Figure 1.

Clearly, the use of androstenedione and related compounds

during that time went well beyond the accumulation

of data that could provide a rational basis for

their use.

PROHORMONE FACTORS

  • 4-Androstenedione: 100 mg

  • 19-Nor-5-Androstenedione: 50 mg

  • 5-Androstenediol: 50 mg

  • DHEA: 50 mg

  • GH/1GF FACTORS

  • L-Arginine Pyroglutamate: 2500 mg

  • L-Ornithine Alpha-Ketoglutarate: 1250 mg

  • Taurine: 750 mg

  • Colostrum: 250 mg

  • LH BOOSTER

  • Tribulus: 250 mg

  • Acetyl-L-Carnitine: 250 mg

  • L-Carnitine: 100 mg

  • DHT BLOCKERS

  • Saw Palmetto: 200 mg

  • Beta Sitosterol: 200 mg

  • Pygeum Africanum: 50 mg

ESTROGEN BLOCKERS

  • Kudzu: 100 mg

  • Chrysin: 250 mg

  • 4-Androstenedione

  • Dehydroepiandrosterone

  • Estrone

  • HCOOH

  • HCOOH

  • 17β-HSD

  • CYP19 (aromatase)

  • CYP19 (aromatase)

  • 17β-HSD

  • 3β-HSD

  • Testosterone Estradiol-17β

BIOCHEMISTRY AND PHYSIOLOGY

Androstenedione is a steroid hormone that is produced

primarily in the adrenals, testes, and ovaries. It is classified

as a “weak androgen” because it binds to the body’s

receptor for androgen hormones in a much less potent

fashion than classic anabolic/androgenic steroids such

as testosterone (2). It is synthesized from the precursor

hormone dehydroepiandrosterone (itself a dietary supplement)

and is the direct precursor to testosterone. In normal

physiological circumstances, androstenedione can also be

converted to potent feminizing hormones such as estrone

and estradiol (both members of the “estrogen” class of hormones).

The relationship between androstenedione, other

steroid hormones, and the enzymes involved in the conversion

of androstenedione to testosterone and estrogens

is shown in Figure 2.

Importantly, the enzymes that convert androstenedione

to potent hormones such as testosterone and estradiol

are active not only in endocrine glands but also in

many peripheral body tissues such as muscle, bone, liver,

and brain (3). Thus, if orally administered androstenedione

has biological activity, it may act either directly or

by conversion to these more potent agents.

ANDROSTENEDIONE USE

There were no precise data concerning the prevalence of

androstenedione use in the general population during the

time that it was widely available. Our best estimates were

based on industry sales figures and extrapolations from

data on classic anabolic/androgenic steroid use in specific

populations. For example, in 1997, it was estimated that

4.9% of male and 2.4% of female adolescents in the United

States had used illegal anabolic steroids (4). Because these

substances were so readily available, there was concern

that androstenedione use in this particularly susceptible

population may have greatly exceeded these numbers.

In fact, in a survey administered in five health clubs in

Boston, Massachusetts, in 2001, 18% of men and 3% of

women respondents admitted to using androstenedione

or other adrenal hormone dietary supplements at least

once. These percentages suggested that as many as 1.5

million U.S. health club members alone may have used

these substances (5).

PHARMACOKINETICS AND HORMONAL EFFECTS OF

ANDROSTENEDIONE IN MEN

Because so many of the claims that surrounded androstenedione

were based on the premise that oral administration

increases serum testosterone levels, it may

be surprising to some that prior to 1999, there was only a

single published study investigating the ability of orally

administered androstenedione to be converted to more

potent steroid hormones (6). In this study, two women

were given a single dose of androstenedione, and the

levels were subsequently measured over the next several

hours. Since 1999, however, numerous small studies

(mostly in men) have investigated the effects of the supplement

(6–16). In general, these studies report that serum

androstenedione levels increase dramatically after oral administration

and thus confirm that a significant portion of

the supplement is absorbed through the gastrointestinal

tract after ingestion. However, the answer to the more important

question, namely, whether it is then converted to

more potent steroid hormones such as testosterone and

estradiol, appears to be complex. In general, these studies

suggest that the ability of oral androstenedione to increase

estrogen and testosterone levels in men is dose dependent

and is possibly related to the age of the study population

as well. Specifically, the bulk of the research indicates that

when androstenedione is administered to men in individual

doses between 50 and 200 mg, serum estrogen levels

increase dramatically. However, larger individual doses

(e.g., 300 mg) are required to increase serum testosterone

levels.

For example, King and colleagues studied the effects

of a single 100-mg oral dose of androstenedione in 10 men

between the ages of 19 and 29 and reported that although

serum androstenedione and estradiol levels increased significantly,

testosterone levels did not change (13). These

investigators then specifically measured the portion of circulating

testosterone that is not bound to protein and considered

the “bioactive” portion (called “free testosterone”)

and similarly saw no effect of the supplement. In a separate

study, Leder and colleagues gave 0, 100, or 300 mg

of androstenedione to normal healthy men between the

ages of 20 and 40 for seven days and took frequent blood

samples on days 1 and 7 (14). As in the study by King, they

also found that men receiving both the 100- and 300-mg

doses of androstenedione experienced dramatic increases

in serum estradiol that were often well above the normal

male range.

Percentage change in serum testosterone and estradiol in healthy

men after a single androstenedione dose (as measured by eight hours of

frequent blood sampling). Source: Adapted from Ref. 14.

did not affect serum testosterone levels. As shown in

Figure 3, however, the novel finding of this study was

that 300 mg of androstenedione increased serum testosterone

levels significantly, even though by only a modest

amount (34%).

Leder and colleagues further observed that there

was a significant degree of variability among men with

regard to their serum testosterone response after androstenedione

ingestion. As shown in Figure 4, some subjects,

even in the 300-mg dose group, experienced relatively

little change in testosterone levels, whereas serum

testosterone levels doubled in other men. This finding

suggests that there may be individual differences in the

way androstenedione is metabolized that could impact

any one person’s physiological response to taking the

supplement.

Brown and colleagues investigated the hormonal response

in a group of men between the ages of 30 and

Figure 4 Individual variability in the peak serum testosterone level

achieved after a single 300-mg dose of androstenedione in men. Each line

represents one study subject. Source: Adapted from Ref. 14.

56 (10). In this study, subjects consuming 100 mg of androstenedione

three times daily experienced increases in

serum estrogens but not in serum testosterone. However,

unlike in the study by King and colleagues discussed in the

previous text, free testosterone did increase significantly

(even though again by only a small amount).

Finally, several studies have compared the hormonal

effects of androstenedione with those of other

“prohormone” dietary supplements. Broeder and colleagues

studied the results of a 100-mg twice-daily dose

of oral androstenedione, androstenediol (a closely related

steroid hormone), or placebo in men between

the ages of 35 and 65 (7). They found that both compounds

increased estrogen levels but neither affected total

serum testosterone levels. Similarly, Wallace and colleagues

studied the effects of 50-mg twice-daily doses

of androstenedione and DHEA in normal men and reported

no increases in serum testosterone levels with

either (16).

EFFECTS ON MUSCLE SIZE AND STRENGTH IN MEN

The results of the studies discussed earlier suggest that

androstenedione use in men would be less likely to promote

the muscle building and performance-enhancing

effects associated with testosterone use and more likely

to induce the undesirable feminizing effects associated

with estrogens. Several studies have assessed the ability

of androstenedione (with or without exercise) to increase

muscle size and strength and have been uniformly

disappointing (7,9,13,15,16). For example, Broeder

and colleagues, in the study described earlier, also

measured changes in body composition and strength

in subjects taking 100 mg androstenedione twice daily

in combination with a 12-week intensive weight-training

program (7). Despite using sensitive methods that can detect

small changes in body composition, they found no

differences in muscle mass, fat mass, or strength in the

subjects receiving androstenedione compared with those

receiving a placebo tablet. Importantly, however, in this

study as well as all of these studies referenced earlier,

the supplement was given in doses that were not sufficient

to increase testosterone levels. It thus remains unknown

whether doses of androstenedione sufficient to increase

testosterone levels enhance muscle mass or athletic

performance.

METABOLISM OF ANDROSTENEDIONE IN MEN

One of the consistent findings of the various androstenedione

studies in men is the inefficiency of conversion of

the supplements to testosterone. Leder and colleagues explored

this issue further by investigating the pattern of

androstenedione metabolism in healthy men (17). Specifically,

they measured the concentration of inactive testosterone

metabolites (also called “conjugates”) in the urine

of subjects ingesting androstenedione and found an increase

of over 10-fold compared with their baseline levels.

This finding was in direct contrast to the much more

modest changes in serum testosterone they had observed.

It suggests that although much of the androstenedione

Figure 5 Serum testosterone levels during 12 hours of frequent blood

sampling in postmenopausal women. Circles represent control subjects receiving

no supplement, triangles those receiving 50 mg of androstenedione,

and squares those receiving 100 mg. Source: Adapted from Ref. 18.

that is absorbed after oral administration is converted to

testosterone, it is then immediately further metabolized

to inactive compounds in the liver. The investigators confirmed

this hypothesis by directly measuring the concentration

of one of these inactive metabolites (testosterone

glucuronide) in the serum of these subjects. As expected,

they found that testosterone glucuronide levels increased

by 500% to 1000% (as opposed to the 34% increase in biologically

active serum testosterone after a single 300-mg

dose of oral androstenedione). Together, these findings

demonstrate the effectiveness of the liver in inactivating

steroid molecules when taken orally.

PHARMACOKINETICS AND HORMONAL EFFECTS OF

ANDROSTENEDIONE IN WOMEN

Since the initial report of androstenedione administration

in two women in 1962 (6), research into the effects of the

supplement has focused largely on the hormonal response

to oral administration in young men. Between 2002 and

2003, however, two studies on women were published.

The first of these studies examined the effects of a single

dose of 0, 50, or 100 mg of androstenedione in postmenopausal

women (18). The findings of this study were

surprising. In contrast to the effects observed in men, even

these low doses increased testosterone levels significantly

in women (Fig. 5).

Also, unlike the results seen in men, estradiol levels

were unaffected by androstenedione administration.

In the other study, 100 mg of androstenedione was administered

to young, premenopausal, healthy women.

Similar to postmenopausal women, these subjects experienced

significant increases in serum testosterone levels

after androstenedione administration (estradiol was

not measured) (19). Importantly, in both of these studies,

the peak testosterone levels achieved by the older and

younger women taking androstenedione were often significantly

above the normal range. Together, these results

predict that the physiological effects of the supplement

may be different in men and women, as might their potential

toxicities. To date, however, there have been no

published reports investigating the long-term physiological

effects in women.

ADVERSE EFFECTS AND TOXICITY

Ever since the publicity surrounding androstenedione exploded

in 1999, many reports in the lay press have focused

on the potential dangerous side effects. Nonetheless,

with the exception of a single case description of a

man who developed two episodes of priapism in the setting

of androstenedione ingestion (20), there have been no

published reports of androstenedione-associated serious

adverse events. This fact should be only partially reassuring,

however, because androstenedione’s prior classification

as a dietary supplement (as opposed to a drug)

allowed manufacturers to avoid responsibility for rigorously

monitoring any potential toxicity of their product.

It is well known that oral administration of certain

testosterone derivatives can cause severe liver diseases,

and anabolic steroid use in general is associated with

anecdotal reports of myocardial infarction, sudden cardiac

death, and psychiatric disturbances (“roid rage”).

Nonetheless, despite androstenedione close chemical

similarity to these substances, it is important to note that it

is not a potent anabolic steroid nor does it have a chemical

structure similar to those specific compounds that cause

liver problems. Thus, the potential of androstenedione to

cause these particular serious side effects appears to be

limited. Of more pressing concern to clinicians are the

possible long-term effects in specific populations. In clinical

trials, the supplement was generally well tolerated,

though several studies did report that it reduces high density

lipoprotein (or “good cholesterol”) levels in men.

Importantly, however, even the longest of these studies

lasted only several months. It thus remains quite possible

that androstenedione use, especially at high doses,

could cause subtle physiological changes over prolonged

periods that could directly lead to adverse health consequences.

In men, for example, the dramatic increase in

estradiol levels observed with androstenedione administration

could, over time, lead to gynecomastia (male breast

enlargement), infertility, and other signs of feminization.

In women, because the supplement increases testosterone

levels above the normal range, it could cause hirsutism

(excess body hair growth), menstrual irregularities, or

male-like changes in the external genitalia. In children,

increases in both testosterone and estrogen levels could

cause precocious puberty or premature closure of growth

plates in bone, thereby compromising final adult height.

PURITY OF COMMERCIALLY AVAILABLE

ANDROSTENEDIONE

During its period of over-the-counter availability, androstenedione

was available from multiple manufacturers

and could be purchased as a tablet, capsule, sublingual

tablet, or even a nasal spray. Often, it was combined

with other products that claimed to limit its potential side

effects (such as chrysin, for example, which is purported

to decrease androstenedione’s conversion to estrogens).

Because the manufacture of dietary supplements was not

Androstenedione

Source: From Ref. 21.

subject to the same regulations as pharmaceuticals, the purity

and labeling of androstenedione-containing products

were often inaccurate. Catlin and colleagues, for example,

reported that urine samples from men treated with

androstenedione contained 19-norandrosterone, a substance

not associated with androstenedione metabolism

but rather with the use of a specific banned anabolic

steroid (21). Further investigation revealed that the androstenedione

product used contained a tiny amount of

the unlabeled steroid “19-norandrostenedione.” Though

the amount of 19-norandrostenedione was not physiologically

significant, it was enough to cause a “positive” urine

test for illegal anabolic steroid use when tested in the standard

fashion. In fact, it is precisely this type of contamination

that may have explained increases in positive tests

for 19-norandrosterone among competitive athletes in the

past decade. Additionally, it is now common for athletes

who test positive for norandrosterone or other androgenic

metabolites to point to dietary supplement contamination

as the potential explanation.

Catlin and colleagues also analyzed nine common

brands of androstenedione and showed that there was

considerable variation and mislabeling among products

in terms of both purity and content (Table 1).

REGULATORY STATUS AND DETECTION

Androstenedione was available over-the-counter from

1994 (when the DSHEA was passed) until it was reclassified

as an anabolic steroid by the Anabolic Steroid Control

Act in 2004. It is important to note that this reclassification

came without any evidence that androstenedione increased

muscle mass or strength, which was the previous

legal definition of an anabolic steroid. Virtually all sports

organizations, including the National Football League, the

National Collegiate Athletic Association, and the International

Olympic Committee, have banned androstenedione.

Despite these prohibitions, detection of androstenedione

has not been standardized. Specifically, the method

used most often to detect testosterone use, measurement of

the urinary testosterone-to-epitestosterone ratio, has not

proven to be reliable in establishing androstenedione use

(22). Further study is still needed to define novel testing

procedures that are able to detect androstenedione use

reliably.

CONCLUSIONS

Androstenedione is a steroid hormone, which, until 2004,

was a popular over-the-counter dietary supplement. Since

then, however, it has been classified as an anabolic steroid,

and hence a controlled substance. It is purported to increase

strength, athletic performance, libido, sexual performance,

energy, and general quality of life. Studies indicate

that when taken orally by men, small doses are

converted to potent estrogens and larger doses to both

testosterone and estrogens. Comparatively, there appears

to be a much more physiologically important increase in

estrogens compared with testosterone in men. In women,

the effects are reversed. Studies have thus far failed to

confirm any effect on muscle size or strength, though the

dosing regimens were modest. Although documentation

of adverse side effects among users of androstenedione is

scarce, there is considerable concern over potential longterm

toxicity, especially in women and adolescents.

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1. Anonymous. Herbal treatments: The promises and pitfalls.

Consum Rep 1999; 64:44–48.

2. Orth DN, Kovacs WJ. The adrenal cortex. In: Wilson D, Foster

DW, Kronenberg HM, et al., eds. Williams Textbook of

Endocrinology. Philadelphia, PA: W.B. Saunders Company,

1998:517–664.

3. Labrie F, Simard J, Luu-The V, et al. Structure, regulation

and role of 3 beta-hydroxysteroid dehydrogenase, 17 betahydroxysteroid

dehydrogenase and aromatase enzymes in

the formation of sex steroids in classical and peripheral

intracrine tissues. Baillieres Clin Endocrinol Metab 1994;

8(2):451–474.

4. Yesalis CE, Barsukiewicz CK, Kopstein AN, et al. Trends

in anabolic-androgenic steroid use among adolescents. Arch

Pediatr Adolesc Med 1997; 151:1197–1206.

5. Kanayama G, Gruber AJ, Pope HG Jr, et al. Over-the-counter

drug use in gymnasiums: An underrecognized substance

abuse problem? Psychother Psychosom 2001; 70(3):137–140.

6. Mahesh VB, Greenblatt RB. The in vivo conversion of dehydroepiandrosterone

and androstenedione to testosterone in

the human. Acta Endocrinol 1962; 41:400–406.

7. Broeder CE, Quindry J, Brittingham K, et al. The Andro

Project: Physiological and hormonal influences of androstenedione

supplementation in men 35 to 65 years old

participating in a high-intensity resistance training program.

Arch Intern Med 2000; 160(20):3093–3104.

8. Brown GA, Vukovich MD, Martini ER, et al. Effects of

androstenedione-herbal supplementation on serum sex hormone

concentrations in 30- to 59-year-old men. Int J Vitam

Nutr Res 2001; 71(5):293–301.

9. Brown GA, Vukovich MD, Reifenrath TA, et al. Effects of anabolic

precursors on serum testosterone concentrations and

adaptations to resistance training in young men. Int J Sport

Nutr Exerc Metab 2000; 10(3):340–359.

10. Brown GA, Vukovich MD, Martini ER, et al. Endocrine responses

to chronic androstenedione intake in 30- to 56-yearold

men. J Clin Endocrinol Metab 2000; 85(11):4074–4080.

11. Earnest CP, Olson MA, Broeder CE, et al. In vivo 4-

androstene-3,17-dione and 4-androstene-3 beta,17 beta-diol

supplementation in young men. Eur J Appl Physiol 2000;

81(3):229–232.

12. Ballantyne CS, Phillips SM, MacDonald JR, et al. The acute effects

of androstenedione supplementation in healthy young

males. Can J Appl Physiol 2000; 25(1):68–78.

20 Leder

13. King DS, Sharp RL, Vukovich MD, et al. Effect of oral androstenedione

on serum testosterone and adaptations to

resistance training in young men. J Am Med Assoc 1999;

281(21):2020–2028.

14. Leder BZ, Longcope C, Catlin DH, et al. Oral androstenedione

administration and serum testosterone concentrations

in young men. J Am Med Assoc 2000; 283(6):

779–782.

15. Rasmussen BB, Volpi E, Gore DC, et al. Androstenedione

does not stimulate muscle protein anabolism in

young healthy men. J Clin Endocrinol Metab 2000; 85(1):

55–59.

16. Wallace MB, Lim J, Cutler A, et al. Effects of dehydroepiandrosterone

vs. androstenedione supplementation in

men. Med Sci Sports Exerc 1999; 31(12):1788–1792.

17. Leder BZ, Catlin DH, Longcope C, et al. Metabolism of

orally administered androstenedione in young men. J Clin

Endocrinol Metab 2001; 86(8):3654–3658.

18. Leder BZ, Leblanc KM, Longcope C, et al. Effects of oral

androstenedione administration on serum testosterone and

estradiol levels in postmenopausal women. J Clin Endocrinol

Metab 2002; 87(12):5449–5454.

19. Kicman AT, Bassindale T, Cowan DA, et al. Effect of androstenedione

ingestion on plasma testosterone in young

women: A dietary supplement with potential health risks.

Clin Chem 2003; 49(1):167–169.

20. Kachhi PN, Henderson SO. Priapism after androstenedione

intake for athletic performance enhancement. Ann Emerg

Med 2000; 35(4):391–393.

21. Catlin DH, Leder BZ, Ahrens B, et al. Trace contamination

of over-the-counter androstenedione and positive urine test

results for a nandrolone metabolite. J Am Med Assoc 2000;

284(20):2618–2621.

22. Catlin DH, Leder BZ, Ahrens BD, et al. Effects of androstenedione

administration on epitestosterone metabolism in men.

Steroids 2002; 67(7):559–564.

Arginine

Glossary, sports nutritionSuccess Chemistry Staff

INTRODUCTION

Arginine was first isolated in 1895 from animal horn. It is

considered a nonessential amino acid under physiological

conditions; however, it may be classified as semi-essential

(or conditioned) in newborns, young children, or other

circumstances characterized by accelerated tissue growth

(e.g., infection, sepsis, trauma) when its production may

be too slow and not sufficient to meet the requirements

(1). Arginine is physiologically active in the L-form (L-Arg)

and participates in protein synthesis in cells and tissues.

It is essential for the synthesis of urea, creatine, creatinine,

and pyrimidine bases. It also strongly influences hormonal

release and has an important role in vascular dynamics,

participating in the synthesis of nitric oxide (NO).

BIOCHEMISTRY

Dietary arginine is particularly abundant in wheat

germ and flour, buckwheat, oatmeal, dairy products

(cottage cheese, ricotta cheese, nonfat dry milk, skimmed

yogurt), chocolate, beef (roasts, steaks), pork, nuts (coconut,

pecans, walnuts, almonds, hazel nuts, peanuts),

seeds (pumpkin, sesame, sunflower), poultry (chicken,

turkey), wild game (pheasant, quail), seafood (halibut,

lobster, salmon, shrimp, snails, tuna), chick peas, and

soybeans (2).

L-Arg, delivered via the gastrointestinal tract, is absorbed

in the jejunum and ileum of the small intestine. A

specific amino acid transport system facilitates this process

and participates also in the transport of the other

basic amino acids, L-lysine and L-histidine. About 60% of

the absorbed L-Arg is metabolized by the gastrointestinal

enterocytes, and only 40% remains intact reaching the

systemic circulation.

An insufficient arginine intake produces symptoms

of muscle weakness, similar to muscular dystrophy (3).

Arginine deficiency impairs insulin secretion, glucose production,

and liver lipid metabolism (4). Conditional deficiencies

of arginine or ornithine are associated with the

presence of excessive ammonia in the blood, excessive

lysine, rapid growth, pregnancy, trauma, or protein deficiency

and malnutrition. Arginine deficiency is also associated

with rash, hair loss and hair breakage, poor wound

healing, constipation, fatty liver, hepatic cirrhosis, and

hepatic coma (4).

Depending on nutritional status and developmental

stage, normal plasma arginine concentrations in humans

and animals range from 95 to 250 mol/L. Toxicity and

symptoms of high intake are rare, but symptoms of massive

dosages may include thickening and coarsening of

the skin, muscle weakness, diarrhea, and nausea.

The proximal renal tubule accounts for much of the

endogenous production of L-Arg from L-citrulline. In the

tubule, arginine reacts via the Krebs cycle with the toxic

ammonia formed from nitrogen metabolism, producing

the nontoxic and readily excretable urea (Fig. 1) (5). If this

mechanism does not efficiently handle metabolic byproducts

and if L-Arg intake is insufficient, ammonia rapidly

accumulates, resulting in hyperammonemia.

L-Arg undergoes different metabolic fates. NO,

L-citrulline, L-ornithine, L-proline, L-glutamate, and

polyamine-like putrescine are formed from L-Arg. Moreover,

the high-energy compound NO-creatinine phosphate,

which is essential for sustained skeletal muscle contraction,

is also formed from L-Arg (Fig. 2).

L-Arg, its precursors, and its metabolites are deeply

involved in the interaction of different metabolic pathways

and interorgan signaling. The amino acid influences

the internal environment in different ways: disposal

of protein metabolic waste; muscle metabolism; vascular

regulation; immune system function; healing and repair

of tissue; formation of collagen; and building of new bone

and tendons.

A leading role for arginine has been shown in the

endocrine system, vasculature, and immune response.

  • CO2 + NH4

  • +

  • NH2

  • NH2

  • C=O

  • 2ATP

  • CARBAMOYL

  • PHOSPHATE

  • 2ADP + Piz

  • GLUTAMATE

  • NH4

  • +-GROUPS

  • GLUTAMINE

  • ORNITHINE CITRULLINE

  • FUMARATE

  • ARGININE ARGININOSUCCINATE

  • ASPARTATE

  • ATP

  • AMP + Ppi + H2O

  • Figure 1 L-Arginine and Krebs cycle in the renal tubule.

  • 21

  • 22 Maccario et al.

  • Nitric Oxide

  • Nitric Oxide

  • Admatine

  • Aldehyde Agmatine

  • Agmatinase

  • Polyamines

  • Ornithine

  • Proline

  • Arginine

  • Group

  • Guanidine

  • CE COCH

  • HN

  • C NH

  • Protein

  • synthesis

  • Glycine

  • Guanidinoacetate

  • Pyrroline-5-carboxylate

  • Glutamyl-γ-′semialdehyde

  • Urea cycle Glutamine Glutamate

  • Creatine

  • Urea

  • Urea

  • NOS

  • NOS

  • Ca2+

  • ADC

  • ADC

  • OAT

  • Arginase-I

  • P-5-C

  • dehydrogenase

  • P-5-C

  • reductase

  • A-GAT

  • GMT

  • Glu synthase

  • DAO

  • NH3

Figure 2 L-Arginine metabolites. Abbreviations: ADC, arginine decarboxylase;

A:GAT, arginine:glycine amidinotransferase; DAO, diamine oxidase; Glu

synthase, glutamine synthase; GMT, guanidinoacetate-N-methyltransferase;

NOS, nitric oxide synthase; OAT, ornithine aminotransferase; P-5-C dehydrogenase,

pyrroline-5-carboxylate dehydrogenase; P-5-C reductase, pyrroline-

5-carboxylate reductase.

PHYSIOLOGY

Endocrine Actions

L-Arg functions as a secretagogue of a number of important

hormones at the pituitary, pancreas, and adrenal levels.

The effects on growth hormone (GH), prolactin (PRL),

adrenocorticotropic hormone (ACTH), and insulin secretion

will be discussed in detail.

GH Secretion

Among the various factors modulating somatotropin

function, arginine is well known to play a primary stimulatory

influence. Arginine has been shown to increase

basal GH levels and to enhance the GH responsiveness

to growth hormone releasing hormone (GHRH) both in

animals and in humans throughout their life span (6–9);

its GH-stimulating activity occurs after both IV and oral

administration and is dose dependent; 0.1 and 0.5 g/kg

are the minimal and the maximal IV effective doses, respectively.

Moreover, a low orally administered arginine

dose has been shown to be as effective as a high IV dose

in enhancing the GH response to GHRH both in children

and in elderly subjects (10,11).

Arginine, directly or indirectly via NO, is likely

to act by inhibiting hypothalamic somatostatin (SS) release.

It has been shown that arginine—but not isosorbidedinitrate

and molsidomine, two NO donors—stimulates

GH secretion (12,13), suggesting that it does not exert its

effects through the generation of NO. Moreover, arginine

does not modify either basal or GHRH-induced GH increase

from rat anterior pituitary (14). On the contrary, it

potentiates the GH response to the maximal GHRH dose

in humans. Arginine can elicit a response even when the

response has been previously inhibited by a GHRH administration,

which induces an SS-mediated negative GH

auto feedback (7,8,15). Moreover, arginine counteracts the

GH-inhibiting effect of neuroactive substances that act by

stimulating SS release; it does not modify the GH-releasing

activity of stimuli acting via SS reduction (8). Again, in favor

of an SS-mediated mechanism is also the evidence

that ornithine, the active form of arginine, is unable to

modify plasma GHRH levels in humans (16). Moreover,

arginine fails to potentiate the increased spontaneous nocturnal

GH secretion, which is assumed to reflect circadian

SS hyposecretion and GHRH hypersecretion, respectively

(8). Arginine does not influence the strong GH-releasing

action of ghrelin, the natural ligand of GH secretagogue

receptors, which is supposed to act as a functional antagonist

of SS at both the pituitary and the hypothalamic levels

(17,18).

The GH-releasing activity of arginine is sex dependent

but not age dependent, being higher in females than

in males but similar in children, young, and elderly subjects

(8,19–23). Moreover, it has been clearly demonstrated

that arginine totally restores the low somatotroph responsiveness

to GHRH in aging, when a somatostatin ergic hyperactivity

is likely to occur (20–23). This evidence clearly

indicates that the maximal secretory capacity of somatotropic

cells does not vary with age and that the age related

decrease in GH secretion is due to a hypothalamic

impairment (20–23). This also points out the possible

clinical usefulness of this substance to rejuvenate the

GH/insulin-like growth factor-I (IGF-I) axis in aging. In

fact, the reduced function of the GH/IGF-I axis in aging

may account for the changes in body composition, structure,

and function. In agreement with this assumption, it

has been reported by some, but not all, authors that elderly

subjects could benefit from treatment with rhGH

to restore IGF-I levels within the young range (21,24). As

it has been demonstrated that the GH releasable pool in

the aged pituitary is basically preserved and that the age related

decline in GH secretion mostly reflects hypothalamic

dysfunction (21,23), the most appropriate, that is,

“physiological,” approach to restore somatotroph function

in aging would be a treatment with neuroactive substances

endowed with GH-releasing action. Among these

GH secretagogues, arginine received considerable attention.

In fact, the coadministration of arginine (even at

low oral doses) with GHRH (up to 15 days) enhanced

the GH responsiveness to the neurohormone in normal

aged subjects (11). However, the efficacy of long-term

treatment with oral arginine to restore the function of the

GH/IGF-I axis in aging has never been shown in elderly

subjects.

Following the evidence that GHRH combined with

arginine becomes the most potent and reproducible stimulus

to diagnose GH deficiency throughout the lifespan

(25), GHRH + arginine is, at present, one of the two gold

standard tests for the diagnosis of GH deficiency (25,26).

In fact, the GH response to a GHRH + arginine test is

approximately threefold higher than the response to classical

tests and does not vary significantly with age (25,26).

Because of its good tolerability and its preserved effect in

aging, the GHRH + arginine test is currently considered

to be the best alternative choice to the insulin-induced

tolerance test (ITT) for the diagnosis of GH deficiency

throughout the lifespan (25).

L-Arginine 23

PRL Secretion

Among the endocrine actions of arginine, its PRL releasing

effect has been shown both in animals and in

humans after IV but not after oral administration (10,27).

The PRL response to arginine is markedly lower than

the response to the classical PRL secretagogues, such

as dopaminergic antagonists or thyrotropin-releasing

hormone (TRH) (6) but higher than that observed after

secretion of GH and other modulators of lactotrope

function (17).

The mechanisms underlying the stimulatory effect

of arginine on PRL secretion are largely unknown, but

there is evidence that this effect is not mediated by galanin,

a neuropeptide with PRL-releasing effect. In fact, galanin

has been shown to potentiate PRL response to arginine,

suggesting different mechanisms of action for the two substances

(28).

ACTH Secretion

Although some excitatory amino acids and their agonists

have been demonstrated to differently modulate

corticotropin-releasing hormone and arginine vasopressin

release in vitro and influence both sympathoadrenal and

hypothalamo-pituitary-adrenal (HPA) responses to hypoglycemia

in animals (29,30), little is known about arginine

influences on HPA axis in humans. Many studies have

shown that mainly food ingestion influences spontaneous

and stimulated ACTH/cortisol secretion in normal subjects

and that central 1-adrenergic-mediated mechanisms

are probably involved (31). In humans free fatty acids inhibit

spontaneous ACTH and cortisol secretion, but no

data exist regarding the effect of each nutrient component

on HPA function. Previous studies demonstrated that

arginine is unable to exert an ACTH-stimulatory effect in

humans via generation of NO (12) and our unpublished

preliminary data failed to demonstrate a significant effect

of arginine (30 g IV) on either ACTH or cortisol secretion

in normal subjects.

Insulin Secretion

Arginine is one of the most effective known insulin secretagogue

and it may be used with glucose potentiation

to determine a patient’s capacity to secrete insulin (32).

Arginine acts synergistically with glucose, and to a much

lesser extent with serum fatty acids, in stimulating insulin

release. A synergistic effect of arginine and glucose on insulin

secretion has been shown in humans (33,34), and the

combined administration of these two stimuli has been

studied in an attempt to test -cell secretory capacity in

diabetic patients (35).

A protein meal leads to a rapid increase in both

plasma insulin and glucagon levels (36). Administration

of arginine has a similar effect. An arginine transport system

is present in the -cell plasma membrane (37). When

arginine enters the cell, it causes ionic changes that depolarize

the cell and trigger Ca2+ uptake and exocytosis

of insulin-containing granules.

Several mechanisms for arginine-induced -cell

stimulation have been proposed. These include the

metabolism of L-Arg leading to the formation of ATP

(38,39), the generation of NO (40,41), and the direct depolarization

of the plasma membrane potential due to the

accumulation of the cationic amino acid (42–44).

A sustained Ca2+ influx is directly related to insulin

secretion following arginine uptake by cells. The

arginine-induced increase in Ca2+ concentration is inhibited

by the activation of ATP-sensitive potassium (K-ATP)

channels with diazoxide and seems dependent on the nutritional

status. These observations suggest that the K-ATP

channels, when fully open, act to prevent membrane depolarization

caused by arginine. The presence of a nutrient,

such as glucose, produces sufficient closure of K-ATP

channels to allow arginine-induced membrane depolarization

and activation of the voltage-activated Ca2+ channels

(37).

Nonendocrine Actions

Cardiovascular System

Increasing interest has been recently focused on NO. This

mediator, which is synthesized from L-Arg (45) by nitric

oxide synthases (NOS) (46), is a potent vasodilator

(47) and inhibitor of platelet adhesion and aggregation

(48). Three isoforms of NOS are described: neuronal NOS

(nNOS—NOS-1), inducible NOS (iNOS—NOS-2), and endothelial

NOS (eNOS—NOS-3). NOS-1 and NOS-3 are

expressed constitutively and they produce NO at low

rates (49). NOS-3 is responsible for a consistent vasodilator

tone and, although constitutive, can be regulated by

endothelial shear stress (50) and substances such as acetylcholine,

histamine, serotonin, thrombin, bradykinin, and

catecholamines. Calcium is required for NOS-3 activation

(51).NO production is mainly dependent on the availability

of arginine and NOS is responsible for the biochemical

conversion of L-Arg to NO and citrulline in the presence

of cofactors such as reduced nicotinamide adenine dinucleotide

phosphate (NADPH), tetrahydrobiopterin (BH4),

flavin mononucleotide, and flavin adenine nucleotide. Reduced production,

leading to vasoconstriction and increases

in adhesion molecule expression, platelet adhesion

and aggregation, and smooth muscle cell proliferation has

been demonstrated in atherosclerosis, diabetes mellitus,

and hypertension (52–54)—conditions known to be associated

with an increased mortality because of cardiovascular

disease. Taken together, these observations lead to the

concept that interventions designed to increase NO production

by supplemental L-Arg might have a therapeutic

value in the treatment and prevention of the endothelial

alterations of these diseases. Besides several actions exerted

mainly through NO production, arginine also has a

number of NO-independent properties, such as the ability

to regulate blood and cellular pH, and the effect on the

depolarization of endothelial cell membranes.

The daily consumption of arginine is normally about

5 g/day. Arginine supplementation is able to increase NO

production, although the Km for L-Arg is 2.9 mol and the

intracellular concentration of arginine is 0.8 to 2.0 mmol.

To explain this biochemical discrepancy, named “arginine

paradox,” there are theories that include low arginine levels

in some diseases (e.g., hypertension, diabetes mellitus,

and hypercholesterolemia), and/or the presence of enzymatic

inhibitors (55), and/or the activity of the enzyme

arginase (which converts arginine to ornithine and urea,

leading to low levels of arginine).

Recently attention has been given to the methylated

forms of L-Arg, generated by the proteolysis of

24 Maccario et al.

methylated proteins; they are represented by asymmetric

dimethylarginine (ADMA) and two symmetric dimethylated

derivatives: symmetric dimethylarginine (SDMA)

and monomethylarginine (MMA) (56). Only ADMA and

MMA, but not SDMA, exert inhibitory effects on NOS-3

activity (57). For this reason, ADMA is now recognized as

a new emerging cardiovascular risk marker and likely as

a causative factor for cardiovascular disease (58).

L-Arginine therapy in cardiovascular pathologies

showed contradictory results. However, it is now clear that

individual response to L-Argmaybe influenced by DMA.

In fact, no effects of L-arg therapy are demonstrated in patients

with low ADMA levels, whereas in patients with

high ADMA level, L-Arg normalizes the L-Arg to ADMA

ratio, thus normalizing the endothelial function (59).

Several studies demonstrated that L-Arg infusion

in normal subjects and patients with coronary heart disease

(60), hypercholesterolemia (61), and hypertension

(62) is able to improve the endothelial function, but the

results, although encouraging, are not conclusive because

of the short-term effects of IV arginine. However, arginine

does not affect endothelial function in patients with

diabetes mellitus. On the other hand, oral L-Arg has a

longer half-life and longer-term effects than L-Arg given

intra-arterially or intravenously (63). Thus, in the setting

of long-term health maintenance or symptom management,

the oral route would be preferred. Studies in animals

documented that oral L-Arg supplementation is able

to reduce the progression of atherosclerosis, preserving

endothelium function (64) and inhibiting circulating inflammatory

cells (65) and platelets (66) in animals with

hypercholesterolemia, and to decrease blood pressure and

wall thickness in animals with experimental hypertension

(67). On the other hand, studies in humans in vivo are

not so widely positive as the animal experimental data.

Actually, although the majority of the data is in normal

subjects, individuals with a history of cigarette smoking

and patients with hypercholesterolemia and claudication

demonstrate beneficial effects of oral L-Arg administration

on platelet adhesion and aggregation, monocyte adhesion,

and endothelium-dependent vasodilation (68,69).

Other studies do not show any benefit (70,71); therefore,

no definitive conclusions can be drawn. Taken together,

the studies show a major effect when L-Arg supplementation

was given in subjects with hypercholesterolemia,

probably because of an increase in NO production via reduction

of the ADMA intracellular concentration, which

is increased in the presence of LDL hypercholesterolemia.

In conclusion, despite several beneficial effects on

intermediate end points, particularly in hypercholesterolemic

patients, there is no evidence for a clinical

benefit in the treatment or prevention of cardiovascular

disease. More data, derived from large-scale prospective

studies evaluating the effect of long-term treatment with

L-Arg, are needed. Future perspectives of pharmacological

intervention are represented by the regulation of the enzyme

dimethylarginine dimethylaminohydrolase responsible

for the ADMA metabolism (57), the arginase (72),

and the endothelial cell L-Arg transporter (73).

Immune System

Many studies, in animals as well as in humans, have

shown that arginine is involved in immune modulation. In

fact, this amino acid is a component of most proteins and

the substrate for several nonprotein, nitrogen-containing

compounds acting as immune modulators.

There is clear evidence that arginine participates in

the cell-mediated immune responses of macrophages and

T lymphocytes in humans through the production of NO

by inducible nitric oxide synthase (iNOS-–NOS-2), which

occurs mostly in the macrophage (74,75), and through the

modulation of T-lymphocyte function and proliferation

(76,77). At intracellular levels, arginine is metabolized by

two different enzymatic pathways: the arginase pathway,

by which the guanidino nitrogen is converted into urea to

produce ornithine, and the NOS pathway, which results

in oxidation of the guanidino nitrogen to produce Land

other substances (78,79).

It has been shown that macrophage superoxide production,

phagocytosis, protein synthesis, and tumoricidal

activity are inhibited by high levels of arginine in vitro and

that sites of inflammation with prominent macrophage

infiltration, such as wounds and certain tumors, are deficient

in free arginine (80). In particular, a decrease in

arginine availability due to the activity of macrophage derived

arginase rather than the arginine/NO pathway

may contribute to the activation of macrophages migrating

at inflammatory sites (80). Arginine metabolism in the

macrophages is activity dependent. At rest, macrophages

exhibit minimal utilization of arginine and lower NOS-2

expression or arginase activity, whereas in activated cells,

arginine is transported into the cell, and NOS-2 expression

and arginase are induced by cytokines and other stimuli

(81). The types of stimuli that induce NOS-2 and arginase

are quite different. In vitro and in vivo studies demonstrated

that NOS-2 is induced by T-helper I cytokines (IL-

1, TNF, and -interferon) produced during activation of

the cellular immune response, such as severe infections or

sepsis (74,75), whereas arginases are induced by T-helper

II cytokines (IL-4, IL-10, and IL-13) and other immune regulators

aimed at inducing the humoral immune response

(82,83). Thus, in disease processes, where inflammatory

response predominates, NOS-2 expression and NO production

prevail. Under biological circumstances where Thelper

II cytokine expression is prevalent, arginase activity

and the production of ornithine and related metabolites

would predominate.

In vitro studies in animals demonstrated depressed

lymphocyte proliferation in cultures containing low levels

of arginine and maximal proliferation when arginine is

added at physiological plasma concentration (77,84), but

the molecular details have not been completely defined.

It has also been shown that supplemental arginine

increased thymic weight in rodents because of increased

numbers of total thymic T lymphocytes. On the other

hand, in athymic mice, supplemental arginine increased

the number of T cells and augmented delayed-type hypersensitivity

responses, indicating that it can exert its effects

on peripheral lymphocytes and not just on those within

the thymus (76).

The immunostimulatory effects of arginine in animal

studies have suggested that this amino acid could be

an effective therapy for many pathophysiological conditions

in humans, able to positively influence the immune

response under some circumstances by restoring cytokine

balance and reducing the incidence of infection.

L-Arginine

In healthy humans, oral arginine supplementation

shows many effects on the immune system, including

increase in peripheral blood lymphocyte mitogenesis,

increase in the T-helper–T-cytotoxic cell ratio and, in

macrophages, activity against microorganisms and tumor

cells (85). Furthermore, the delayed-type hypersensitivity

response as well as the number of circulating natural

killer (NK) and lymphokine-activated killer cells are

increased (85–87). Therefore, it has been hypothesized

that arginine could be of benefit to patients undergoing

major surgery after trauma and sepsis and in cardiovascular

diseases, HIV infection, and cancer (88). In

fact, short-term arginine supplementation has been shown

to maintain the immune function during chemotherapy;

arginine supplementation (30 g/day for 3 days) reduced

chemotherapy-induced suppression of NK cell activity,

lymphokine-activated killer cell cytotoxicity, and lymphocyte

mitogenic reactivity in patients with locally advanced

breast cancer (89). It must be noted that chronic administration

of arginine has also been shown to promote cancer

growth by stimulating polyamine synthesis in both animal

and human studies (89). On the other hand, NO has

been shown to inhibit tumor growth. Thus, the real effect

on cancer processes depends on the relative activities of

NOS and arginase pathways that show variable expression,

depending on the stage of carcinogenesis (91).

These data clearly indicate the involvement of arginine

in immune responses in both animals and humans.

Large clinical trials are needed to clarify the clinical application

and efficacy of this amino acid in immunity and

immunopathology.

SUPPLEMENTAL ARGININE

The available form of supplemental L-Arg is represented

by the free base, the Cl− salt (L-Arg hydrochloride-–L-Arg-

HCl) and the aspartate salt of the amino acid (92).

L-Arg is stable under sterilization condition and its

administration is safe for mammals in an appropriate dose

and chemical form (91).

Oral L-Arg (up to 9 g of Arg-HCl per day for adults)

has no adverse effects on humans but higher doses can

lead to gastrointestinal toxicity, theoretically increasing

local production of NO and impairing intestinal absorption

of other basic amino acids (91). Moreover, the local

NOproduction may be particularly dangerous if intestinal

diseases are present (92).

Oral L-Arg supplement is commonly used to increase

GH release and consequentially physical performance;

moreover, it has been hypothesized that L-Arg

supplement could lead to improved muscular aerobic

metabolism and less lactate accumulation, enhancing NOmediated

muscle perfusion.

However, in a clinical trial, arginine supplement in

endurance-trained athletes did not show any difference

from placebo in endurance performance (maximal oxygen

consumption, time to exhaustion), endocrine (GH,

glucacon, cortisol, and testosterone concentrations), and

metabolic parameters (93).

In another study, the association “arginine plus exercise”

produced a GH response approximately 50% lower

than that observed with exercise alone, suggesting that

the acute use of oral L-Arg prior to exercise blunts the GH

response to subsequent exercise (94).

No effects on NO production, lactate and ammonia

metabolism, and physical performance in intermittent

anaerobic exercise were shown in well-trained male athletes

after short-term arginine supplementation (95). It has

been hypothesized thatNOproduction is not modified by

arginine supplementation in athletes because they may

have higher basal concentrations of NO than general population;

in fact, basal NO production can be increased by

regular exercise training, without any pharmacological intervention

(95).

There are many interesting clinical perspectives on

arginine supplementation therapy, especially in critical

care setting (96), treatment and prevention of pressure

ulcers (97), hypertension (59), and asthma and chronic obstructive

pulmonary disease (98), but further studies are

required to clarify which categories of patients may benefit

from this treatment (99).

CONCLUSIONS

From an endocrinological point of view, the simple classification

of arginine as an amino acid involved in peripheral

metabolism is no longer acceptable. In fact, besides other

nonendocrine actions, it has been clearly demonstrated

that arginine plays a major role in the neural control of

anterior pituitary function, particularly in the regulation

of somatotrophin secretion. One of the most important

concepts regarding arginine is the existence of an arginine

pathway at the CNS level, where this amino acid represents

the precursor of NO, a gaseous neurotransmitter of

major importance. On the other hand, NO does not necessarily

mediate all the neuroendocrine or the peripheral

arginine actions.

In the past years, new discoveries have led to a rapid

increase in our knowledge of the arginine/NO system,

from a neuroendocrine and nonendocrine point of view.

Up to now, there is no evidence for the utility of L-Arg

supplement for muscle strength or exercise performance

in humans. However, several other aspects still remain to

be clarified; the potential clinical implications for arginine

have also never been appropriately addressed and could

provide unexpected results both in the endocrine and in

the cardiovascular fields.

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Astragalus

Herbs, GlossarySuccess Chemistry Staff

INTRODUCTION

Astragalus root (Astragalus membranaceus and Astragalus

mongholicus) (Figs. 1 and 2; flowers are shown in Fig.

2) is one of the most important plant products used

in traditional Chinese medicine (TCM) for supporting

immune resistance ( ; wei qi) and energy production

( ; bu qi). Astragalus is also one of the most popular

ingredients in botanical dietary supplements for its

putative effect of supporting healthy immune function.

Despite the widespread use of this botanical among

TCM practitioners and its extensive use in botanical

supplements, there are few clinical trials supporting

its use, though those that are available are positive.

Numerous preclinical studies provide evidence for a

number of pharmacological effects that are consistent

with the traditional and modern use of astragalus.

BACKGROUND

Traditional and Modern Uses

In Asia, astragalus is commonly used according to both

its traditional Chinese medical indications as a general

tonifier and specifically for immune enhancement and for

modern biomedical indications such as immune, liver, and

cardiovascular support. It has been used for the prevention

of the common cold and upper respiratory tract infections

and is widely prescribed to children for prevention

of infectious disease, though formal clinical English language

studies regarding this use are lacking. In the West,

astragalus is primarily used as an immune modulator.

Astragalus potentiates recombinant interleukin-2 (rIL-2)

and recombinant interferon-1 and -2 (rIFN-1 and -2) immunotherapy

and by lowering the therapeutic thresholds,

may reduce the side effects normally associated with these

therapies. The data and opinion of those expert with the

use of the botanical suggest that astragalus is useful as a

complementary treatment during chemotherapy and radiation

therapy and in immune deficiency syndromes. There

is some modern evidence for its use in hepatitis and the

treatment of cardiovascular disease.

In TCM and Western clinical herbal medicine, astragalus

is most commonly used in combination with other

botanicals and is very seldom used as a single agent. There

are numerous studies of some of the classic combinations

of astragalus (e.g., astragalus and Angelica sinensis). These

have not been reviewed, but use of formulas is more consistent

with the use of the astragalus than with the use of

the herb alone according to traditional Chinese medical

principles.

CHEMISTRY AND PREPARATION OF PRODUCTS

The primary compounds of interest in astragalus are triterpenes,

polysaccharides, and flavonoids. The triterpene astragaloside

IV is a relatively unique marker for astragalus

species used in Chinese medicine. A variety of preparations

are utilized in clinical practice and in herbal supplements.

A number of preparations, including crude extracts,

isolated polysaccharides, and triterpene saponins,

have been subject to study and correlated with activity.

Clinically, in China and among some practitioners in the

United States, decoctions are frequently given. However,

due to the time required for cooking and the subsequent

smell and taste of Chinese herb preparations in general

(though astragalus is very agreeable), many consumers

and practitioners prefer crude powder or extract preparations

(capsules, tablets), freeze-dried granules, or liquid

extracts. Astragalus is also used as a relatively common

ingredient in soups, especially during winter months.

Polysaccharides (12–36 kD) have been most often

correlated with immune activity, while triterpene

saponins have been predominantly associated with cardiovascular

and hepatoprotective effects. Astragalus

polysaccharides are generally composed of a mixture of

D-glucose, D-galactose, and L-arabinose or D-glucose

alone. The glucose units appear to be primarily -(1,4)-

linked with periodic -(1,6)-linked branches (1,2). The

triterpene glycosides vary by position, number, and type

of sugar residues at positions 3, 6, and 25. Several of

these “astragalosides” (e.g., astragaloside IV; Fig. 3) are

composed of a single xylopyranosyl substituent at the 3-

position, which may or may not be acetylated. Others possess

either disaccharide or trisaccharide substituents (3–

5). Primary flavonoids of astragalus for which activity has

been reported include calycosin, formononetin (Fig. 3),

and daidzein (Fig. 3) and additionally include isorhamnetin,

kaempferol, and quercetin, among others (6).

PRECLINICAL STUDIES

Pharmacokinetics

Pharmacokinetic data available in English language publications

on astragalus, its crude extracts, or its constituents

are very limited. In the most detailed study to

date, the pharmacokinetics of a decoction of astragalus,

the preparation most used traditionally were investigated

in four models: four complement in silico, a cacao-2 intestinal

cell model, an animal, and a human volunteer

(n = 1). Intestinal absorption was demonstrated for several

flavonoids including calycosin and formononetin,

along with their aglycone metabolites in all four

models. Triterpene saponins, used as chemical markers

of astragalus (e.g., astragaloside I and IV) in the Pharmacopoeia

of the People’s Republic of China and the American

Herbal Pharmacopoeia, were lacking, likely due to

their low concentrations in the preparation. In the human

volunteer, nine flavonoids, including calycosin, formononetin,

and the isoflavone daidzein, were detected

Figure 2 Astragalus flowers. Source: Photo courtesy of Bill Brevoort, American

Herbal Pharmacopoeia.

in urine (7). In animal models (rats and dogs), astragaloside

IV, which has demonstrated cardioprotective activity,

showed moderate-to-fast elimination. The half-life in male

rats was from 67.2 to 98.1 minutes, in female rats 34.0 to

131.6 minutes, and was linear at the intravenous doses

given. The highest concentration of astragaloside IV was

found in the lungs and liver. Only 50% of the compound

was detected in urine and feces. Binding to plasma protein

was also linear at the concentration of 250–1000 ng/mL.

Slow systemic clearance of astragaloside IV occurred via

the liver at approximately 0.004 L/kg/min (8).

In another pharmacokinetic study, a two compartment,

first-order pharmacokinetic model was

used to describe the pharmacokinetics of intravenously administered

astragaloside IV. Systemic clearance of this

triterpene was reported as moderate and distribution into

peripheral tissues was limited (9).

Pharmacodynamics

A large percentage of research on astragalus has focused

on its immunostimulatory activity and its purported

ability to restore the activity of a suppressed immune

system. More recently, interest in its potential as a cardioprotective

agent has been shown. Reviews of a limited

number of clinical trials and preclinical data provide some

evidence for its usefulness in the prevention of the common

cold and as an adjunct to cancer therapies. There is

limited evidence to suggest a benefit to the cardiovascular

Astragalus 31

  • O

  • O

  • O

  • OH

  • OH

  • HO

  • O

  • H

  • OH

  • O

  • OH

  • OH

  • HO

  • OH

  • HO

  • O

  • O

  • OCH3

  • HO

  • O

  • OH

  • O

  • HO

  • Astragaloside IV

  • Formononetin

  • Daidzein

Figure 3 Some major constituents of Astragalus.

system, with improvement in clinical parameters associated

with angina, congestive heart failure, and acute myocardial

infarct. There is also some indication from animal

studies supporting its use in the treatment of hepatitis and

diabetes.

Immunomodulatory Effects

There are relatively strong preclinical data of pharmacological

mechanisms that provide support for the putative

immunomodulatory effects of astragalus.

Cancer

In a rat study, animals were pretreated orally for 50 days

with a low or high dose of astragalus extract (3.3 or

10 g/kg/day) prior to IP injection of doxorubicin (cumulative

dose of 12 mg/kg over a 2-week period). After 5 weeks

of the final injection of doxorubicin, a significant inhibition

of cardiac diastolic function was observed. This was

accompanied by ascites, catexia, decreased heart weight,

and increased mortality. Treatment with astragalus at both

doses significantly attenuated the negative effects of doxorubicin

on cardiac functions and ascites, while the high

dose also improved survival. This protective effective

was at least partially associated with the ability of astragalus

to attenuate changes in cardiac SERCA2a mRNA

expression (10).

A broad array of immunomodulatory effects has

been demonstrated in numerous preclinical studies that

suggest a substantial value of astragalus in conjunction

with conventional cancer therapies. The most relevant

of these was a series of investigations conducted

by researchers at the MD Anderson Cancer Center

that found that astragalus extract restored to normal

the immune response of patients’ mononuclear cells

that were grafted into rats immunocompromised by cyclophosphamide.

These researchers concluded that astragalus

and its polysaccharide fraction reversed the immunosuppressive

effect of cyclophosphamide (11–15). In

other studies, astragalus and its various fractions were

shown to stimulate macrophage phagocytosis (16) and

hematopoiesis (17).

One study reported on the gastroprotective effects

of astragalus extract (characterization not reported) in human

peritoneal mesothelial cells (HPMCs) subjected to

gastric cancer cell lines. Upon incubation with cancer cell

lines, apoptosis of the HPMC cells was observed. The astragalus

preparation, via regulation of Bcl-2 and Bax, partially

inhibited apoptosis. The authors interpreted these

findings as a potential that astragalus may slow down the

metastasis of the primary cancer and is therefore a potential

treatment for gastric cancer (18).

The ability of an astragalus fraction to potentiate the

effects of rIL-2 has been demonstrated in in vitro assays.

Lymphokine-activated killer (LAK) cells were treated with

a combination of the astragalus fraction and 100 units/mL

of IL-2. The combination therapy produced the same

amount of tumor-cell-killing activity as that generated

by 1000 units/mL of rIL-2 on its own, thus suggesting

that the astragalus fraction elicited a 10-fold potentiation

of rIL-2 in this in vitro model (19). These findings were

confirmed in a follow-up study by the MD Anderson researchers

using LAK cells from cancer and AIDS patients.

In this study, the cytotoxicity of a lower dose of 50 g/mL

of rIL-2 given with the astragalus fraction was comparable

to that of a higher dose of 500 g/mL of rIL-2 alone

against the Hs294t melanoma cell line of LAK cells. With

the combination, the effector-target cell ratio could be reduced

to one-half to obtain a level of cytotoxicity that was

equivalent to the use of rIL-2 alone. In addition, the astragalus

fraction was shown to increase the responsiveness

of peripheral blood lymphocytes that were not affected by

rIL-2. In this study, and in another by the same researchers,

it was concluded that the fraction potentiated the activity

of LAK cells and allowed for the reduction in rIL-2, thus

minimizing the toxicity of rIL-2 therapy (20). Other groups

of researchers reported almost identical findings (a 10-fold

potentiation) and concluded that astragalus is effective in

potentiating IL-2-generated LAK cell cytotoxicity in vitro

(21,22). Astragalus was also found to enhance the secretion

of tumor necrosis factor (TNF) from human peripheral

blood mononuclear cells (PBMCs). A polysaccharide

fraction (molecular weight 20,000–25,000) increased secretion

of TNF- and TNF- after isolation of adherent and

non adherent mononuclear cells from PBMCs (23).

Other investigations support the role of astragalus

polysaccharides as immunomodulating agents.

In an in vitro study, astragalus polysaccharides significantly

induced the proliferation of BALB/c mouse

splenocytes resulting in subsequent induction of interleukin

1 and tumor necrosis factor- and the activation

of murine macrophages. The researchers concluded

32 Upton

that astragalus had an intermediate-to-high affinity

for membrane immunoglobulin (Ig) of lymphocytes

(24).

Cardiovascular Effects

In animal studies, astragalus or its compounds were reported

to elicit antioxidant (25), mild hypotensive (26),

and both positive (27) (50–200 g/mL) and negative

(30 g/mL) inotropic activity (28). The inotropic activity

was reported to be due to the modulation of Na+–K+

ATPase in a manner similar to strophanthin K. Antioxidant

(29), calcium channel blocking (30), and fibrinolytic

activity (31) have been reported in in vitro studies. Astragaloside

IV was studied for potential cardioactivity.

Various effects have been reported. Intravenous administration

of astragaloside IV reduced the area of myocardial

infarct and reduced plasma creatine phosphokinase

release in dogs subjected to 3-hour ligation and increased

coronary blood flow in anesthetized dogs. In isolated rat

heart perfusion investigations, astragaloside IV significantly

improved (P < 0.01) postischemic heart function

and reduced creatine phosphokinase release from the myocardium.

In addition, coronary blood flow during baseline

perfusion and reperfusion in ischemic rat hearts was

increased, while reperfusion damage was decreased. This

activity was shown to be at least partially attributable to

coronary dilation via an increase in endothelium-derived

nitric oxide. Antioxidant activity via an increase in superoxide

dismutase (SOD) activity has also been reported for

astragalus and is considered to contribute to its cardioprotective

effects (32). Astragaloside IV was also shown

to significantly attenuate blood–brain barrier permeability

in a rat ischemia/reperfusion model (33).

Hepatoprotective Effects

Hepatoprotective effects against numerous hepatotoxic

agents (e.g., acetaminophen, carbon tetrachloride, and Escherichia

coli endotoxin) have been reported in both animal

and in vitro studies. In these experiments, improvement

in histological changes in hepatic tissue, including

fatty infiltration, vacuolar degeneration, and hepatocellular

necrosis, was reported. These effects may be associated

with saponin fractions (34). In one clinical study of

hepatitis B patients, concomitant use of astragalus with

lamivudine and -2b interferon showed greater efficacy

than with lamivudine alone (35).

Systemic Lupus Erythematosus

Astragalus was also studied for its ability to affect natural

killer (NK) cell activity, using an enzyme-release assay.

The NK cell activity of PBMCs from 28 patients with systemic

lupus erythematosus (SLE) was increased after in

vitro incubation with an undefined astragalus preparation.

Low levels of NK cell activity were correlated with

disease activity. PBMCs from patients with SLE had significantly

decreased NK cell activity as compared with

those from healthy donors. The extent of stimulation by

the astragalus preparation was related to the dose and

length of the preincubation period (36). Despite its use as

an immune-enhancing agent, which would normally be

considered contraindicated in autoimmune disorders, investigation

of astragalus may be warranted as evidence

suggests that it elicits significant anti-inflammatory activity

and improves ratios and function of T lymphocytes in

SLE (37).

Viral Infections

Prophylaxis against flu and modulation of endogenously

produced interferon have been reported in several animal

studies utilizing astragalus alone (6).

Other Effects

In a new line of investigation for astragalus, two triterpenes

(astragaloside II and isoastragaloside I) were

shown to alleviate insulin resistance and glucose intolerance

in mice. The two compounds selectively increased

adiponectin secretion on primary adipocytes and potentiated

the effects of the insulin-sensitizer rosiglitazone.

Chronic administration of the compounds (specific details

lacking) to both dietary and genetically obese mice

resulted in a significant increase in serum adiponectin, resulting

in an alleviation of hyperglycemia, glucose intolerance,

and insulin resistance. These effects were diminished

in mice lacking adiponectin (38).

One study showed that a liquid extract of astragalus

(2 g/mL/intravenous) retarded the progression of renal

fibrosis in a manner similar to the angiotensin-II-receptor

antagonist losartan. The study reported that like losartan,

astragalus decreased deposition of fibronectin and

type-I collagen by significantly reducing the expression of

transforming growth factor-1 and -smooth muscle actin

(P < 0.05) (39).

Astragalus was investigated for its potential effect

of reducing atopic dermatitis in mice. Using prednisolone

(3 mg/kg/day) as a comparator, an astragalus water extract

was administered orally at 100 mg/kg. Astragalus

significantly reduced the severity of chemically induced

inflammation (2,4-dinitrofluorobenzene) to a degree similar

to the comparator but, unlike prednisolone, did not

inhibit interleukin-4 production (40).

CLINICAL STUDIES

There are both English and Chinese language studies on

astragalus. As with much of the literature regarding Chinese

herbs, there are few clinical data of high methodological

quality. In addition, a positive publication bias

regarding Chinese literature has been reported (41), while

in primary American medical literature, a negative publication

bias against dietary supplement studies has been

reported (42).

Immunomodulatory Effects

Cancer

Among modern herbal practitioners, astragalus is recommended

as an immune supportive botanical in conjunction

with conventional chemo and radiation therapies for

cancer. There is a common belief and some clinical and

preclinical evidence that astragalus both reduces side effects

associated with conventional cancer therapies and

can potentiate the effects of certain therapies. The available

evidence is not strong enough to recommend astragalus

as a standard part of conventional cancer care. However,

Astragalus 33

its demonstrated safety, lack of negative interaction with

conventional therapies, and its putative benefit in building,

preserving, and restoring immunocompetency before

and after conventional therapies warrant specific study.

There is also potential for use of both oral and injectable

preparations, the latter of which are not approved in North

America but are widely used throughout Asia.

In one clinical study, an astragalus drip (20 mL in

250 mL saline solution daily for 84 days) was administered

to cancer patients (n = 60). Compared with the control

group (no astragalus), those in the astragalus group

showed a slower rate of tumor progression, a lower rate

of reduction in peripheral leukocytes and platelets, reduction

in suppressor CD8s, improved CD4/CD8 ratios,

increased IgG and IgM, and better Karnofsky scores (43).

In addition to its use alone, both as a primary treatment

and as an adjunct to conventional cancer therapies,

astragalus is most often combined with other similar acting

immune-enhancing plants. A number of randomized

prospective clinical studies of cancer patients were conducted

using a combination of astragalus and ligustrum

(Ligustrum lucidum) (undisclosed quantities) with positive

results, such as mortality reduction in breast and lung

cancer patients (44). These effects, of course, must be considered

to be due to the cumulative effects of the two

botanicals and cannot be presumed to occur with astragalus

alone but are more consistent with the manner in

which astragalus is used in TCM.

An early clinical trial reported that 53 cases of

chronic leukopenia responded favorably to an astragalus

extract (1:1; 2 mL daily intramuscularly for 1–2 weeks).

Improvements in symptoms and white blood cell counts

were observed, but specific data were lacking (34).

Cardiovascular Effects

Various cardioactive properties have been reported for astragalus,

and astragalus is widely used in the treatment of

both chronic and acute cardiovascular disease in China.

In one study, 92 patients with ischemic heart disease were

given an unidentified preparation of astragalus. Marked

relief from angina pectoris and other improvements as

measured by electrocardiogram (ECG) and impedance

cardiogram were reported. Improvement in the ECG index

was reported as 82.6%. Overall improvement was significant

as compared with the control group (P<0.05) (45).

A similar result in cardiac performance was reported by

other groups of researchers. In one study, 43 patients were

hospitalized within 36 hours of acute myocardial infarct.

After administration of an astragalus preparation (undefined

profile), the ratio of pre ejection period/left ventricular

ejection time was decreased, the antioxidant activity of

SOD of red blood cells was increased, and the lipid peroxidation

content of plasma was reduced (46). In another experiment,

20 patients with angina pectoris were given an

undefined astragalus preparation. Cardiac output, as measured

by Doppler echocardiogram, increased from 5.09 °æ

0.21 to 5.95 °æ 0.18 L/min 2 weeks after administration

of astragalus (P < 0.01). In this study, neither improvement

in left ventricular diastolic function nor inhibition of

adenosine triphosphate was observed (47). Intravenous

administration of astragalus (undefined preparation) was

reported to significantly shorten the duration of ventricular

late potentials in cardiac patients (39.8 °æ 3.3 ms vs.

44.5 °æ 5.9 ms; P < 0.01) (48).

In another investigation, astragaloside IV (intravenous;

unspecified amount) was given to patients with

congestive heart failure for 2 weeks. Improvement in

symptoms such as tightness in the chest, difficult breathing,

and reduced exercise capacity were reported. Radionuclide

ventriculography showed that left ventricular

modeling improved and left ventricular end-diastolic and

left ventricular end-systolic volume diminished significantly.

The authors concluded that astragaloside IV is an

effective positive inotropic agent (49), an action supported

by others (27).

Hepatoprotective Effects

In China, astragalus is widely used in the treatment of

chronic hepatitis where reductions in elevated liver enzymes

and improvements in symptoms in humans have

been reported. This activity is stated to be associated with

polysaccharides that increase interferon production (35).

Viral Infections

According to one English language review of the Chinese

literature, a prophylactic effect against the common cold

was reported in an epidemiological study in China involving

1000 subjects. Administration of astragalus, given either

orally or as a nasal spray, reportedly decreased the

incidence of disease and shortened cold duration. Studies

exploring this protective effect found that oral administration

of the preparation to subjects for 2 weeks enhanced

the induction of interferon by peripheral white blood cells.

Levels of IgA and IgG antibodies in nasal secretions were

reported to be increased following 2 months of treatment

(34). The effect of astragalus on the induction of interferon

was studied in a placebo-controlled study involving 28

people. Fourteen volunteers were given an extract equivalent

to 8 g of dried root per day and the rest were given

placebos. Blood samples were drawn before treatment,

then 2 weeks and 2 months after treatment. Interferon

production by leukocytes was statistically increased after

both time periods (P < 0.01) in the astragalus group but

not the control group (50). In another study, astragalus

was shown to potentiate the effects of interferon (rIFN-1)

in patients with chronic cervicitis (51).

Dosages

Crude root: 9–30 g daily as a decoction (52).

Decoction: 0.5–1 L daily.

SAFETY PROFILE

Side Effects

None cited in the literature.

Contraindications

None cited in the literature.

Precautions

There is some evidence to suggest that astragalus and its

putative anti-inflammatory effects are beneficial in those

with autoimmune conditions such as lupus. However, astragalus

should be used cautiously for the treatment of

34 Upton

autoimmune diseases or in conjunction with immunosuppressive

therapies. Because immunostimulating polysaccharides

may stimulate histamine release, allergic symptoms

may be aggravated by the use of astragalus. This,

however, has not been reported in the literature or from

clinical use. According to the principles of TCM, astragalus

should not be used during acute infectious conditions

unless under the care of a qualified TCM practitioner.

Interactions

Both positive and negative interactions may occur. Astragalus

potentiates the effects of acyclovir (53); IL-2, -20,

-21; and rIFN-1 and -2 therapies (50,51). Because of its immuno promoting

effects, astragalus may be incompatible

with immunosuppressive agents in general.

Pregnancy, Mutagenicity, and Reproductive Toxicity

According to one review, astragalus is reported to have no

mutagenic effects (54).

Lactation

Based on an authoritative review of the available pharmacologic

and toxicologic literature, no limitation is to be

expected (6,34,54).

Carcinogenicity

Studies suggest an anticarcinogenic activity.

Influence on Driving

Based on the available pharmacologic and toxicologic literature,

no limitation is to be expected (6,34,54).

Overdose and Treatment

Specific data are lacking.

Toxicology

Based on a review of the available data and the experience

of modern practitioners, astragalus can be considered

a very safe herb even when taken within its large

dosage range. Investigations of specific fractions including

flavonoids, polysaccharide, and triterpene similarly

show little toxicity (14,34,54).

Regulatory Status

In the United States, astragalus is regulated as a dietary

supplement.

CONCLUSIONS

Astragalus is one of the most frequently used herbal

medicines throughout Asia and is a very popular botanical

used in western herbal supplements. In China, astragalus

is used for a myriad of purposes relating to its

high regard as a strengthening tonifier, immune modulator,

anti-inflammatory, and anti-hepatotoxic. In the West,

astragalus figures prominently in immune supportive formulas.

Despite its popularity, there are few clinical trials

regarding its use. There is some evidence to support

the oral administration of astragalus for the prevention of

colds and upper respiratory infections, and as an adjunct

to conventional cancer therapies. These are very common

indications for which astragalus is applied by herbal practitioners.

For its use in cancer therapies, there are no definitive

guidelines. The modern experience of practitioners

together with the limited clinical and preclinical data

pointing to an immunomodulatory effect suggests that

there may be some value for these indications, including

the concomitant use of astragalus to reduce doxorubicininduced

immune suppression. However, more specific investigation

in this area is needed.

Regarding its putative immunomodulating effects,

the following mechanisms of action have been proposed:

restoration of immune function, increased stem cell generation

of blood cells and platelets, lymphocyte proliferation,

rise in numbers of antibody-producing spleen cells,

potentiation of rIL-2 and rIFN-1 and -2 immunotherapy,

enhancement of phagocytic activity by macrophages and

leukocytes, and increased cytotoxicity by NK cells.

Potential benefits to cardiovascular health, including

relief from angina and congestive heart failure and

improvement in clinical parameters following acute myocardial

infarct, have been reported. Limited animal studies

suggest that astragalus enhances coronary blood flow,

may potentiate the release of nitric oxide, and potentiates

the effects of endogenous antioxidant systems (e.g., SOD).

In Asia, astragalus is also used in conjunction with

conventional medical treatments for hepatitis. Both animal

and in vitro studies offer support for such treatment.

As in the use of astragalus in cancer therapies, further

clinical trials are required.

Though methodologically sound clinical trials for

astragalus are generally lacking, natural health practitioners

have a generally high regard for its use as a prophylactic

against infectious disease and for its ability to build,

maintain, and restore immunocompetency when used as

a part of conventional cancer therapies. In addition to the

very limited number of formal clinical studies that are

available in English language sources, the published medical

literature on astragalus has to be considered cautiously,

as a number of the supporting studies utilize injectable

preparations of isolated fractions that are not consistent

with the oral use of astragalus supplements. Still, the existing

data do support many of the traditional uses for

which astragalus has been employed for centuries.

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Bilberry

Beauty, Nutrition, GlossarySuccess Chemistry Staff

INTRODUCTION

Bilberry, Vaccinium myrtillus L., is a shrub with edible

fruits that is native to Circumboreal regions from Europe

to Asia as well as the Rocky Mountains in North America.

Bilberries are related to other edible berries including

blueberry, cranberry, huckleberry, and lingonberry.

Bilberry fruits contain anthocyanins, which are natural

pigments, responsible for the dark blue color of the fruits

and for many of the health benefits. In vitro studies have

shown that bilberry extracts have antioxidant activity, inhibit

platelet aggregation, prevent degradation of collagen

in the extracellular matrix surrounding blood vessels and

joints, and have a relaxing effect on arterial smooth muscle.

Bilberry extracts have also demonstrated anticancer

and antibacterial actions, in vitro. Pharmacokinetic studies

in animals and humans show that a small percentage

of the anthocyanins is absorbed into the body and widely

distributed. Human clinical studies have been conducted

evaluating the potential benefits of bilberry preparations

in treating venous insufficiency and visual disorders ranging

from night vision to diabetic retinopathy as well as

cancer prevention. No serious toxicities have been associated

with preparations of the fruits in animal screens and

no serious side effects have been identified in humans.

BACKGROUND

Bilberries are edible fruits from V. myrtillus L. of the family

Ericaceae. Bilberry is the standardized common name for

the fruit in the United States, but the fruit is also known

as European blueberry, huckleberry, and whortleberry (1).

Related to bilberry, and in the same genus of Vaccinium, are

other edible berries including blueberry, cranberry, huckleberry,

and lingonberry.

Bilberry is a shrub, 1–6 dm high, found in heaths,

meadows, and moist coniferous forests in Circumboreal

regions from Europe to Asia, with populations in the

American and Canadian Rocky Mountains (2).

The blue-black berries are harvested when ripe,

usually during the months of July through September.

The berries are oblate-globose, 5–9 mm diameter, with

4–5 locules containing many seeds. The seeds are approximately

1 mm long with a yellow/brown-dimpled

surface (2).

Both the leaves and fruits of bilberry have been used

medicinally since the Middle ages.

The leaves were used topically for inflammation, infections,

and burns, as well as internally as a treatment for

diabetes. According to the herbalist Grieve, the fruits were

used to treat dysentery, diarrhea, gastrointestinal inflammation,

hemorrhoids, vaginal discharges, scurvy, urinary

complaints, and to dry up breast milk. More recently, it

was found that bilberry was used by World War II pilots

to improve their night vision (2,3).

Bilberry fruit preparations are still used to improve

vision as well as for their benefits to the circulatory system

treating fragility and altered permeability of blood

vessels that is either primary or secondary to arterial hypertension,

arteriosclerosis, or diabetes (3).

CHEMISTRY AND PREPARATION

Bilberry fruits contain anthocyanins that are natural pigments

in the chemical class known as flavonoids. Anthocyanins

are glycosides or compounds with sugars

attached at the 3 position, while anthocyanidins are aglycones

(the same basic structure without the sugars attached)

(see chapter 74, “Polyphenol Overview”). The majority

(64%) of anthocyanins in the fruit are glycosides of

cyanidin and delphinidin (Fig. 1). The quantity of anthocyanin

in the fruit ranges from 300 to 700 mg per 100 g.

Bilberry fruits also contain flavonols, tannins, phenolic

acids, organic acids, sugars, vitamins, and volatile compounds

(2).

The primary commercial source of bilberry fruits is

“wild harvest” from regions in Europe and Scandinavia.

The fruits are sold fresh, frozen, or dried. Besides the

whole fruit, commercial products include cold macerates,

decoctions, and dry extracts. The dry extracts are commonly

prepared using alcohol, methanol or ethanol (2).

Until recently, a single-wavelength spectrophotometric

technique (UV) was commonly used to standardize

the anthocyanin content of bilberry products. However,

this technique did not detect adulteration of bilberry

preparations with substances of similar color (4). A high performance

liquid chromatographic technique that can

detect and quantitate both anthocyanins and anthocyanidins

has recently been developed enabling a better assurance

of product identity and quality (5).

Most studies on bilberry have been conducted using

extracts characterized as containing 36% anthocyanins or

25% anthocyanidins.

PRECLINICAL STUDIES

In vitro studies have shown that bilberry extracts have

antioxidant activity, inhibit platelet aggregation, prevent

  • Cyanidin 3-O-glycoside OH OH H arabinose, glucose, or galactose

  • Delphinidin 3-O-glycoside OH OH OH arabinose, glucose, or galactose

  • Malvidin 3-O-glycoside OCH3 OH OCH3 arabinose, glucose, or galactose

  • Peonidin 3-O-glycoside OCH3 OH H arabinose, glucose, or galactose

  • Petunidin 3-O-glycoside OH OH OCH3 arabinose, glucose, or galactose

  • OH

  • HO

  • O R3

  • R2

  • R1

  • R

  • O+

degradation of collagen in the extracellular matrix

surrounding blood vessels and joints, and have a relaxing

effect on arterial smooth muscle. These actions are vasoprotective,

increasing capillary resistance and reducing

capillary permeability (3). Bilberry extracts have also

demonstrated anticancer and antibacterial actions in

vitro. There is no evidence of toxicity in animals at the

effective doses.

Antioxidant Activity

Bilberry fruits have demonstrated antioxidant activity in

in vitro and in animal models. In the oxygen radical absorbance

capacity(ORAC)assay, an in vitro test measuring

free radical quenching, bilberry fruits had potent activity

compared with other fresh fruits and vegetables (44.6 +

2.3 mol Trolox equivalents (TE)/g) (6). In another assay,

a bilberry extract (25% anthocyanins) exhibited antioxidant

activity in protecting keratinocytes in culture from

damage due to UVA and UVB light (7,8). The bilberry

extract attenuated UVA-induced reactive oxygen species

formation, peroxidation of membrane lipids, and depletion

of intracellular glutathione in concentrations of 10–50

mg/L (7). In the same concentration range, the extract inhibited

UVB-induced generation of reactive oxygen and

nitrogen species, DNA strand breaks, as well as caspase-

3 and caspase-9 activity (mediators that execute apoptotic

cell death) (8).

In an animal study, a bilberry extract characterized

as containing 38% anthocyanins reduced oxidative stress

caused in mice by removal of the animal’s whiskers. The

extract administered orally at a dose of 100 mg/kg for

7 days ameliorated the increase in oxygen radicals (thiobarbituric

acid reactive substances), protein carbonyl formation,

and lipid peroxidation in the brain, heart, kidney,

and liver. The extract also suppressed the stress-induced

changes in dopamine levels (9).

A bilberry extract (42% anthocyanins) reduced oxidative

stress to the liver in a restraint-stress mouse model.

The extract, administered at a dose of 200 mg/kg for

5 days, ameliorated the increase in plasma levels of alanine

aminotransferase, a liver enzyme. The extract also reduced

plasma and liver ORAC levels and increased plasma glutathione

and vitamin C levels in the liver (10). A similar

study was conducted in mice with kidney damage induced

by potassium bromate. Oral administration of the

same extract ameliorated the increase in blood urea nitrogen

levels and the decreases in kidney malondialdehyde,

nitric oxide, and xanthine oxidase levels. The bilberry extract

also improved the kidney ORAC levels (11).

MyrtoSelectTM, an extract containing approximately

40% anthocyanins, was tested for its effects on gene expression

(DNAmicroarray) in a macrophage cell line stimulated

with lipopolysaccharide (LPS). The extract, at a

concentration of 75 g/mL, appeared to mitigate the effect

of LPS, targeting genes involved in inflammation and

immune defense. Pretreatment with the bilberry extract

affected 45% of the genes downregulated by LPS and 36%

of genes upregulated by LPS (12).

Circulation

Bilberry extracts have demonstrated beneficial effects on

the circulation.

including inhibiting platelet aggregation,

reducing capillary permeability, facilitating vasodilation,

and inhibiting the development of atherosclerosis and angiogenesis.

Myrtocyan R (also known as MirtoSelectTM, containing

36% anthocyanins) inhibited platelet aggregation in

vitro induced by adenosine diphosphate (ADP), collagen,

and sodium arachidonate in rabbit platelet-rich plasma

with IC50 values ranging from 0.36 to 0.81 mg/mL. Myrtocyan

administered orally to rats (400 mg/kg) prolonged

the bleeding time in the animals, without affecting coagulation

pathways. The same dose administered to mice reduced

the adhesiveness of platelets to glass (3). Myrtocyan

administered to healthy human subjects, 480 mg/day

(173 mg anthocyanins/day) for 30–60 days, reduced the

aggregation response ex vivo to ADP and collagen (13).

In a rabbit skin model, oral treatment with 400 mg

anthocyanins per kilogram body weight 30 minutes before

topical application of chloroform reduced the capillary

permeability caused by the irritant by 66%. In rats, administration

of bilberry anthocyanins, 200 mg/kg orally,

decreased bradykinin-induced capillary permeability by

39%. The same dose reduced carrageenin-induced rat paw

edema by 45% (14). In a rat model of experimentally induced

hypertension, 500 mg anthocyanins per kilogram

body weight given orally for 12 days completely ameliorated

the increase in blood–brain barrier permeability and

reduced the increase in aortic vascular permeability by

40% (15). In a hamster cheek pouch model, 100 mg bilberry

extract per kilogram daily for 4 weeks reduced the

circulatory damage due to ischemic reperfusion (16).Arat

model suggested that bilberry anthocyanins (50mg/kgIP)

inhibited the enzymatic degradation of collagen, decreasing

the permeability of the blood–brain barrier caused by

proteases (17).

Bilberry preparations are reported to relax arterial

tissues in vitro.

Preliminary experiments pointed

to a mechanism involving prostaglandins. However, a

more recent study using porcine coronary arteries demonstrated

a mechanism involving nitric oxide (endothelialderived

relaxing factor) (18).

A bilberry extract was reported to inhibit the development

of atherosclerosis in apolipoprotein E–deficient

mice. The mice received diets supplemented with 0.02%

of a bilberry extract (52% anthocyanins) for 16 weeks. The

Bilberry 39

extract reduced lipid deposits and the development of

lesions. It did not affect plasma antioxidant capacity or

plasma lipid levels (19).

A bilberry extract (25% anthocyanins) was tested

for its effect on angiogenesis both in vitro and in vivo.

The extract at concentrations of 0.3–30 g/mL inhibited

tube formation and the migration of human umbilical

vein endothelial cells induced by vascular endothelial

growth factor A. The extract also inhibited the induction

of retinopathy in newborn mice, which was induced

with oxygen. Intravitreal administration of 300 ng extract

per eye significantly inhibited the area of neovascular

tufts (20).

Anticancer

Anthocyanins have been reported to mediate several

physiological functions that ultimately may result in cancer

suppression. Anthocyanins suppress the growth of

cancer cell lines in vitro, including HL60 human leukemia

calls and HCT116 human colon cancer cells. A bilberry extract

induced apoptotic cell bodies and nucleosomal DNA

fragmentation in HL60 cells (21). A bilberry extract (Mirtocyan)

has also been shown to suppress the activity of

receptor tyrosine kinases, which are thought to play a crucial

role in carcinogenesis and tumor progression. When

tested over a number of tyrosine kinases, the activity was

consistent but not specific (22).

Antibacterial

Phenolic compounds in bilberry have demonstrated in

vitro antimicrobial effects against strains of Salmonella and

Staphylococcus possibly through interfering with adhesion

of the bacteria. Treatment of bilberry preparations with

pectinase released phenolics from the cell wall matrix and

increased the antibacterial activity (23). In experiments

with Neisseria meningitidis, the bacteria that causes meningitis

and septicemia, a bilberry juice fraction was reported

to inhibit the binding of the bacteria to epithelial cells

in culture. Fractions of the juice also bound to the bacterial

pili. The authors concluded that anthocyanins were

partly responsible for the activity but that there appeared

to be other compounds in bilberry that may also interact

directly with the pili or act synergistically with the anthocyanins

(24).

Safety Studies (Animal Toxicology)

Myrtocyan (25% anthocyanins) has been tested for acute

and chronic toxicity in animal studies. There were no

deaths with an acute dose in rats up to 20 g/kg orally

and in mice up to 25 g/kg. Six months treatment with

doses of 125–500 mg/kg in rats and 80–320 mg/kg in

dogs found no evidence of toxicity. The preparation was

tested in guinea pigs for 2 weeks and in rats for 6 weeks

with doses up to 43 mg/kg without incident (2,3).

PHARMACOKINETICS

Animal studies show that bilberry anthocyanins are absorbed

intact, or after methylation. This is unlike other

flavonoid glycosides which are hydrolyzed to their aglycones

and metabolized to glucuronidated or sulfated

derivatives (25). Following administration of 400 mg/kg

orally to rats, peak blood levels of anthocyanins were detected

within 15 minutes and afterwards declined rapidly.

Only 1% of the anthocyanins was eliminated in the urine

and 4% in the bile. The absolute bioavailability of bilberry

anthocyanins was estimated to be 1.2–5% (26). A study in

mice reported that malvidin 3-glucoside and malvidin 3-

galactoside were the principal anthocyanins in the plasma

60 minutes after oral administration of 100 mg/kg. When

the mice were maintained on a diet containing 0.5% bilberry

extract, plasma levels of anthocyanins reached 0.26

M. Anthocyanins were detected in the liver, kidney,

and lung. They were not detected in the spleen, thymus,

heart, muscle, brain, white fat, or eyes (25).

A pharmacokinetic study with six human subjects

detected anthocyanins in the plasma 1.5–6 hours following

intake of a bilberry–lingonberry puree. The study

examined the production of urinary phenolic acids and

found the greatest increase in methylated compounds.

The amount of urinary phenolic acids was low, and the authors

suggested that the fragmentation of anthocyanins

to phenolic acids was not a major metabolic pathway

(27). Another pharmacokinetic study with 20 subjects

that consumed 100 g/day of berries, including black currant,

lingonberries, and bilberries, for 8 weeks reported

an increase in serum quercetin (up to 51% higher) compared

with control subjects who did not consume berries

(28). A study with 25 subjects administered 1.4–5.6 g

Mirtocyan (25% anthocyanins) daily for 7 days reported

detection of anthocyanins as well as methyl and glucuronide

metabolites in the plasma and urine but not in the

liver (29).

CLINICAL STUDIES

Human clinical studies have been conducted evaluating

the potential benefits of bilberry preparations in treating

venous insufficiency and visual disorders ranging from

night vision to diabetic retinopathy as well as cancer prevention.

Vascular Health

Clinical studies have been conducted evaluating the potential

benefits of bilberry preparations in treating venous

insufficiency. A review of studies conducted between 1970

and 1985 included 568 patients with venous insufficiency

of the lower limbs who were treated with bilberry preparations

(30). The studies reported an improvement in circulation

and in lymph drainage resulting in a reduction

in edema. A more recent placebo-controlled study which

included 60 participants with varicose veins reported improvement

in edema in the legs and ankles, sensation of

pressure, cramps, and tingling or “pins and needles” sensations

with a dose of 160 mg Tegens R , three times daily

for 1 month (31). Tegens (Inverni della Beffa, Italy) contains

a bilberry extract named Myrtocyan or MirtoSelect

(25% anthocyanins), manufactured by Indena SpA, Italy.

Visual Health

Asystematic review was conducted on placebo-controlled

studies on the effects of bilberry preparations on night

40 Barrett

vision. Literature searches identified 30 clinical studies,

and 12 of those met the inclusion criteria of being placebo

controlled. Of the 12 studies, 5 were randomized. Healthy

subjects with normal or above average eyesight were

tested in 11 out of the 12 studies. Many of the studies

were acute, using a single dose, and the longest treatment

period was 28 days. Full characterization of the products

used in the studies was not available, but assuming

25% anthocyanin content, the doses of anthocyanin

ranged from 12 to 2880 mg. The techniques used to measure

the extent and rate of dark adaptation ranged from

visual acuity, contrast sensitivity, and critical flicker fusion

to electroretinographic monitoring of response to light

flashes. The four most recent randomized controlled studies

with rigorous methodology reported negative results.

One randomized controlled study and all seven of the nonrandomized

studies reported positive effects. The authors

concluded that the present studies do not support the use

of bilberry by those who are healthy with normal vision to

improve their night vision. However, uncontrolled studies

report a benefit for those with eye disorders, including

retinal degeneration, myopia, simple glaucoma, and

pathological fundus. Furthermore, studies with synthetic

anthocyanins suggest a positive benefit for those with myopia,

central retinal lesions, and night blindness (32).

Two studies on diabetic retinopathy, using a dose of

160 mg Tegens twice daily, demonstrated a trend toward

improvement in mild cases of the disease. The first study

was a 1-month, placebo-controlled study that included

36 subjects. At the end of the month, 10 of 13 patients in

the Tegens group with ophthalmoscopic ally detectable

retinal abnormalities (microaneurysms, hemorrhagic foci,

exudates) were improved, while all 15 patients with these

abnormalities in the placebo group remained unchanged.

A similar trend was observed among those patients with

fluoroangiography abnormalities (33). The second study

lasted 1 year and included 40 subjects who were given

Tegens or placebo in addition to the usual therapy for

retinopathy. As a result, in 50% of patients given bilberry,

the retinal lesions and associated edema were improved

compared with 20% in the control group (34).

A mixture of vitamin E and bilberry (FAR-1, Ditta

Farmigea SpA, Italy) showed a trend toward prevention

of senile cataracts after 4 months of 180 mg bilberry

anthocyanins (25% anthocyanidins) and 100 mg DLDL tocopheryl

acetate twice daily. When the placebo group

was changed from placebo to the bilberry preparation,

and the trial continued for an additional 4 months, there

was no statistical difference between the two groups. The

rationale for this study was previous indications that

antioxidants might prevent the development of senile

cataracts (35).

A mechanistic study using Myrtocyan examined

changes in pupillary reflexes to light following a single

high dose of 240 mg anthocyanosides or placebo in

40 healthy volunteers. The study was conducted to explore

the use of bilberry in work situations where exposure

to high light intensities dampens pupillary reflexes

and leads to vision fatigue. The authors of the study suggested

that the pigments in bilberry might increase sensitivity

to light and improve blood flow in the capillaries of

the eye. Improvement in pupillary reflexes was observed

in both groups, with the improvement in the treatment

group being only slightly better than that in the placebo

group (36).

Cancer Prevention

In an open label study, 25 colorectal cancer patients scheduled

to undergo surgery were given 1.4, 3.8, or 5.6 g

bilberry extract (Mirtocyan) containing 0.2–2.0 g anthocyanins

for 7 days before surgery. Availability of anthocyanins

was determined by detection in the plasma,

colorectal tissue, and urine but not in the liver. Anthocyanins

detected in the body were unaltered, or products

of metabolic glucuronidation and O-methylation. Proliferation

of cells in the tumor tissue was decreased by 7%

compared with before the bilberry intervention (29).

Side Effects and Adverse Effects

No side effects were reported in the clinical studies mentioned

earlier. In a 1987 postmarketing surveillance study

with 2295 subjects, only 94 (4.1%) complained of minor

side effects, most of which involved the gastrointestinal

track. Most of the participants took 160 mg Tegens twice

daily for 1–2 months (3).

Observed Drug Interactions and Contraindications

No drug interactions or contraindications have been reported

in the literature for bilberry.

CONCLUSIONS

Bilberry fruit extracts and anthocyanins have been the

subject of pharmacological studies and human clinical trials.

In vitro and in vivo studies demonstrate good evidence

for the antioxidant activity of bilberry extracts

along with strong indications of benefit to the cardiovascular

system. Animal and human pharmacokinetic studies

demonstrate bioavailability of anthocyanins, but absorption

appears to be limited. Human clinical studies

on the effects of bilberry extracts on eyesight and vascular

diseases suffer from poor methodology, including

small sample sizes and short-term exposures. While it

appears doubtful that bilberry preparations benefit the

night vision of healthy subjects, the benefit for those with

diabetic retinopathy and other eye disorders merits exploration.

Another area that appears promising is that of

benefits to the cardiovascular system, specifically vasculitis

or venous insufficiency. Bilberry products have been

safely consumed, without significant adverse events or

side effects.

REGULATORY STATUS

Bilberry is a food and preparations are also used medicinally

In the United States, preparations of bilberry are

sold as foods and dietary supplements. The U.S. Pharmacopoeia

has published a standard monograph for powdered

bilberry extract (37). The German Commission

E completed a monograph for bilberry fruits in which

preparations of the ripe fruit are indicated orally for

Bilberry 41

nonspecific, acute diarrhea and topically for mild inflammation

for the oral and pharyngeal mucosa (38). The European

Scientific Cooperative on Phytotherapy (ESCOP)

monograph lists the internal use of bilberry fruit preparations

(enriched in anthocyanins) for symptomatic treatment

of problems related to varicose veins, such as painful

and heavy legs. The ESCOP monograph also lists the dried

fruit as supportive treatment of acute, nonspecific diarrhea

(39). In Canada, bilberry products are approved as natural

health products for traditional use orally as an astringent

and as a source of antioxidants as well as for use as a

gargle to relieve mild inflammation of the mouth and/or

throat (40).

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apoptosis in cancer cells by bilberry (Vaccinium myrtillus)

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25. Sakakibara H, Ogawa T, Koyanagi A, et al. Distribution and

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anthocyanosides pharmacokinetics in rats. Arzneimittelforschung

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Biotin

Nutrition, GlossarySuccess Chemistry Staff

INTRODUCTION

Biotin is usually classified as a B-complex vitamin. “Biotin”

is by far the most widely used term for this vitamin.

However, discovery of biotin by different approaches has

also led to names such as Bios IIB, protective factor X, vitamin

H, coenzyme R, factor S, factorS, and vitamin BW.

This entry reviews the biochemistry of biotin and summarizes

the clinical findings of deficiency. Readers are encouraged

to use the references for further information.

SCIENTIFIC NAMES AND STRUCTURE

The molecular weight of biotin is 244.31 Da. The structure

of biotin was elucidated independently by Kogl and du

Vigneaud in the early 1940s and is shown in Figure 1 (1).

Biotin is a bicyclic compound. The imidazolidone contains

an ureido group (–N–CO–N–). The tetrahydrothiophene

ring contains sulfur and has a valeric acid side chain attached

to the C2 carbon of the sulfur-containing ring. This

chain has a cis configuration with respect to the ring that

contains the nitrogen atoms. The two rings are fused in

the cis configuration, producing a boat-like structure.With

three asymmetric carbons, eight stereoisomers exist; only

one [designated D-(+)-biotin or, simply, biotin] is found in

nature and is active when covalently joined via an amide

bond between the carboxyl group of the valeric acid side

chain of biotin and the ε-amino group of a lysine residue of

an app carboxylase. Biocytin (ε-N-biotinyl-L-lysine) is the

product of digestion of protein-bound dietary biotin and

cellular turnover of biotin-containing carboxylases and histones;

biocytin is as active as biotin on a molar basis in

mammalian growth studies.

Goldberg/Sternbach synthesis or a modification

thereof is the method by which biotin is synthesized commercially

(1). Additional stereospecific methods have been

published (2,3).

HISTORY

Biotin was discovered in nutritional experiments that

demonstrated a factor present in many foodstuffs that was

capable of curing the scaly dermatitis, hair loss, and neurologic

signs induced in rats fed dried egg white.

Avidin, a glycoprotein found in egg white, binds biotin very specifically

and tightly. From An evolutionary standpoint, avidin

probably serves as a bacteriostat in egg white. Consistent

with this hypothesis is the observation that avidin is resistant

to a broad range of bacterial proteases in both free

and biotin-bound form. Because avidin is also resistant to

pancreatic proteases, dietary avidin binds to dietary biotin

(and probably any biotin from intestinal microbes)

and prevents absorption, carrying the biotin on through

the gastrointestinal tract.

Biotin is definitely synthesized by intestinal microbes;

however, the contribution of microbial biotin to

absorbed biotin, if any, remains unknown. Cooking denatures

avidin, rendering this protein susceptible to pancreatic

proteases and unable to interfere with the absorption

of biotin.

BIOCHEMISTRY

Biotin acts as an essential cofactor for five mammalian

carboxylases.

Each has the vitamin covalently bound to a

polypeptide. For monomeric carboxylases, this polypeptide

is the apo carboxylase. For the dimeric carboxylases,

this monomer with a biotinylation site is designated the

chain. The covalent attachment of biotin to the app carboxylase

protein is a condensation reaction catalyzed by

holocarboxylase synthetase (EC 6.3.4.10). These apo carboxylase

regions contain the biotin motif (methionine–

lysine–methionine), a specific sequence of amino acids

present in each of the individual carboxylases; this sequence

tends to be highly conserved within and between

species. One interpretation concerning conservation

of this amino acid sequence is that these residues

allow the biotinylated peptide to swing the carboxyl (or

acetyl) group from the site of activation to the receiving

substrate.

All five of the mammalian carboxylases catalyze the

incorporation of bicarbonate as a carboxyl group into a

substrate and employ a similar catalytic mechanism. In

the carboxylase reaction, the carboxyl moiety is first attached

to biotin at the ureido nitrogen opposite the side

chain. Then the carboxyl group is transferred to the substrate.

The reaction is driven by the hydrolysis of ATP

to ADP and inorganic phosphate. Subsequent reactions

in the pathways of the five mammalian carboxylases release

CO2 from the product of the enzymatic reaction.

Thus, these reaction sequences rearrange the substrates

into more useful intermediates but do not violate the classic

observation that mammalian metabolism does not result

in the net fixation of carbon dioxide (4).

The five carboxylases are pyruvate carboxylase

(EC 6.4.1.1), methylcrotonyl-CoA carboxylase (EC 6.4.1.4),

propionyl-CoA carboxylase (EC 6.4.1.3), and two isoforms

of acetyl-CoA carboxylase (EC 6.4.1.2), denoted I

and II, which are also known as ACC and ACC. Each

  • 43

  • 44 Mock

  • HN

  • C

  • O

  • NH

  • HC CH

  • CH

  • O

  • S

  • H2C (CH2) C N (CH2) 4

  • O

  • H

  • 4

  • N-H

  • C-H

  • C=O

  • amino group

Figure 1 Protein-bound biotin with arrow showing the amide bond to the

-amino acid.

carboxylase catalyzes an essential step in intermediary

metabolism.

Pyruvate carboxylase mediates in the incorporation

of bicarbonate into pyruvate to form oxaloacetate, an intermediate

in the Krebs tricarboxylic acid cycle. Thus, pyruvate

carboxylase catalyzes an anaplerotic reaction. In gluconeogenic

tissues (i.e., liver and kidney), the oxaloacetate

can be converted to glucose. Deficiency of this enzyme (denoted

by a block in the metabolic pathway) is likely the

cause of the lactic acidosis and hypoglycemia observed in

biotin-deficient animals and humans.

Methylcrotonyl-CoA carboxylase catalyzes an essential

step in the degradation of the branch-chained

amino acid leucine. Deficient activity of this enzyme

leads to metabolism of 3-methylcrotonyl CoA to 3-

hydroxyisovaleric acid and 3-methylcrotonyl glycine by

an alternate pathway. Thus, increased urinary excretion

of these abnormal metabolites reflects deficient activity of

this carboxylase.

Propionyl-CoA carboxylase catalyzes the incorporation

of bicarbonate into propionyl CoA to form methylmalonyl

CoA, which undergoes isomerization to succinyl

CoA and enters the tricarboxylic acid cycle. In a fashion

analogous to methylcrotonyl-CoA carboxylase deficiency,

inadequacy of this enzyme leads to increased urinary excretion

of 3-hydroxypropionic acid and 3-methylcitric acid

and enhanced accumulation of odd-chain fatty acids C15:0

and C17:0. The mechanism is likely the substitution of propionyl

CoA for acetyl CoA during fatty acid elongation.

Although the proportional increase is large (e.g., 2- to 10-

fold), the absolute composition relative to other fatty acids

is quite small (<1%) and likely produces little or no functional

consequences.

Acetyl-CoA carboxylases, I and II both, catalyze the

incorporation of bicarbonate into acetyl CoA to form malonyl

CoA. Acetyl-CoA carboxylase I is located in the

cytosol and produces cytosolic malonyl CoA, which is

rate limiting in fatty acid synthesis (elongation). Acetyl-

CoA carboxylase II is present on the outer mitochondrial

membrane. As demonstrated by the pioneering work of

Wakil and colleagues, acetyl-CoA carboxylase II controls a

separate mitochondrial pool of malonyl CoA that, in turn,

controls fatty acid oxidation in mitochondria through the

inhibitory effect of malonyl CoA on fatty acid transport

into mitochondria.

  • isoleucine

  • methionine

  • propionyl CoA

  • d-methylmalonyl CoA

  • succinyl CoA

  • glucose

  • oxaloacetate pyruvate acetyl CoA

  • lactate

  • malonyl CoA

  • tricarboxylic acid cycle

  • 3-methylglutaconyl CoA

  • 3-methylcrotonyl CoA

  • Methylcrotonyl-CoA

  • Carboxylase

  • Acetyl-CoA

  • Carboxylase

  • Pyruvate

  • Carboxylase

  • Propionyl-CoA

  • Carboxylase

  • leucine

  • 3-hydroxyisovalerate

  • 3-methylcrotonylglycine

  • 3-hydroxypropionate

  • methylcitrate

  • odd-chain fatty acid

  • fatty acid elongation

Pathways involving biotin-dependent carboxylases. Deficiencies (hatched bar) of pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA

carboxylase, and acetyl-CoA carboxylase lead to increased blood concentrations and urinary excretion of characteristic organic acids denoted by ovals.

Biotin 45

In the normal turnover of cellular proteins, holocarboxylase

are degraded to biocytin or biotin linked

to an oligopeptide containing at most a few amino acid

residues. Because the amide bond between biotin and

lysine (Fig. 1) is not hydrolyzed by cellular proteases,

the specific hydrolase biotinidase [biotin amide hydrolase

(EC 3.5.1.12)] is required to release biotin for recycling.

Biotin exists in free and bound pools within the cell

that are responsive to changes in its status (5). The pool

size is likely determined by a balance between cellular uptake

and cellular release, incorporation into apo carboxylases

and histones, release from these biotinylated proteins

during turnover, and catabolism to inactive metabolites.

Regulation of intracellular mammalian carboxylase activity

by biotin remains to be elucidated.

Genetic deficiencies of holocarboxylase synthetase

and biotinidase cause the two distinct types of multiple

carboxylase deficiency that were previously designated

the neonatal and juvenile forms. The genes for holocarboxylase

synthetase and human biotinidase have been

cloned, sequenced, and characterized (6). The gene coding

for holocarboxylase synthetase is located on chromosome

21q22.1 and consists of 14 exons and 13 entrons in a span of

240 kilobase (kb). Studies of human mutant holocarboxylase

synthetase indicate that all forms of holocarboxylase

synthetase are likely encoded by one gene. Biotinidase

deficiency is particularly relevant to understanding biotin

inadequacy because the clinical manifestations appear to

result largely from secondary biotin depletion.

PHYSIOLOGY

Digestion of Protein-Bound Biotin

The content of free and protein-bound forms of biotin in

foods is variable, but the majority in meats and cereals

appear to be protein bound via an amide bond between

biotin and lysine. Neither the mechanisms of intestinal hydrolysis

of protein-bound biotin nor the determinants of

bioavailability have been clearly delineated.Wolf et al. (7)

have postulated that biotinidase plays a critical role in the

release of biotin from covalent binding to protein. Doses

of free biotin that do not greatly exceed the estimated

dietary intake (e.g., 50–150 g/day) appear adequate to

prevent the symptoms of biotinidase deficiency. This suggests

that biotinidase inadequacy in patients causes biotin

deficiency, at least in part, through impaired intestinal digestion

of protein-bound biotin.

Intestinal Absorption

At physiologic pH, the carboxylate group of biotin is negatively

charged. Thus, the vitamin is at least modestly water

soluble and requires a transporter to cross the membranes

of enterocytes for intestinal absorption, of somatic cells for

utilization, and of renal tubule cells for reclamation from

the glomerular filtrate.

An excellent in-depth review of intestinal uptake of

biotin has been published recently (8). Two biotin transporters

have been described: (i) a multivitamin transporter

present in many tissues including the intestine and (ii) a

biotin transporter identified in human lymphocytes.

The transporter responsible for absorption of free biotin

in the small and large intestine is saturable and Na+

dependent. The transporter also transports pantothenic

acid and lipoate and is deemed the sodium-dependent

multivitamin transporter (SMVT). SMVT was discovered

in 1997 by Prasad et al. (9) in human placental choriocarcinoma

cells. This transporter is widely expressed in

human tissues (10). SMVT system has been cloned and

demonstrated to be exclusively expressed at the apical

membrane of enterocytes. SVMT is the main biotin uptake

system that operates in human intestinal epithelial

cells. The 5-regulatory region of the SMVT gene has also

been cloned and characterized both in vitro and in vivo

(8). Intestinal biotin uptake is adaptively upregulated in

biotin deficiency via a transcriptionally mediated mechanism

that involves KLF4 sites. The cytoplasmic C-terminal

domain of the polypeptide is essential for its targeting to

the apical membrane domain of epithelial cells (8).

In rats, biotin transport is upregulated during maturation

after weaning and by biotin deficiency (11). Carrier Mediated

transport of the vitamin is most active in the

proximal small bowel of the rat and humans (8). However,

absorption from the proximal colon is still significant, supporting

the potential nutritional significance of biotin synthesized

and released by enteric flora (11). Clinical studies

have provided some evidence that biotin is absorbed from

the human colon (12). In contrast, more rigorous studies

in swine indicate that biotin absorption from the hindgut

is much less efficient than that from the upper intestine;

furthermore, biotin synthesized by enteric flora may not

present at a location or in a form in which bacterial biotin

contributes importantly to absorbed biotin.

Exit of biotin from the enterocyte (i.e., transport

across the basolateral membrane) is also carrier mediated

(11). However, basolateral transport is independent

of Na+, is electrogenic, and does not accumulate biotin

against a concentration gradient.

Transport in Blood

Biotin dissolved in blood is carried from the site of absorption

in the intestine to the peripheral tissues and

the liver.

(1). Wolf et al. (13) originally hypothesized that

biotinidase might serve as a biotin-binding protein in

plasma or perhaps even as a carrier protein for the movement

of biotin into the cell. Based on protein precipitation

and equilibrium dialysis using 3H-biotin, Chauhan

and Dakshinamurti (14) concluded that biotinidase is the

only protein in human serum that specifically binds biotin.

However, using 3H-biotin, centrifugal ultrafiltration,

and dialysis to assess reversible binding in plasma from

the rabbit, pig, and human, Mock and Lankford (15)

found that less than 10% of the total pool of free plus reversibly

bound biotin is reversibly bound to plasma protein;

the biotin binding observed could be explained by

binding to human serum albumin. Using acid hydrolysis

and 3H-biotinyl-albumin, Mock and Malik (16) found

additional biotin covalently bound to plasma protein.

The percentages of free, reversibly bound, and covalently

bound biotin in human serum are approximately 81%,

7%, and 12%. A biotin-binding immunoglobulin has been

identified in human serum. An approximately fivefold

higher concentration of this biotin-binding immunoglobulin

was reported in patients with Graves disease than

in normal and healthy controls (17). The role of plasma

proteins in the transport of biotin remains to be definitively

established.

Biotin concentrations in erythrocytes are equal to

those in plasma

(D.M. Mock, unpublished observation).

However, transport into erythrocytes is very slow, consistent

with passive diffusion (18).

Uptake by the Liver

Studies in a variety of hepatic cell lines indicate that

uptake of free biotin by the liver is similar to intestinal

uptake and is mediated by SMVT (19–21). Transport is

mediated by a specialized carrier system that is Na+ dependent,

electroneutral, and structurally specific for a free

carboxyl group. At large concentrations, movement is carried

out by diffusion. Metabolic trapping, for example,

biotin bound covalently to intracellular proteins, is also

important. After entering the hepatocyte, biotin diffuses

into the mitochondria via a pH-dependent process.

The biotin transporter identified in lymphocytes

is also Na+ coupled, saturable, and structurally specific

(22). Recent studies by Daberkow and coworkers provide

evidence in favor of monocarboxylate transporter 1 as the

lymphocyte biotin transporter (23).

A child with biotin dependence due to a defect in

the lymphocyte biotin transporter has been reported (18).

The child became acutely encephalopathic at the age of

18 months. Urinary organic acids indicated deficiency

of several biotin-dependent carboxylases. Symptoms improved

rapidly following biotin supplementation. Serum

biotinidase activity and biotinidase gene sequence were

normal. Activities of biotin-dependent carboxylases in

lymphocytes and cultured skin fibroblasts were normal,

excluding biotin holocarboxylase synthetase deficiency

as the cause. Despite extracellular biotin sufficiency, biotin

withdrawal caused recurrence of abnormal organic

aciduria, indicating intracellular biotin deficiency. Biotin

uptake rates into fresh lymphocytes from the child and

into his or her lymphocytes transformed with Epstein–

Barr virus were about 10% of normal fresh and transformed

control cells, respectively. For fresh and transformed

lymphocytes from his or her parents, biotin uptake

rates were consistent with heterozygosity for an autosomal

recessive genetic defect. SMVT gene sequence was

normal; regulatory regions of the SMVT gene have not

been characterized. These investigators speculated that

lymphocyte biotin transporter is expressed in additional

tissues such as the kidney and may mediate some critical

aspect of biotin homeostasis, but the complete molecular

etiology of this child’s biotin transporter deficiency

remains to be elucidated.

Ozand et al. (24) recently described several patients

in Saudi Arabia with biotin-responsive basal ganglia disease.

Symptoms include confusion, lethargy, vomiting,

seizures, dystonia, dysarthria, dysphagia, seventh nerve

paralysis, quadriparesis, ataxia, hypertension, chorea, and

coma. A mutation in SLC19A3 was identified, and defect

in the biotin transporter system across the blood–brain

barrier was postulated (25). However, in an elegant set

of experiments, Said and coworkers demonstrated that

SLC19A3 is the apical thiamine transporter and renamed

SLC19A3 appropriately as THTR2 (26), in contrast to the

basolateral thiamine transporter THTR1. The explanation

for the documented biotin responsiveness of these patients

remains unknown.

Renal Handling

Specific systems for the reabsorption of water-soluble vitamins

from the glomerular filtrate contribute importantly

to conservation of these vitamins (27). Animal studies using

brush border membrane vesicles from human kidney

cortex indicate that biotin is reclaimed from the glomerular

filtrate against a concentration gradient by a saturable,

Na+-dependent, structurally specific system (28). Using

human-derived proximal tubular epithelial HK-2 cells as

a model, Said and coworkers demonstrated that biotin uptake

by human renal epithelial cells occurs via the SMVT

system and that the process is regulated by intracellular

protein kinase C and Ca++/calmodulin-mediated pathways

(29). The uptake process is adaptively regulated by

extracellular biotin concentrations via transcriptional regulatory

mechanisms (29) consistent with previous studies

demonstrating reduced biotin excretion early in experimentally

induced biotin deficiency in human subjects

(30,31).

Subsequent egress of biotin from the tubular cells

occurs via a basolateral membrane transport system that

is not dependent on Na+. Biocytin does not inhibit tubular

reabsorption of biotin (28). Studies in patients with

biotinidase deficiency suggest that there may be a role for

biotinidase in the renal handling of biotin (32,33).

Transport into the Central Nervous System

A variety of animal and human studies suggest that biotin

is transported across the blood–brain barrier (1,34,35).

The transporter is saturable and structurally specific for

the free carboxylate group on the valeric acid side chain.

Transport into the neuron also appears to involve a specific

transport system as well as subsequent trapping of

biotin by covalent binding to brain proteins, presumably

the biotin-dependent carboxylases and histones.

Recently, concentrations of biotin were determined

initially as total avidin-binding substances in cerebrospinal

fluid (CSF) from 55 children, and biotin, biotin

sulfoxide, and bisnorbiotin were quantitated by highperformance

liquid chromatography (HPLC) and avidinbinding

assay in CSF samples from a subset of 11 children

(36). Concentrations of total avidin-binding substances

averaged 1.6 nmol/L with substantial variability, SD =

1.3 nmol/L. CSF concentrations of biotin and biotin analogous

varied widely, but substantial amounts of biotin

sulfoxide were detected in every sample. Of the total, biotin

accounted for 42% °æ 16%, biotin sulfoxide for 41% °æ

12%, and bisnorbiotin for 8% °æ 14%. Surprisingly, the molar

sum of biotin plus biotin sulfoxide and bisnorbiotin on

average exceeded the total avidin-binding substances concentrations

fromthe same CSF sample by>200-fold. These

investigators found no masking of detection or degradation

of biotin or biotin sulfoxide. Gel electrophoresis

and streptavidin Western blot detected several biotinylated

proteins in CSF leading to the conclusion that biotin

is bound to protein covalently, reversibly, or both; they

speculated that biotin bound to protein likely accounts

for the increase in detectable biotin after HPLC and that

Biotin 47

 

protein-bound biotin plays an important role in biotin nutriture

of the brain.

Placental Transport

Biotin concentrations are 3- to 17-fold greater in plasma

from human fetuses compared with their mothers in the

second trimester, consistent with active placental transport

(37). Specific systems for transport of biotin from the

mother to the fetus have been reported recently (10,38–

40). The microvillus membrane of the placenta contains a

saturable transport system for biotin that is Na+ dependent

and actively accumulates biotin within the placenta,

consistent with SMVT (10,38–40).

 

Transport into Human Milk

More than 95% of the biotin is free in the skim fraction of

human milk (41). The concentration of biotin varies substantially

in some women (42) and exceeds that in serum

by one to two orders of magnitude, suggesting that there

is a transport system into milk. The biotin metabolite bisnorbiotin

(see discussion of metabolism under pharmacology

section) accounts for approximately 50%. In early

and transitional human milk, the biotin metabolite and

biotin sulfoxide accounts for about 10% of the total biotin

plus metabolites (43). With postpartum maturation,

the biotin concentration increases, but the bisnorbiotin

and biotin sulfoxide concentrations still account for 25%

and 8% at 5 weeks postpartum. The concentration of biotin

in human milk exceeds the plasma concentration by

10- to 100-fold, implying that a transport system exists.

Current studies provide no evidence for a soluble biotin binding

protein or any other mechanism that traps biotin

in human milk. The location and the nature of the

biotin transport system for human milk have yet to be

elucidated.

PHARMACOLOGY

Studies in which pharmacologic amounts of biotin were

administered orally and intravenously to experimental

subjects and tracer amounts of radioactive biotin were

administered intravenously to animals show that biotin

in pure form is 100% bioavailable when administered

orally. The preponderance of dietary biotin detectable

by bioassays is bound to macromolecules. Likely biotin

is bound to carboxylases and perhaps to histones. The

bioavailability of biotin from foodstuffs is not known,

whereas that from animal feeds varies but can be well

below 50%. After intravenous administration, the vitamin

disappears rapidly from plasma; the fastest phase of the

three-phase disappearance curve has a half-life of less than

10 minutes.

An alternate fate to being covalently bound to

protein (e.g., carboxylases) or excretion unchanged in

urine is catabolism to an inactive metabolite before excretion

in urine (4). About half of biotin undergoes

metabolism before excretion. Two principal pathways of

biotin catabolism have been identified in mammals. In

the first pathway, the valeric acid side chain of biotin

is degraded by -oxidation. This leads to the formation

of bisnorbiotin, tetranorbiotin, and related intermediates

Table 1 Normal Range of Urinary Excretion of Biotin and Major

Metabolites (nmol/24 hr; n = 31 Males and Females)

Biotin Bisnorbiotin Biotin sulfoxide

that are known to result from -oxidation of fatty acids.

The cellular site of this -oxidation of biotin is uncertain.

Nonenzymatic decarboxylation of the unstable -

keto-biotin and -keto-bisnorbiotin leads to formation of

bisnorbiotin methyl ketone and tetranor biotin methylketone,

which appear in urine. In the second pathway, the

sulfur in the thiophene ring of biotin is oxidized, leading

to the formation of biotin L-sulfoxide, biotin D-sulfoxide,

and biotin sulfone. Combined oxidation of the ring sulfur

and -oxidation of the side chain lead to metabolites such

as bisnorbiotin sulfone. In mammals, degradation of the

biotin ring to release carbon dioxide and urea is quantitatively

minor.

On a molar basis, biotin accounts for approximately

half of the total avidin-binding substances in human

serum and urine (Table 1). Biocytin, bisnorbiotin, bisnorbiotin

methyl ketone, biotin sulfoxide, and biotin sulfone

form most of the balance. Biotin metabolism is accelerated

in some individuals by anticonvulsant therapy and

during pregnancy, thereby increasing the ratio of biotin

metabolites to biotin excreted in urine.

 

OCCURRENCE AND DIAGNOSIS OF BIOTIN DEFICIENCY

The fact that normal humans have a requirement for biotin

has been clearly documented in two situations: prolonged

consumption of raw egg white and parenteral nutrition

without biotin supplementation in patients with

short-gut syndrome and other causes of malabsorption

(1). Deficiency of this member of the vitamin B

group also has been clearly demonstrated in biotinidase

deficiency (6).

The clinical findings and biochemical abnormalities

in cases of biotin deficiency include dermatitis around

body orifices, conjunctivitis, alopecia, ataxia, and developmental

delay (1). The progression of clinical findings in

adults, older children, and infants is similar. Typically, the

symptoms appear gradually after weeks to several years

of egg white feeding or parenteral nutrition. Thinning of

hair progresses to loss of all hair, including eyebrows and

lashes. A scaly (seborrheic), red (eczematous) skin rash

was present in the majority of reports. In several reports,

the rash was distributed around the eyes, nose, mouth,

and perineal orifices. The appearance of the rash was similar

to that of cutaneous candidiasis; Candida albicans could

often be cultured from the lesions. These manifestations

on skin, in conjunction with an unusual distribution of

facial fat, have been dubbed “biotin deficiency facies.”

Depression, lethargy, hallucinations, and paresthesias of

the extremities were prominent neurologic symptoms in

the majority of adults, while infants showed hypotonia,

lethargy, and developmental delay.

In cases severe enough to produce the classic cutaneous

and behavioral manifestations of biotin deficiency,

urinary excretion rates and plasma concentrations
of biotin are frankly decreased. Urinary excretion of the

organic acids discussed in biochemistry section and

shown in Figure 2 is frankly increased. The increase is

typically 5- to 20-fold or more. However, such a severe

degree of biotin deficiency has never been documented to

occur spontaneously in a normal individual consuming a

mixed general diet.

Of greater current interest and debate are the health

consequences, if any, of marginal biotin deficiency. Concerns

about the teratogenic effects have led to studies

of biotin status during human gestation (44–48). These

studies provide evidence that a marginal degree of deficiency

develops in at least one-third of women during

normal pregnancy. Although the degree of biotin deficiency

is not severe enough to produce overt manifestations,

the deficiency is severe enough to produce metabolic

derangements. A similar marginal degree of biotin deficiency

causes high rates of fetal malformations in some

mammals (30,49,50). Moreover, data from a multivitamin

supplementation study provide significant, albeit indirect,

evidence that the marginal degree of deficiency

that occurs spontaneously in normal human gestation is

teratogenic (44).

Valid indicators of marginal biotin deficiency have

been reported. Asymptomatic biotin shortage was induced

in normal adults housed in a general clinical

research center by egg white feeding. Decreased urinary

excretion of biotin, increased urinary excretion

of 3-hydroxyisovaleric acid, and decreased activity of

propionyl-CoA carboxylase in lymphocytes from peripheral

blood are early and sensitive indicators of biotin deficiency

(30,31,51). On the basis of a study of only five

subjects, 3-hydroxyisovaleric acid excretion in response to

a leucine challenge appears to be an even more sensitive

indicator of marginal biotin status (31). The plasma concentration

of biotin and the urinary excretion of methylglycine,

3-hydroxypropionic acid, and 3-methylcitric acid

do not appear to be good indicators of marginal biotin

deficiency (52). In a biotin repletion study, the resumption

of a mixed general diet produced a trend toward normalization

of biotin status within 7 days. This was achieved

when the supplement was started immediately at the time

of resuming a normal diet. However, supplementation

of biotin at 10 times the dietary reference intake (DRI)

(300 g/day) for 14 days reduced 3-hydroxyisovaleric

acid excretion completely to normal in only about half

of pregnant women who were marginally biotin deficient

(47) suggesting a substantial depletion of total

body biotin, a substantially increased biotin requirement,

or both.

On the basis of decreased lymphocyte carboxylase

activities and plasma biotin levels, Velazquez et al. (53)

have reported that biotin deficiency occurs in children

with severe protein-energy malnutrition. These investigators

have speculated that the effects of biotin inadequacy

may be responsible for part of the clinical syndrome of

protein-energy malnutrition.

Long-term treatment with a variety of anticonvulsants

appears to be associated with marginal biotin

deficiency severe enough to interfere with amino acid

metabolism (54–56). The mechanism may involve both

accelerated biotin breakdown (56–58) and impairment of

biotin absorption caused by the anticonvulsants (59).

Biotin deficiency has also been reported or inferred

in several other circumstances including Leiner disease

(60–62), sudden infant death syndrome (63,64), hemodialysis

(65–69), gastrointestinal diseases and alcoholism (1),

and brittle nails (70). Additional studies are needed to confirm

or refute an etiologic link of these conditions to the

vitamin’s deficiency.

The mechanisms by which biotin deficiency produces

specific signs and symptoms remain to be completely

delineated. However, several studies have given

new insights on this subject. The classic assumption for

most water-soluble vitamins is that the clinical findings of

deficiency result directly or indirectly from deficient activities

of the vitamin-dependent enzymes. On the basis

of human studies on deficiency of biotinidase and isolated

pyruvate carboxylase, as well as animal experiments regarding

biotin deficiency, it is hypothesized that the central

nervous system effects of biotin deficiency (hypotonia,

seizures, ataxia, and delayed development) are likely mediated

through deficiency of brain pyruvate carboxylase

and the attendant central nervous system lactic acidosis

rather than by disturbances in brain fatty acid composition

(71–73). Abnormalities in metabolism of fatty acids

are likely important in the pathogenesis of the skin rash

and hair loss (74).

Exciting new work has provided evidence for a potential

role for biotin in gene expression.

These findings

will likely provide new insights into the pathogenesis of

biotin deficiency (75,76). In 1995, Hymes and Wolf discovered

that biotinidase can act as a biotinyl transferase;

biocytin serves as the source of biotin, and histones are

specifically biotinylated (6). Approximately 25% of total

cellular biotinidase activity is located in the nucleus. Zempleni

and coworkers have demonstrated that the abundance

of biotinylated histones varies with the cell cycle,

that these histones are increased approximately twofold

compared with quiescent lymphocytes, and that these are

biotinylated enzymatically in a process that is at least

partially catalyzed by biotinidase (77–79). These observations

suggest that biotin plays a role in regulating DNA

transcription and regulation.

Biotinylation of histones is emerging as an important

histone modification. Recent studies from Hassan

and Zempleni provide evidence that biotinylation likely

interacts with other covalent modification of histones to

suppress gene expression and gene transposition (80). Although

the relative importance in biotinidase and holocarboxylase

synthetase in the biotinylation and biotinylation

of histones has yet to be fully elucidated, Gravel

and Narang have produced evidence that holocarboxylase

synthetase is present in the nucleus in greater quantities

than in the cytosol or the mitochondria and that holocarboxylase

synthetase likely acts in the nucleus to catalyze

the biotinylation of histones (81). Moreover, fibroblasts

from patients with HCLS deficiency are severely deficient

in histone biotinylation (82). Zempleni and coworkers

have shown that biotinylation of lysine-12 in histone H4

(K12BioH4) causes gene repression and have proposed

a novel role for HCS in sensing and regulating levels

of biotin in eukaryotic cells (83). They have hypothesized

that holocarboxylase synthetase senses biotin and

that biotin regulates its own cellular uptake by participating

in holocarboxylase synthetase–dependent chromatin

Biotin 49

 

remodeling events at an SMVT promoter locus. Specifically,

they hypothesize that nuclear translocation of HCS

increases in response to biotin supplementation and then

biotinylated histone H4 at SMVT promoters, silencing biotin

transporter genes. This group has shown that nuclear

translocation of HCS is a biotin-dependent process potentially

involving tyrosine kinases, histone deacetylases,

and histone methyltransferases. The nuclear translocation

of holocarboxylase synthetase correlates with biotin concentrations

in cell culture media and is inversely linked to

SMVT expression. Moreover, biotin homeostasis by holocarboxylase

synthetase–dependent chromatin remodeling

at an SMVT promoter locus is disrupted in holocarboxylase

synthetase knockdown cells.

 

Transposable elements such as retrotransposons

containing long-terminal repeats constitute about half of

the human genome, and the transposition events associated

with these elements impair genome stability. Epigenetic

mechanisms are important for transcriptional repression

of retrotransposons, preventing transposition events,

and abnormal regulation of genes. Zempleni and coworkers

have provided evidence that the covalent binding of

biotin to lysine-12 in histone H4 and lysine-9 in histone

H2A mediated by holocarboxylase synthetase is an epigenetic

mechanism to repress retrotransposon transcription

in human and mouse cell lines and in primary cells from a

human supplementation study. Abundance of biotinylation

at those sites depended on biotin supply and on holocarboxylase

synthetase activity and was inversely linked

with the abundance of long terminal repeat transcripts.

Knockdown of holocarboxylase synthetase in Drosophila

enhanced retrotransposition. Depletion of biotinylation

at those sites in biotin-deficient cells correlated with increased

production of transposition events and decreased

chromosomal stability.

Recently, controversy has arisen concerning the role

of biotin as an in vivo covalent modifier of histones. Bailey

and coworkers have reported that streptavidin binds

to histones independently of biotinylation (84). To further

investigate this phenomenon, 293T cells were grown in

14C-biotin; in contrast to the ready detectability of 14Cbiotin

in carboxylases, 14C-biotin was undetectable in histones

(i.e., represented no more than 0.03% of histones)

(84). In a subsequent study, Healy and coworkers demonstrated

that histone H2A is nonenzymatically biotinylated

by biotinyl-5-AMP and provided evidence that these enzymes

promotes biotinylation of histone H2A by releasing

biotinyl-5-AMP, which then biotinylated lysines in histone

H2A somewhat nonspecifically (85). Recently, this

group has proposed that biotin is not a natural histone

modifier at all. On the basis of studies that fail to find

in vivo biotin incorporation into histones using 3H-biotin

uptake, Western blot analysis of histones, or mass spectrometry

of affinity purified histone fragments, these investigators

concluded that biotin is absent in native histones

to a sensitivity of 1 part per 100,000 and that the

regulatory impact on gene expression must occur through

a mechanism other than histone modification (86). These

conclusions are likely to generate a lively debate until

definitive evidence is provided using mass spectrometric

analysis of in vivo histones harvested at various phases

of the cell cycle and at specific locations within particular

histones.

INDICATIONS AND USAGE

In 1998, the United States Food and Nutrition Board of the

National Academy of Sciences reviewed the recommendations

for biotin intake (87). The committee concluded that

the data were inadequate to justify setting an estimated

average requirement. However, adequate intake (AI) was

formulated (Table 2). The AI for infants was based on

an empirical determination of the biotin content of human

milk. Using the value for free biotin determined microbiologically

(6 g/L) and an average consumption of

0.78 L/day by infants of age 0–6 months, an AI of 5g/day

was calculated. The AI for lactating women has been increased

by 5 g/day to allow for the amount of biotin

secreted in human milk. Using the AI for 0–6-month-old

infants, the reference body weight ratio method was used

to extrapolate AIs for other age groups (see Table 2).

 

TREATMENT OF BIOTIN DEFICIENCY

If biotin deficiency is confirmed, biotin supplementation

should be undertaken and effectiveness should be documented.

Doses between 100 g and 1 mg are likely to

be both effective and safe on the basis of studies supplementing

biotin deficiency during pregnancy, chronic

anticonvulsant therapy, and biotinidase deficiency.

TOXICITY

Daily doses of up to 200 mg orally and up to 20 mg intravenously

have been given to treat biotin-responsive inborn

errors of metabolism and acquired biotin deficiency.

Toxicity has not been reported.

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Bitter Orange

Glossary, NutritionSuccess Chemistry Staff

INTRODUCTION

Citrus aurantium (C. aurantium) is the Latin name for a

plant commonly referred to as bitter orange, sour orange,

Neroli, Chongcao, or Seville orange. It is a source

of synephrine and several other biogenic amines, as well

as other bioactive phytochemicals and has been used in dietary

supplements for weight loss. In this entry,we discuss

the available evidence pertaining to safety and efficacy of

C. aurantium for weight loss, as examined in animal studies,

clinical trials, and case reports.

GENERAL DESCRIPTION

Bitter orange is a member of the Rutaceae family, a hybrid

between Pummelo, Citrus grandis, and Mandarin, Citrus

reticulata.

Native to Asia, various parts of the plant are

used throughout the world for a variety of indications.

Bitter orange and its components are commercially available

in herbal weight loss supplements, ostensibly for their

adrenergic agonistic properties (1), often in combination

with other ingredients hypothesized to promote weight

loss. Its constituent p-octopamine and synephrine alkaloids

(SAs) are usually cited as the active ingredients in

such products (2). With the banning of ephedra in the

United States in 2004, bitter orange has been increasingly

included in weight loss supplement formulations. Because

of similarities in their constituents and possible mechanisms

(both sources of natural alkaloids with sympathomimetic

activity), concerns have been raised that bitter

orange may carry risks similar to those hypothesized to

exist for ephedra (3).

HISTORICAL USE

Bitter Orange origin is in China and appears in writing

as far back as 300 BC.

Its ancient use has also been documented

in Japan and Rome (4). It is native to eastern

Africa, Arabia, and Syria and is cultivated in various European,

North American, and South American regions. The

leaf was historically used as a tonic, laxative, or sedative

in Mexico and South America and for insomnia, palpitations,

or stomachaches by the European Basque people

(5,6). The fruit and peel are also used for stomach aches, as

well as high blood pressure (BP), spasm, and a variety of

gastrointestinal conditions by both the Basque and practitioners

of traditional Chinese medicine (7). While the

practice arose in Ancient Egypt, neroli oil is still currently

used for aromatherapy and bergamot, a subspecies of

C. aurantium, is used for flavoring and aroma in Earl Grey

teas (8). Modern uses for C. aurantium include digestive,

cardiovascular, neuromuscular, and antiseptic indications

in countries such as China, Curacao, Haiti, India, Mexico,

Trinidad, Turkey, and the United States (9). The most

common current western use, however, is as a dietary

supplement for weight loss.

POTENTIAL CONSTITUENTS

Some authors (10) state that C. aurantium contains meta synephrine

(m-synephrine, m-s), whereas others (11) state

that it contains only para-synephrine (p-synephrine, ps).

However, research (I.A. Khan, oral communication,

2005) has shown that C. aurantium naturally contains

p-synephrine and does not contain m-synephrine. Allison

and colleagues reported that at least one over-the counter

(OTC) product purportedly containing SAs from

C. aurantium contains both p-synephrine andm-synephrine

(12), raising concerns about possible adulteration and

mislabeling. There is also an ortho isomer of synephrine

(o-synephrine), whose content in C. aurantium is unknown.

p-, m-, and o-synephrine can each exist in D or L forms.

p-Synephrine, an undisputed component of C. aurantium,

is typically referred to simply as synephrine (13).

It is an -adrenergic agonist (14) that also has some -

adrenergic properties (15). p-Synephrine occurs naturally

in the human body in small quantities and might act as

a neurotransmitter (16). Under the name oxedrine, it has

been used since 1927 (17) in eyedrops. p-Synephrine is

thought to be the ingredient in C. aurantium primarily

responsible for weight loss. However, neither this nor

whether C. aurantium actually produces weight loss in

humans is firmly established.

m-Synephrine, often referred to as phenylephrine,

is an isomer of p-synephrine. To the best of our knowledge,

m-synephrine is not contained naturally in C. aurantium.

m-Synephrine is also an -adrenergic agonist that has

some -adrenergic agonist properties. It has been studied

more extensively than p-synephrine and is one of the

two most widely used OTC decongestants today (Fig. 1)

(13). p-Synephrine and m-synephrine have similar structure

to ephedrine, as well as other substances that have

some effects on reducing food intake and/or body weight

such as epinephrine and norepinephrine (Fig. 1), supporting

the conjecture that, to the extent that function follows

structure, p-synephrine and m-synephrine, may also reduce

food intake and or body weight.

The -adrenergic sympathomimetic amine, octopamine,

is also present in C. aurantium, though possibly

at appreciable levels (2). Like both forms of

synephrine, it is an -adrenergic agonist with some

Chemical structures of (A) p-synephrine, (B) m-synephrine,

(C) ephedrine, (D) epinephrine, and (E) norepinephrine.

-adrenergic properties. It is used to treat hypertension

and as a cardiotonic (13) and has also been examined for

its potential role in promoting weight loss (18).

Because of their similar properties and the overlap

of their inclusion in supplements, we will refer to these

substances collectively as synephrine alkaloids (SAs). SAs

are used clinically as decongestants (1), during surgical

procedures as a vasopressor (19), for acute treatment of

priapism (20), and in ophthalmological examinations for

pupil dilation (21). Products that contain C. aurantium or

its derivatives, including OTC weight loss supplements,

will be referred to as C. aurantium products (CAPs).

Regulatory oversight for dietary supplements is

much less rigorous than for pharmaceuticals, and extensive

evidence is not required prior to release of a product

on the public market. While a phase of requirements for

meeting good manufacturing practices is currently underway,

this may help to explain why the quality and quantity

of the evidence we have available to evaluate the safety

and efficacy of C. aurantium is minimal.

POTENTIAL MECHANISMS

As sympathomimetic agents with both - and -

adrenergic receptor agonist properties, SAs might increase

energy expenditure and/or decrease food intake (22). In

addition, there is some evidence that adrenergic agonists,

including SAs, decrease gastric motility (23). Similar to

compounds such as cholecystokinin and other gut peptides

which both decrease gastric motility and food intake

(24), one might conjecture that SAs may also decrease food

intake via reducing gut motility. Activation of lipolysis is

a known -adrenergic activity (25) that may be fueled by

these components of C. aurantium.

ANIMAL STUDIES

Bitter Orange Weight Loss

SAs reduce food intake in rodents (26), and some studies

indicate that SAs can reduce rodent body weight

(13,26). SAs have also been shown to promote lipolysis

in adipocytes through -adrenergic stimulation (27)

and to increase lipoprotein lipase activity in the parametrial

fat pad of female hamsters (28). However, among

monosodium glutamate–treated obese mice, SAs reduced

weight gain but had no effect on body fat percent (29).

Toxicity and Mortality

Data suggest that m-synephrine (not present in bitter orange)

may prolong life in rodents. A 2-year study by the

National Toxicology Program (13) evaluated the effects

of m-synephrine on spontaneous food intake of rats and

mice. At 2 years, there were no significant differences in

survival among mice or female rats. However, for male

rats, there was a significant reduction in mortality rate,

although there was increased mortality in the early phase

of the study at the highest dose. It should be noted that

too few deaths occurred during the 2-year trial to provide

the degree of precision and power desired for a rigorous

longevity study (30). Nonetheless, similar results have

been reported for ephedrine, another sympathomimetic

amine (31).

Arbo et al. (32) conducted a subchronic toxicity

study in mice and the effects of p-synephrine and C. aurantium

L. extract on oxidative stress biomarkers that are

believed to be indicators of cell membrane injury (malondialdehyde)

and (glutathione and the enzyme glutathione

peroxidase) indicative of amphetamine-induced toxicity.

The study evaluated adult male CF1 mice treated with

400, 2000, or 4000 mg/kg C. aurantium dried extract and

p-synephrine 30 or 300 mg/kg over the course of 28 days.

Results showed a reduction in glutathione in mice treated

with C. aurantium 400 mg/kg and p-synephrine 30 and

300 mg/kg. Inhibition of glutathione peroxidase activity

occurred within mice treated with C. aurantium 400 and

2000 mg/kg and p-synephrine 30 and 300 mg/kg; however,

no change occurred within malondialdehyde levels.

These two findings suggest the possibility of subchronic

toxicity. No significant change in weight occurred in any

of the groups, suggesting on the positive side a lack of

severe toxicity, and on the negative side a lack of efficacy

in producing weight loss.

With regard to adverse effects, a study (33) of male

Sprague-Dawley rats reported what was believed to be

evidence of cardiotoxicity when C. aurantium fruit extracts

standardized to 4% and 6% SAs were administered. Increased

mortality has been observed among CAPs-treated

rats (33) as well as a strain of mice selected to be uniquely

susceptible to the effects of adrenergic stimulation (34).

CLINICAL TRIALS Bitter Orange Weight Loss

Few clinical trials have examined the effects of CAPs alone

or in combination with other ingredients on body weight

and/or body composition (Table 1). It should be kept in

mind that these trials are of short duration and the sample

sizes are frequently quite small. Nonetheless, these

trials suggest that body weight and/or fat loss may be

enhanced by CAPs or SAs. The mechanisms involved are

unclear but may be partially due to a suppressing effect

of appetite and/or a moderate increase in resting energy

expenditure.

Armstrong et al. (37) evaluated exercise and herbal

preparation containing Ma Huang, bitter orange (5 mg

SAs), and guarana over 6 weeks in a randomized, controlled

trial. Compared with controls, the intervention

group obtained significant reductions in fat mass and a

nearly significant reduction in body mass index (kg/m2)

54 Haaz et al.

Table 1 Summary of Clinical Weight Loss Trials

Reference Treatment Design Sample size Duration Results Comments

Colker et al. (10) 975 mg Citrus

aurantium, +528

caffeine and 900 mg

St. John’s wort;

placebo (with pill) and

control (no pill)

Blinded parallel

groups RCT

Supplement n = 9;

placebo, n = 7;

control group (no

pills), n = 4

6 wk Supplement group lost

more fat (3.1 kg; P <

0.05) than other

groups and increased

RMR (2–3%)

Citrus aurantium may

assist individuals in

losing body fat, due to

increased energy and

reduced energy intake

expenditure. No

adverse events were

reported

Kalman et al. (36) Ephedrine and

synephrine alkaloids

(SAs) (5 mg twice

daily) based product

vs. placebo with

exercise and diet

Prospective,

randomized,

double blind

30 overweight

subjects; BMI > 27

8 wk 3.4 kg weight loss in

experimental group vs.

2.05 kg in placebo

(P < 0.05)

No adverse events;

findings indicate

apparent short-term

safety and efficacy of

ephedrine and

synephrine-based

compound

Armstrong et al.

(37)

Exercise program with

assignment to drug

(Ma Huang, bitter

orange, and guarana)

or placebo. Bitter

orange standardized

for 5 mg synephrine

Randomized

trial—unclear if

study is blinded

Five overweight

males/14 females

44 days Supplement increased

fat loss (2.5 kg; P =

0.033) more than

placebo (0.5 kg))

Low statistical power,

no marked side effects

Greenway et al.

(38): Pilot 1

Two capsules

containing

pantothenic acid,

40 mg; green tea leaf

extract, 200 mg;

guarana extract,

550 mg; bitter orange,

150 mg; white willow

bark extract, 50 mg;

ginger root, 10 mg;

proprietary charge

thermoblend

(L-tyrosine, L-carnitine,

naringin), 375 mg

Prospective,

randomized,

double blind

Eight subjects (1:1

ratio) between

supplement group and

placebo group

8 wk Supplement group

gained more weight

(1.04 °æ 0.27 kg; P <

0.04) than placebo

and increased RMR

(but not at 8 wk)

CAP was not

efficacious for weight

loss

Greenway et al.

(38): Pilot 2

m-Synephrine 20 mg Prospective,

randomized,

double blind

Twenty subjects (1:1

ratio) between

supplement group and

placebo group

8 wk Supplement group lost

weight (0.8 °æ 3.4 kg;

not significant) in 8

wk, and increased

RMR in 8 wk. No

control group was

used (Greenway,

written

communication,

November 1, 2009)

m-Synephrine was not

efficacious for weight

loss

Abbreviations: BMI, body mass index; CAP, Citrus aurantium product; RCT, randomized, controlled trial; RMR, resting metabolic rate.

and fat percentage. No significant changes were noted in

resting energy expenditure, blood chemistries, or dietary

intake between the placebo and experimental groups.

In a double-blind, placebo-controlled, randomized

trial, Colker et al. found that subjects receiving a

combination of C. aurantium, caffeine, and St. John’s wort,

along with diet and exercise protocols, lost a statistically

significant amount of body weight. Analysis comparing

changes in this group with those in placebo or control

groups on the same diet and exercise regimen did not

show significant differences, though loss of fat mass was

significantly greater in the experimental group (35). BP,

heart rate, electrocardiographic, blood, and urine analyses

were not significantly different between the groups.

Another randomized trial (36) of 30 overweight

adults investigated the effects of supplementation, along

with a cross-training exercise regimen and dietary education

program compared with exercise and dietary

education alone on body composition. Supplementation

included ephedrine, SAs, caffeine, and calicine.

Greater weight and fat loss occurred for the supplement

group compared with the exercise–diet only

group.

Overall, studies indicate a weight loss of 2.4–3.4 kg

among participants using SAs, while placebo groups lost

0.94–2.05 kg, suggesting the plausibility of some weight

loss benefit from SA supplementation, beyond diet and

exercise alone. However, these studies do not separate

Bitter Orange 55

the effects of C. aurantium or SAs from other ingredients,

particularly ephedrine and caffeine.

Metabolic Rate and Cardiovascular Effects

Several studies have evaluated the effects of acute administration

of SAs on cardiovascular indicators. Kalman et al.

(39) tested a product containing 335 mg Ma Huang standardized

for 20 mg ephedrine alkaloids, 910 mg guarana

standardized for 200 mg caffeine, and 85 mg bitter orange

standardized for 5 mg SAs per two capsules. Twentyseven

overweight adults were randomized to treatment

or placebo for 14 days. BP, heart rate, electrocardiogram,

and Doppler echocardiograms were evaluated before and

after treatment. Ingestion of this commercial weight loss

supplement did not produce any detectable cardiovascular

side effects.

Penzak et al. (10) examined cardiovascular outcomes

in 12 normotensive individuals who were administered

8 oz of Seville orange juice (containing 13–14 mg SAs)

and water in a crossover fashion, followed by a repeat

ingestion 8 hours later. No changes in cardiovascular indices

(BP, maximal arterial pressure, and heart rate) were

detected.

Thomas et al. (40) evaluated the cardiovascular effects

of 10 mg oral SAs in healthy volunteers over a 4-hour

period on impedance cardiography and forearm plethysmography.

Elevation in total peripheral resistance was observed

30–60 minutes after dosing, although other hemodynamic

indexes were not affected.

Hemodynamic effects were observed in a crossover

design, placebo-controlled study (41) with the administration

of Xenadrine, a CAP that contains a variety of other

potentially bioactive substances, including green tea extract,

cocoa extract, yerba mate, ginger root, grape seed

extract, and others. However, these increases in heart rate,

and systolic and diastolic BP were not observed with administration

of Advantra Z, which contains C. aurantium

alone, even at an eightfold higher dose.

Haller et al. (42) evaluated a dietary supplement

[Ripped Fuel Extreme Cut, containing synephrine from

C. aurantium (presumably p-synephrine) and caffeine] in

10 healthy adults (three women) aged 20–31 years. Each

subject was given one dose of the dietary supplement

under three conditions: (i) resting conditions (without

placebo); (ii) moderately intense exercise; and (iii) placebo

plus moderately intense exercise in a three-arm, randomized,

crossover study. Greater postexercise diastolic BP

was seen with the dietary supplement plus exercise than

with placebo plus exercise. There were no obvious supplement

effects on postexercise HR, systolic BP, or body

temperature.

Bui et al. (43) reported the effect on BP (systolic

and diastolic) and heart rate over 6 hours after one

dose of a CAP (Nature’s Way Bitter Orange) on 15

young, healthy adults in this prospective, randomized,

double-blind, placebo-controlled, crossover study. Systolic

and diastolic BP increased significantly within the

1–5 hours time period in comparison with the placebo

group with the peak being 7.3 °æ 4.6 mm Hg, while the

4–5 hours time period increase was 2.6 °æ 3.8 mm Hg after

consumption in comparison with the placebo group

with the peak being 4.2 °æ 4.5 beats/minute, while

heart rate was significantly elevated 2–5 hours after

ingestion.

In one study of obese adults, increases in resting

metabolic rate (RMR) were observed with C. aurantium,

both alone and with food, beyond the thermic effect of

food (TEF) alone (44). (RMR is a measure of the energy

required to maintain basic physiological function while

the body is at rest.) However, another recent investigation

(45) found that the thermic response to CAPs increased in

women only, who had lower TEF than men at baseline.

After the intervention, TEF did not differ by gender. BP

and pulse rate were not affected, but epinephrine secretion

increased. In normal weight adults, an increase in RMR

was also found when the extract was taken with a meal

(46).Noadverse changes in pulse rate or BP were reported.

Finally, the effects of two dietary supplement formulas

onRMRand other metabolic indicators were evaluated

(47). When compared with placebo, Formula A (containing

ephedra, guarana, green tea, yohimbe, and quercetin)

and Formula B (containing C. aurantium, jing jie, fang feng,

guarana, green tea, yohimbe, and quercetin) resulted in increased

total RMR, decreased respiratory exchange ratio

toward fat burning, and increased body core temperature.

Heart rate and RMR increased at each 15-minute interval

with Formula A only. BP increased with both, but to a

greater extent with Formula A.

 

CASE REPORTS OF ADVERSE EVENTS

Nykamp et al. (48) describe a case of acute lateral-wall

myocardial infarction co-occurring with consumption of

CAPs in a 55-year-old woman with undetected coronary

vascular disease. She reported taking a multicomponent

dietary weight loss supplement containing 300 mg of bitter

orange over the preceding year.

Consumer Reports article (49) describes a 21-yearold

woman who took ephedra-free Xenadrine EFX (which

contains C. aurantium). After 3 weeks on the supplement,

she suffered a seizure. Her neurologist believes the bitter

orange in the supplement was the most likely the cause,

though the basis for this conclusion is unknown.

Nasir et al. (50) described exercise-induced syncope

in a healthy 22-year-old woman that occurred 1 hour after

a second dose of Xenadrine EFX, a weight loss supplement

that contains, among other compounds, ephedrine and

synephrine. The electrocardiography revealed prolongation

of the QT interval, which resolved in 24 hours.

Bouchard et al. (51) report a case of a 38-year-old

male patient with ischemic stroke that occurred after taking

a CAP for 1 week. The patient reportedly had no relevant

medical history or major atherosclerotic risk factors

and took no other medications.

Gray andWoolf (52) reported a case of CAPs use by

an adolescent with anorexia nervosa and raised concerns

that the SAs may have masked bradycardia and hypotension

while exacerbating her weight loss. Firenzuoli et al.

(53) report a case of a 52-year-old woman that had an

allergic reaction after taking a CAPs product.

Sultan et al. (54) reported a case of a 52-year-old

woman with ischemic colitis that occurred 1 week after

consumption of a CAP (Natural Max Skinny Fast,

containing bitter orange). She reported no known drug

56 Haaz et al.

Table 2 Summary of Effects, Safety, and Efficacy of Citrus aurantium

Physiological effects Effects on weight Effects on body composition Safety

Variable changes in BP in animals;

generally stable BP, heart rate, pulse

rate, blood and urine measures in

humans; inconsistent changes to

resting metabolic rate

Weight loss documented in rodents;

weakly supported in humans, as

studies used multiple supplements or

did not find significant difference

from controls

Limited support for loss of fat mass in

human studies, noting a trend or

using multiple supplements; for

animals, some increased lipase

activity

Inconsistent mortality data in

rodents; some evidence of

elevated BP. Results not

consistent from study to study,

but this may be a function of

small sample sizes used in most

studies. Several case reports of

serious adverse events

allergies and took no other medications. Symptoms resolved

over 24–48 hours with conservative management

after the supplement was discontinued.

Health Canada reported that fromJanuary 1, 1998, to

February 28, 2004, it received 16 reports in which products

containing bitter orange or synephrine were suspected

of being associated with cardiovascular events, including

tachycardia, cardiac arrest, ventricular fibrillation, transient

collapse, and blackout. All cases were considered

serious (55).

Adverse events from CAPs are currently fairly rare

in scientific literature. As CAPs are used more widely in

place of ephedrine-containing products, any potentially

harmful effects may be clarified over time.

DISCUSSION

The Safety of CAPs

Some have hailed the potential therapeutic value of CAPs

(1), while others have warned about possible safety concerns

(33). The safety concerns pertain primarily to adverse

cardiovascular and cerebrovascular effects. Information

on the safety of CAPs comes from the three sources

described above: animal studies, clinical trials, and case

reports. To date, no large epidemiologic (case control or

cohort) studies have evaluated the safety of CAPs.

Of course, one cannot extrapolate the safety of CAPs

from short-term studies used for one indication (e.g., several

days for relief of nasal congestion among the general

population) to long-term studies use for another indication

(e.g., several months or years for weight loss

among obese individuals). Although substantial safetyrelated

data exist for CAPs (13,56), there is no published

human weight loss trial of CAPs with more than 20 participants

or for a duration of more than 7 weeks.

It is important to note that the majority of

studies evaluating the safety of CAPs are performed

with normotensive subjects. However, because hypertension

is a common comorbidity associated with overweight/

obesity, studies that evaluate the effects of CAPs

on BP should also be conducted with obese hypertensive

adults.

While C. aurantium extracts have been used in a variety

of cultures for thousands of years, they have not been

traditionally utilized for long periods of time, or specifically

for weight loss (1). As such, there is little, if any, basis

for making definitive statements about the intermediate

or long-term safety/risk of CAPs used for weight loss.

Table 2 summarizes the physiological effects, safety, and

weight loss efficacy of C. aurantium.

 

DOSE CONSIDERATIONS

Given the dearth of weight loss trials, the optimal dose

(if one exists) of C. aurantium or its SA constituents for

weight loss is unknown. Table 3 highlights some relevant

dosage information. Although generalizing across species

and compounds is difficult and can only provide a limited

basis for conjecture, the following comparisons with

ephedrine can be made. We analyzed data (12) in which

ephedrine or SAs was given to mice. Regression of weight

and food intake on dose of ephedrine or SAs yielded

slopes (in absolute value) that were approximately four

to six times greater for ephedrine than for SAs. Based on

linear projections, it would take four to six times the dose

of SAs (in these mice) to achieve equivalent reduction in

intake and body weight as for ephedrine. In human studies

of ephedrine, doses of about 50 mg per day begin to be

effective (57). Although an extrapolation, this might suggest

a useful clinical dose for SAs as high as 240–360 mg

Table 3 Dosage Information on Citrus aurantium or Synephrine Alkaloids (SAs)

Dose

5–14 mg/day Citrus aurantium extract with SAs has been used (34–36) and no serious adverse events were reported. These doses purportedly

showed efficacy, but products tested included substances beyond C. aurantium, notably ephedrine which we know to be effective for

weight loss. We believe that these doses of SAs are very unlikely to be effective when used without ephedrine

32 mg/day The nasal decongestant Endal (60) contains 20 mg of m-s per tablet and two tablets per dose twice per day are recommended

120 mg/day Via C. aurantium extract, SAs are marketed in over-the-counter (OTC) products for weight loss. In products, such as Nutres Lipo 6

(61), the directions suggest that for “extreme fat loss” a recommended dosage is two capsules three times per day. The SA content

per capsule is 20 mg; this provided a maximal recommended dose of 120 mg/day

300 mg/day According to Clarke’s Analysis of Drugs and Poisons (62), oxedrine (p-synephrine) is used clinically at ∼300 mg/day

1000 mg/day Minimum adult lethal dose of m-s (63)

Bitter Orange 57

per day. From a safety point of view, SAs (per equal

weight) have lower potential to raise BP than ephedrine;

however, nearly all commercial preparations of SAs also

contain caffeine, which might compound any cardiovascular

effects. In the absence of caffeine, human studies

suggest that 15–30 times the dose of SAs are required to

elevate BP to the same degree as ephedrine (58,59). This

suggests that such high doses might be well tolerated,

but clearly more data are needed, particularly regarding

potential synergistic effects of CAPs components.

SAs appear to be readily absorbed after oral administration

(63). About 80% of oral doses are excreted in

the urine within 24 hours. After single oral doses, peak

plasma concentrations are typically reached in 1–2 hours.

Plasma half-life is ∼2–3 hours. Sympathomimetic drugs

for weight loss are typically given TID before meals (64)

reducing the evening dose if sleep problems arise.

CONTRAINDICATIONS

Topical application (as with aromatherapy or antifungal

uses) of CAPs may result in photosensitivity for fair skinned

individuals (65) (possibly due to photosensitizing

furanocoumarins that occur in the rinds of certain

citrus species, especially immature fruits). Although

rare, this has also occurred after oral ingestion. To reduce

this risk, exposure to ultraviolet light can be minimized.

Caution is recommended for use in children, as it

may conceivably produce toxic effects (66). Some sources

advise that CAPs should be avoided by women who are

pregnant or breastfeeding (7,67), while others claim that

CAPs can be used safely during pregnancy (66). While

effects on BP are unclear, those with hypertension, tachyarrhythmia,

or narrow-angled glaucoma may consider

refraining from use of CAPs until further evidence confirms

their safety (67).CAPs could also possibly exacerbate

symptoms for those with stomach or intestinal ulcers (68).

DRUG INTERACTIONS

Because CAPs may increase stomach acid, they could potentially

reduce the efficacy of acid-lowering drugs, such

as antacids and ulcer medications (69). Although a speculative

precaution, those taking medications containing

SAs, including some cold medications and monoamine oxidase

inhibitors (MAOIs), should consider the combined

dose of these products with the SAs present in CAPs formulations

and possible multiplicative effects (68,69). It has

been suggested that CAPs could interfere with the activity

of drugs that are metabolized by the liver enzyme cytochrome

P450-3A, CYP3A (70,71). A recent comment in

Experimental Biology and Medicine noted that some research

on drug effects have utilized parts of the plant or

methods of administration that may not be applicable to

oral consumption of currently marketed dietary supplements

FUTURE RESEARCH

The safety and efficacy of CAPs and SAs for weight

loss are not well established. While existing literature

demonstrates plausibility for reducing weight, previous

trials were not designed to rigorously evaluate safety and

efficacy. Doing so will require better-designed randomized

clinical trials with large sample sizes, reliable well established

outcome measures, and active surveillance of

side effects and adverse events. To better understand the

effects of CAPs or SAs specifically, studies will need to test

these components without combining them with other ingredients

postulated to have antiobesity effects. It would

also be worthwhile to examine differences between the

types of synephrine-containing compounds that are derived

from various sources and how this influences the

consistency and potency of supplements.

ACKNOWLEDGMENTS

The writing of this entry was supported in part

by NIH grant nos. P30DK056336, AR49720–01A1, and

T32HL072757. The opinions expressed are solely the responsibility

of the authors and do not necessarily represent

the official views of the NIH or any other organization

with which the authors are affiliated.

Disclosure: Dr. Allison has received grants, honoraria,

consulting fees, and donations from numerous

companies, government agencies, and nonprofit organizations

with interests in obesity in general and dietary

supplements in particular, including organizations litigating

cases involving C. aurantium.

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Black Cohosh

Glossary, HerbsSuccess Chemistry Staff

Black cohosh is a native eastern North American plant that

was used as traditional medicine by Native Americans.

 

Extracts of the roots and rhizomes were used for analgesic,

sedative, and anti-inflammatory properties.More recently,

root and rhizome black cohosh preparations have had a

rich clinical history, spanning almost 60 years of study.

These studies have primarily focused on relieving climacteric

symptoms associated with menopause as a possible

alternative to classical hormone or estrogen replacement

therapy.

 

BACKGROUND

The common name for black cohosh [Actaea racemosa L.

syn., Cimicifuga racemosa (L.) Nutt. (Ranunculaceae, Buttercup

Family)] originated with North American Indians.

The term cohosh is thought to be an Algonquian

word meaning “rough,” with reference to the texture

of the thick, knotted roots and underground stems (rhizomes).

A New World plant used by Native Americans,

it was most abundant in the Ohio River Valley, but it

could also be found from Maine to Wisconsin, south

along the Allegheny Mountains to Georgia, and west to

Missouri.

Various common names have been used to refer to

black cohosh, including black snakeroot, bugbane, rattleroot,

squawroot, and macrotys. It is a member of the Ranunculaceae

or Buttercup family, which includes other

medicinal plants such as aconite, goldenseal, and pulsatilla.

It has been known by the scientific name C. racemosa

and recently has been assigned to A. racemosa. The

generic name Cimicifuga derives from the Latin cimex

(a kind of bug) and fugare (to put to flight), which is

perhaps indicative of the use of some strongly smelling

close relatives to repel insects. The specific epithet racemosa

refers to the flowering stalk, termed a raceme. The

name rattleroot is indicative of the rattling sound made by

the dry seeds in their pods. This plant prefers the shade

of rich open hardwood forests, but it will tolerate some

sunny spots.

 

Black cohosh has been used clinically for relief of

climacteric symptoms for more than 60 years, and its popularity

in the United States as a botanical dietary supplement

has increased due to the recently recognized potential

risks associated with classical estrogen replacement

therapy or hormone replacement therapy (1,2). The part

of the black cohosh plant used in medicinal preparations

is the root and rhizome. It was officially recognized in the

United States Pharmacopeia (USP) from the first edition in

1820 to 1936 and in the National Formulary from 1936 to

1950. The eclectic physicians used a preparation of black

cohosh called macrotys. It was considered one of the bestknown,

specific medicines for heavy, tensive, and aching

pains as it was noted to have a direct influence on the

female reproductive organs.

While the mechanism of action has not been completely

elucidated, recent literature suggests that alleviation

of climacteric symptoms is mediated through neurotransmitter

regulation and not through classical estrogen

receptor (ER) endocrine pathways (3,4).

CHEMISTRY

More than 60 triterpene glycosides, most with a 9,19

cycloartane skeleton, and unique to Actaea spp., have

been reported from the roots and rhizomes of A. racemosa

(5,6). The compound 23-epi-26-deoxyactein (formerly

27-deoxyactein) is the constituent usually selected for

standardization of commercial products based on its

abundance in the roots and rhizomes (7–12). The pharmacokinetics

of 23-epi-26-deoxyactein in serum and urine has

recently been reported (13). While triterpenes are structurally

similar to steroids and possess a broad range of

biological activity (14–17), no significant ligand binding

affinity was found toward ER- in the evaluation of 23-

epi-26-deoxyactein, cimiracemoside F and cimicifuga,

and their respective aglycones (18). This, coupled with

the lack of demonstrated estrogenic activity in A. racemosa

extracts, has called into question the notion that black cohosh

acts through direct ER binding by the triterpenes, as

has been hypothesized (19–23).

In addition to the triterpene saponins, the roots and

rhizomes of black cohosh also contain a number of aromatic

acids/polyphenols that possess a wide array of biological

activities (5,24–26). Caffeic acid, which is found

widely across all species of flowering plants, has shown

pregnant mare anti gonadotropin activity (27–29), rat uterine

antispasmodic activity (30), and smooth muscle relaxant/

antispasmolytic activity in rats (31) and guinea

pig ileum (32). Ferulic acid, also more or less ubiquitous

among flowering plants, has demonstrated luteinizing

hormone (LH) release inhibition (33), follicle-stimulating

hormone (FSH) release stimulation (33), antiestrogenic activity

(34), prolactin stimulation in cows (35) and inhibition

in rats (33), and uterine relaxant/antispasmolytic

activity in rats (36). Fukinolic acid produced an estrogenic

effect on MCF-7 cells with reference to estradiol

Black Cohosh

(37). A more recent study refuted this effect and demonstrated

a lack of estrogenic effect for 10 other phenolic

esters, many of which are unique to Actaea spp. (caffeoyl glycolic

acid; 2-caffeoylquinic acid (cimicifuga acid

D); 3,4-dihydroxyphenyl caffeate (petasiphenone); 3,4-

dihydroxyphenyl-2-oxopropyl isoferulic (cimici phenol);

3,4-dihydroxyphenyl isoferulic (cimici phenone); cimicifuga

acids A, B, E, F; and folic acid) from black

cohosh (38).

Studies on the phenolic acid constituents of black

cohosh have shown antioxidant activity (24,39) that may

correlate with or prove useful in the determination of the

mechanism of action of black cohosh. In addition, a number

of plant sterols and fatty acids, generally regarded as

ubiquitous in the plant kingdom, are contained in the roots

and rhizomes for which the biological activities probably

do not relate to the mechanism of action of black cohosh

(5). In the past 5 years, novel guanidine alkaloids have

been isolated from A. racemosa underground parts (40,41).

New phytochemical methodology called pH zone refinement

gradient centrifugal partitioning chromatography

coupled with a sensitive liquid chromatography–mass

spectral dereplication method led to the identification of

N-(omega)-methylserotonin as a potential active principle

with serotonergic properties (41). Alkaloids have also been

reported from other Actaea spp. roots and rhizomes (42,43).

There has been some debate over the occurrence

of the weakly estrogenic compound formononetin in the

plant (44–49). Although there has been at least one report

of its occurrence in A. racemosa (46), prior studies using

plant material collected from different sites in the Eastern

United States at different times of the year failed to find

formononetin (47,48). More recent studies on both commercial

black cohosh products and wild-crafted material,

incorporating both high-performance liquid chromatography

with mass spectral and photodiode array detection,

confirmed the prior findings of no detectable formononetin

in black cohosh (8,49).

BOTANICAL DESCRIPTION

A. racemosa syn. C. racemosa is an erect, smooth-stemmed

perennial 1–2.5 m in height. Large compound leaves are

alternately arranged and triternate on short clasping petioles.

Basal leaf petioles are grooved in young specimens.

This shallow, narrow sulcus in A. racemosa disappears as

the petiole enlarges, whereas it remains present throughout

the life of the two related eastern North American

species, A. cordifolia DC syn. C. rubifolia Kearney and

A. podocarpa DC syn. C. americana Michx (50). Terminal

leaflets of A. racemosa are acute and glabrous with sharp

serrated margins, often trilobate, occasionally bilobed.

Fruits are ovoid follicles occurring sessile on the pedicel.

The flowering portion, the raceme, is a long wandlike

structure with showy white flowers. The flowers possess

numerous characteristic stamen and slender filaments

with distinctive white anthers (51). The roots and rhizomes

are branched and knotted structures with a dark brown

exterior and are internally white and mealy or brown and

waxy. The upper rhizome surface has several buds and numerous

large stem bases terminated frequently by deep,

cup-shaped, radiating scars, each of which show a radiate

structure or less frequently fibrous strands. Lower and lateral

surfaces exhibit numerous root scars and a few short

roots. The fracture is horny, the odor slight, and the taste

bitter and acrid (52).

Black Cohosh EFFECTS ON CLIMACTERIC SYMPTOMS

RELATED TO MENOPAUSE

With a history of clinical study spanning almost 60 years,

mainly in Europe (53), black cohosh is one of the more popular

alternatives to hormone replacement therapy. Most

of the clinical research over this span has been performed

on the product known as Remifemin R , whose formula

has changed over the years. However, a number of other

commercial formulations are also available. In 2007, black

cohosh was the 50th best-selling dietary supplement in

the United States with sales of approximately $52 million

(USD), according to the Nutrition Business Journal (54).

Black cohosh clinical study outcomes have been

evaluated using a variety of tools, including self- or physician

assessments of symptom scores and physiological

parameters. Typical measurements include psychological,

neurovegetative, somatic, and physiological markers of

menopause or relief from the climacteric symptoms of

menopause. As in all clinical trials, study design is vital,

so studies that are adequately powered, incorporate

proper controls, and are designed to address confounders

relevant to climacteric symptoms such as the placebo

effect and botanical product quality should be given

more weight than studies that are not as well designed

(55–59).

Placebo effects in menopausal trials are generally

large (60) and reflect underlying fluctuations of symptoms.

Therefore, any well-designed study must adjust

the appropriate variables (i.e., study duration, number

of subjects (n), and/or dosage) to account for such an effect.

In the evidence-based medicine model, the gold standard

in terms of efficacy involves randomized, controlled

trials (RCTs). Many RCTs on black cohosh exist. When

high-quality studies are combined, more than 3000 subjects

have been randomized, with the more recent studies

adding layers of design sophistication. For example,

double-blind, multicenter, placebo-controlled trials that

provide details regarding clinical material specifications

are becoming more prevalent (55–60).

A recent phase III, double-blind, randomized,

placebo-controlled crossover trial of the effectiveness of

black cohosh for the management of hot flashes was conducted

over two 4-week periods (one capsule, 20 mg bid)

(61). The study used a daily hot flash diary and found

that subjects receiving the black cohosh material reported

a mean 20% decrease in hot flash score (comparing the

fourth treatment week to the baseline week) versus a 27%

decrease for patients on placebo (P = 0.53), mean hot flash

frequency was reduced 17% in the black cohosh group and

26% on placebo (P = 0.36). Thus, the authors concluded

that the study did not provide any evidence that black

cohosh reduced hot flashes more than the placebo. Critics

of the study point to the short duration and low dose as

potential confounders of the results.

The Herbal Alternatives for Menopause trial or

HALT trial compared the efficacy of 160 mg daily black

62 Fabricant et al.

cohosh against several other interventions (200 mg daily

multi botanical with black cohosh and nine other ingredients;

200 mg daily multi botanical plus dietary soy counseling;

0.625 mg daily conjugated equine estrogen with or

without 2.5 mg medroxyprogesterone acetate daily; and

placebo) in 351 menopausal and postmenopausal women

of ages 45–55 years with two or more vasomotor symptoms

per day. Results did not suggest efficacy for any of

the herbal interventions when compared with placebo at

any time point over the 1-year course of the study (62).

The Jacobson study (63), spanning only 60 days of

treatment, suggests that the short study duration may

have limited the findings (60). In addition, all the study

participants had a history of breast cancer. The authors

reported that the median number of hot flashes decreased

27% in both the placebo and black cohosh groups. No significant

differences were observed between groups. Thus,

black cohosh, on the basis of this study, was no more effective

than placebo in the treatment of hot flashes. The

source and formulation of the extract used in this study

was not specified. A more recent open-label study that

treated breast cancer survivors with either Tamoxifen R or

a combination of BNO 1055, a proprietary black cohosh extract,

with Tamoxifen suggested a reduction in the number

and severity of hot flashes in the combination treatment

group (64).

In another randomized, double-blind, placebo controlled

study that lasted 12 weeks, black cohosh was

compared with standard conjugated estrogen (CE) therapy

(0.625 mg/daily). Patients’ physical and psychological

symptoms were measured every 4 weeks. The end

result of the study was that the patients treated with

black cohosh had significantly lower index scores on

both the Kupperman menopausal (KM) and the Hamilton

menopausal (HAM-A) scales compared with placebo,

indicating a decrease in severity and frequency of hot

flashes. In addition, this study showed an increase in the

number of estrogenized cells in the vaginal epithelium in

the black cohosh treatment arm, which could indicate an

estrogenic action in this tissue (65).

In 2003, a similar study compared effects of two

different preparations of BNO 1055 extract and CE therapy

on climacteric symptoms and serum markers of

bone metabolism (66). The study outcomes were evaluated

using patient self-assessment (diary and menopause

rating scale), CrossLaps (to measure bone resorption),

bone specific alkaline phosphatase (marker of bone formation),

and endometrial thickness (measured by ultrasound).

Both BNO 1055 extracts were equipotent to CE

therapy and significantly greater than placebo at reducing

climacteric complaints. In addition, the study showed

that both BNO 1055 preparations had beneficial effects

on bone metabolism in serum. Specifically, an increase in

bone-specific alkaline phosphatase and no reduction in

bone resorption were noted indicating an increase in bone

turnover formation. No change in endometrial thickness

was observed in either BNO 1055 treatment groups, but it

was significantly increased with CE therapy. An increase

in superficial vaginal cells was observed in the CE and

both BNO 1055 treatment groups. The authors of the study

hypothesized that the activity of both BNO 1055 preparations

was similar to the effects of selective estrogen receptor

modulating (SERM), that is, Raloxifene R therapy on

bone and neurovegetative climacteric symptoms, without

any uterotrophic effects (66).

A recent high-quality, double-blind, randomized

study evaluated the effects of two dosages (low, 39 mg;

high, 127 mg) of a Remifemin extract on menopausal

symptoms. Effectiveness was measured using the KM index,

self-assessment depression scale (SDS), clinical global

impression scale (CGI), serum levels of LH and FSH,

sex hormone–binding globulin, prolactin, 17--estradiol,

and vaginal cytology. Reductions in the KM and SDS indices

were significant. Global efficacy (CGI) was scored

at good to very good in 80% (low dosage) and 90%

(high dosage) of the patients in the treatment groups (67).

No effect on serum hormone levels or vaginal cytology

was shown, prompting the authors of the study to suggest

that black cohosh does not have a direct estrogenic

effect on the serum hormone levels or vaginal epithelium

(68). Two recent open-label studies using unspecified

types of extracts reported reduced KM index scores.

One study reported a significant reduction in 1 month

(69), while the other, which also used the HAM-A scale,

recorded a 90% improvement in climacteric symptoms

in menopausal women after 3 months of black cohosh

administration (70).

Chung and colleagues (71) examined a combination

of black cohosh and St. John’s wort (Gynoplus R ) inamulti center

RCT in 89 peri- or postmenopausal women with

climacteric symptoms. Subjects were treated for 12 weeks

with either the Gynoplus extract or placebo. In addition

to climacteric complaints, investigators also examined effects

on vaginal atrophy, serum hormone levels (FSH, LH),

and lipid profiles [total cholesterol, high-density lipoprotein

(HDL) cholesterol, low-density lipoprotein cholesterol,

and triglyceride]. Significant improvements in climacteric

symptoms and hot flashes, as well as an increase

in HDL, were observed in the Gynoplus group by 4 weeks

and maintained after 12 weeks, but there was no significant

impact on vaginal atrophy.

In a 12-month, randomized, four-arm, double-blind

clinical trial of standardized black cohosh, red clover,

placebo, and 0.625 mg conjugated equine estrogens plus

2.5 mg medroxyprogesterone acetate (conjugated equine

estrogens (CEE) and medroxyprogesterone acetate (MPA);

n = 89), black cohosh did not significantly reduce the

frequency of vasomotor symptoms as compared with

placebo. The primary outcome measures were reduction

in vasomotor symptoms (hot flashes and night sweats) by

black cohosh and red clover compared with placebo; secondary

outcomes included safety evaluation, reduction in

somatic symptoms, relief of sexual dysfunction, and overall

improvement in quality of life. Reductions in number of

vasomotor symptoms after a 12-month intervention were

as follows: black cohosh (34%), red clover (57%), placebo

(63%), and CEE/MPA (94%), with only CEE/MPA differing

significantly from placebo. Secondary measures indicated

that both botanicals were safe as administered. In

general, there were no improvements in other menopausal

symptoms (72).

A 12-week trial investigating the effects of black cohosh

on menopause-related anxiety disorder found no

statistically significant anxiolytic effect of black cohosh

versus placebo.

However, small sample size, choice of

black cohosh preparation, and dosage used may have

Black Cohosh 63

contributed to the negative results according to the study’s

authors (73).

More details of the human studies discussed here,

as well as others, are presented in Table 1.

BIOCHEMISTRY AND FUNCTIONS

Despite the extensive clinical research, the mechanism of

action of black cohosh on menopausal and other symptoms

remains unclear, which is consistent with the varied

results from clinical trials.A Majority of the older literature

suggest a direct estrogenic effect. More recent hypotheses

have proposed an effect on the limbic system (hypothalamus)

or an effect on the neurotransmitters involved in

regulation of this system as being responsible for the activity

of black cohosh. Data fall into the following categories.

Estrogen Receptor Competitive Binding

The first report of ER-binding activity of black cohosh

indicated this as a possible mechanism of action (74).

Additional studies were carried out to substantiate this

purported endocrine activity (75,76). However, a factor

frequently overlooked regarding black cohosh receptor

binding studies is the lipophilic nature of the extracts

tested. Chemically, lipophilic extracts and fractions that

display ER-binding activity are significantly different

from the typical hydroalcoholic extracts used to make

products for human consumption. A lipophilic extract of

the plant showed relatively weak (35 g/mL) ER binding

on rat uteri (75). Another study also confirmed the ERbinding

activity of an unspecified lipophilic subfraction

on ovariectomized (ovx) rat uterine cells, with no binding

activity seen with a hydroalcoholic extract (76).

Recent reports have contradicted the ER-binding

affinity of black cohosh extracts (4,20,22,77,78). A root extract

tested in an in vitro competitive cytosolic ER (from

livers of ovx rat) binding assay with diethylstilbestrol

(50), an inhibitor of estrogen binding, showed a significant

inhibition of estradiol binding in the presence of diethylstilbestrol

(77). However, no binding was demonstrated

for the black cohosh extract. A hydroalcoholic A. racemosa

rhizome extract (50% aqueous ethanol) was assayed for

ER binding in intact human breast cancer cell lines MCF-7

and T-47-D. Again no binding affinity was shown for the

black cohosh extract. However, binding activity was evident

for other hydroalcoholic plant extracts, such as red

clover (78). In another study, a high concentration (200g/

mL) methanol extract of black cohosh displayed no binding

affinity for recombinant diluted ER- and ER- (20).

A study using BNO 1055 showed contrasting results

(79). The extract displayed dose-dependent competition

with radio-labeled estradiol in both a porcine and human

endometrial cytosolic ER ligand-binding assay system.

However, the extract did not displace human recombinant

ER- and ER-. These contradictory findings prompted

the authors to suggest that their product contains estrogenic

compounds that have binding affinity for a putative

ER-. The absence of a direct estrogenic effect was again

confirmed in a human study (21). Postmenopausal Women

took black cohosh extract for 12 weeks followed by a 12-

week washout. Black cohosh demonstrated no effect on

estrogenic markers in serum and no effect on pS2 or cellular

morphology in nipple aspirate fluid (21).

Receptor Expression

As with the receptor-binding assays, the nature of the extract

or fraction is a decisive factor in the expression of

ERs. A lipophilic and hydrophilic black cohosh extract

was studied for luciferase expression in a MCF-7 - and

-ER expressing subclone (80). The lipophilic extract at

35 g/mL activated transcription of the estrogen regulated

genes, while the hydrophilic extract showed no

activity. A recent study measuring an extract at a low concentration

(4.75 g/L) increased ER levels in human MCF-

7 cells as did estradiol (81). An unspecified black cohosh

extract tested in a transient gene expression assay using

HeLa cells co-transfected with an estrogen-dependent reporter

plasmid in the presence of human ER- or ER-

cDNA failed to show transactivation of the gene (82).

Plasma Hormone Levels

The effect of black cohosh on serum concentrations of FSH

and LH has been studied extensively. Crude alcoholic extracts

suppressed plasma LH with no effect on FSH in

ovx rats (75,77). Further fractionation of the crude extract

resulted in activity of the lipophilic fraction while

the hydrophilic fractions were devoid of this activity (74).

A later study in rats using lipophilic and hydrophilic extracts

at high doses (140 and 216 mg/rat, IP) resulted in

LH suppression with a single injection administration of

the lipophilic but not the hydrophilic extract (75). Another

study reported LH suppression in ovx rats with

an unspecified dose of black cohosh extract (83). A recent

study compared the effect of BNO 1055 with that

of estradiol on LH levels (79). Extract administered subcutaneously

at a dosage of 60 mg/day for 7 days was

reported to reduce LH levels in the treated animals. However,

another study reported no estrogen agonistic effects

on FSH, LH, or prolactin levels in ovx rats using the 7,12-

Dimethylbenz(a)anthracene model following 7 weeks of

daily administration of a 40% isopropanolic extract of the

plant (Remifemin) (84).

Hormonal Secretion

The effect of black cohosh on prolactin secretion in pituitary

cell cultures was measured using an unspecified

extract (85). Basal and Thyrotropin-releasing hormone

(TRH)-stimulated prolactin levels were significantly reduced

at doses of 10 and 100 g/mL. This effect was

reversed by the addition of haloperidol (D2-antagonist) to

the cell cultures, suggesting dopaminergic regulation of

hormone secretion by black cohosh.

Osteopenia Inhibition

The black cohosh extract BNO 1055 (60 mg/rat, SC) has

been shown to increase the expression of collagen I and

osteocalcin in rats in a manner similar to that produced

by 8 g of estradiol in ovx rats (79). An additional study

using BNO 1055 demonstrated an osteoprotective effect

as shown by a reduced loss of bone mineral density in

rat tibia after 3 months of administration (81). A study

using an unspecified isopropanol extract of black cohosh

showed reduced urinary markers of bone loss. The authors

64 Fabricant et al.

Table 1 Selected Black Cohosh Clinical Studies

Author (reference no.) Year Extract/formulation/dosage Study length N Outcome measure/result Study design

Kessel Kaul (110) 1957 Remifemin R

60 drops 2 wk 63 Alleviation of climacteric complaints in 95%

of patients

Case series

Schotten (111) 1958 Remifemin 20 drops 3–4 wk 22 Alleviation of neurovegetative and psychic

complaints associated with menopause and

premenopause

Case series

Foldes (53) 1959 Remifemin, 3 tablets/day Unknown 41 31 patients of the verum group responded

to the treatment with a decrease in

menopausal complaints

Placebo,

controlled, open,

crossover, patient

self-assessment

Starfinger (112) 1960 Remifemin, 3–20 drops/day 1 yr 105 Decreased climacteric complaints without

incidence of side effects or resulting in

non physiological bleeding

Case series

Brucker (113) 1960 Remifemin, tablets, variable

dose

Variable 87 (517) Alleviation of menopausal complaints Case series

Heizer (114) 1960 Remifemin, tablets 3–6/day 2–18 mo 66 Alleviation of menopausal (neurovegetative

and psychic) complaints in 47% of patients

with intact uteri and 35% with

hysterectomies

Case series

Gorlich (115) 1962 Remifemin, tablets, variable

dose

Variable 41 (258) Alleviation of climacteric and vascular

symptoms in 85% of patients

Case series

Schildge (116) 1964 Remifemin, fluid extract

60 drops/day

Variable 135 Euphoric and mild sedative-calming effects

in all pts

Case series

Stolze (117) 1982 Remifemin, fluid extract

80 drops/day

6–8 wk 629 Alleviation of neurovegetative and

psychological menopausal symptoms in

80% of patients

Open, physician

and patient

self-assessment

Daiber (118) 1983 Remifemin, fluid extract

80 drops/day

12 wk 36 Alleviation of climacteric complaints (hot

flashes, insomnia, sweating, and

restlessness)

Open, KMI, CGI

Vorberg (119) 1984 Remifemin, fluid extract

80 drops/day

12 wk 50 Significant or highly significant alleviation of

menopausal (neurovegetative and psychic)

complaints; study included subjects

contraindicated to hormone therapy

Randomized,

open, KMI, CGI,

POMS

Warnecke (120) 1985 Remifemin, fluid extract

80 drops/day

12 wk 20 Significant alleviation of symptoms (psychic

and neurovegetative) in the black cohosh,

conjugated estrogen, and diazepam groups.

Vaginal cytology of treatment group was

comparable to estrogenic stimulation

Randomized,

open, KMI, HAM-A,

SDS, CGI,

karyopyknosis

index, eosinophil

index

Stoll (121) 1987 Remifemin, tablets

equivalent to 8 mg

extract/day

12 wk 26 Significant alleviation of climacteric

symptoms (vaginal atrophy, neurovegetative

and psychic complaints) in comparison with

estrogen and placebo groups

Double-blinded,

randomized,

placebo controlled,

KMI,

HAM-A, VMI

(vaginal

epithelium)

Petho (122) 1987 Remifemin, tablets,

unspecified dose

6 mo 50 KMI decreased significantly from 17.6 to

9.2, correlates with a significant reduction

in neurovegetative symptoms. Severity of

subjective self-assessments of subjects

physical and psychological symptoms

decreased

Open, KMI, patient

self-assessment

Lehman-Willenbrock

and Riedel (123)

1988 Remifemin, tablets

equivalent to 8 mg

extract/day

6 mo 15 Significant alleviation of climacteric

symptoms in black cohosh and drug

treatment groups. No significant change in

gonadotropin (FSH, LH) levels

Randomized,

open, KMI

Duker et al. (75) 1991 Remifemin, tablets

equivalent to 40 mg dried

herb/day

2 mo 110 LH suppression In vitro study using

blood from

menopausal

women taking

black cohosh

Black Cohosh 65

Table 1 Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.) Year Extract/formulation/dosage Study length N Outcome measure/result Study design

Baier-Jagodinski

(124)

1995 Cimisan R

T Tropfen,

variable dose

4–8 wk 157 89% of patients showed symptom improvement

after 4 wk. At final visit, the efficacy was assessed

as very good, 40%; good, 41%; sufficient, 12%;

inadequate, 7%

Open, uncontrolled

Mielnik (69) 1997 Uncharacterized extract,

4 mg daily

6 mo 34 Alleviation of climacteric (neurovegetative)

symptoms in 76% of patients after 1 mo

Open, KMI

Georgiev and

Iordanova (70)

1997 Uncharacterized extract,

unspecified dose

3 mo 50 Alleviation of climacteric symptoms in 90% of

patients. Increase in vaginal cell proliferation

(VMI) in 40% of treated women

Open, KMI,

HAM-A, VMI

Nesselhut and Liske

(125)

1999 Remifemin, tablets,

equivalent to 136 mg dried

herb/day

3 mo 28 Good to very good alleviation of 10 menopausal

symptoms in 80% of study participants

Open, postmarket

surveillance

Jacobson, et al. (63) 2001 Remifemin, tablets

equivalent to 40 mg dried

herb/day

60 days 42a No change in median number or intensity of hot

flashes

Double blinded,

randomized,

placebo

controlled, patient

self-assessment,

VAS, MSS

Liske et al. (67) 2002 Unique Cimicifuga

racemosa preparation,

equivalent to 39 or

127.3 mg/day

6 mo 152 No direct systemic estrogenic effect on serum

levels of FSH, LH, SHBG, prolactin, and 17-

estradiol. No change in vaginal cytology. Higher

dose had a more significant reduction in KM

index after 6 mo. Significant reduction with both

doses in neurovegetative and psychic complaints

Drug equivalence

trial, KMI, SDS,

CGI

Hernandez Munoz and

Pluchino (66)

2003 BNO 1055 12 mo 136 Combination therapy with tamoxifen (20 mg)

reduced severity and incidence of hot flashes

Open, randomized,

patient

self-assessment

Wuttke et al. (64) 2003 Klimadynon R

/BNO 1055 3 mo 62 Equipotent to 0.6 CE for relief of climacteric

complaints and for bone resorption. No effect on

endometrial thickness

Randomized,

double blinded,

placebo

controlled,

multicenter, MRS

Verhoeven et al.

(126)

2005 125 mg soy extract daily

(providing 50 mg

isoflavones including 24 mg

genistein and 21.5 mg

daidzein), 1500 mg evening

primrose oil extract

(providing 150 mg gamma

linoleic acid), 100 mg

Actaea racemosa L. extract

(providing 8 mg

deoxyacetein), 200 mg

calcium, 1.25 mg vitamin D,

and 10 IU vitamin E,

placebo group received

2000 mg olive oil daily

12 wk 124 Subjects were experiencing at least five

vasomotor symptoms every 24 hr at study entry.

At weeks 6 and 12, all scores in both groups had

improved compared with baseline, though the

overall difference in scores between the groups

was not statistically significant

Multicenter,

randomized,

placebo controlled,

double-blind

study, Kupperman

index and Greene

Climacteric scale

Nappi et al. (127) 2005 Aqueous isopropanolic

extract 40 mg/day

3 mo 64 Postmenopausal women were recruited. Both CR

and low-dose TTSE2 significantly reduced the

number of hot flushes per day (P < 0.001) and

vasomotor symptoms (P < 0.001), starting at

the first month of treatment. Such a positive

effect was maintained throughout the 3 mo of

observation, without any significant difference

between the two treatments. An identical effect

was evident also for both anxiety (P < 0.001)

and depression (P < 0.001), which were

significantly reduced following 3 mo of both CR

and low-dose TTSE2. Total cholesterol was

unchanged by CR treatment but significantly

(P < 0.033) reduced by 3 mo of low-dose TTSE2.

A slight but significant increase of HDL cholesterol

Randomized,

controlled, clinical

study

(continued)

66 Fabricant et al.

Table 1 Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.) Year Extract/formulation/dosage Study length N Outcome measure/result Study design

(P < 0.04) was found only in women treated with

CR, while LDL-cholesterol levels were significantly

lowered by 3 mo of both CR (P < 0.003) and

low-dose TTSE2 (P < 0.002). Triglyceride levels

were not affected by both treatments nor was liver

function. FSH, LH, and cortisol were not

significantly affected after the 3-mo treatment,

while PRL (P < 0.005) and 17--E2 (P <

0.001) were increased slightly only by low-dose

TTSE2. Endometrial thickness was not affected by

either CR or low-dose TTSE2

Frei-Kleiner et al.

(128)

2005 6.5 mg dry rhizome extract;

60% ethanol extraction

solvent. Dose = 1 cap daily

12 wk 122 Menopausal women were recruited. The primary

efficacy analysis showed no superiority of the

tested black cohosh extract compared with

placebo. However, in the subgroup of patients

with a Kupperman index > or = 20 a significant

superiority regarding this index could be

demonstrated (P < 0.018). A decrease of 47%

and 21% was observed in the black cohosh and

placebo group, respectively. The weekly weighted

scores of hot flashes (P < 0.052) and the

Menopause Rating Scale (P < 0.009) showed

similar results. Prevalence and intensity of the

adverse events did not differ in the two treatment

groups

Multicenter,

randomized,

placebo controlled,

double-blind,

parallel group

study

Pockaj et al. (61) 2006 20 mg C. racemosa and

rhizome extract standardized

to contain 1 mg of triterpene

glycosides as calculated by

27-deoxyacetin, placebo

Two 4-wk

crossover

treatment

periods

132 Toxicity was minimal and not different by

treatment group. Patients receiving black cohosh

reported a mean decrease in hot flash score of

20% (comparing the fourth treatment week with

the baseline week) compared with a 27%

decrease for patients on placebo (P = 0.53).

Mean hot flash frequency was reduced 17% on

black cohosh and 26% on placebo (P = 0.36).

Patient treatment preferences were measured

after completion of both treatment periods by

ascertaining which treatment period, if any, the

patient preferred. Thirty-four percent of patients

preferred the black cohosh treatment, 38%

preferred the placebo, and 28% did not prefer

either treatment

Double-blind,

randomized,

crossover clinical

trial. Primary end

point was the

average

intrapatient hot

flash score (a

construct of

average daily hot

flash severity and

frequency)

difference between

the baseline week

and the last study

week of the first

treatment period.

Green Climacteric

scale

Newton et al. (HALT)

(62)

2006 (i) Black cohosh, 160 mg

daily; (ii) multi botanical with

black cohosh, 200 mg daily,

and 9 other ingredients;

(iii) multi botanical plus

dietary soy counseling;

(iv) conjugated equine

estrogen, 0.625 mg daily,

with or without

medroxyprogesterone

acetate, 2.5 mg daily; or

(v) placebo

1 yr 351 Women aged 45–55 yr with two or more

vasomotor symptoms per day were recruited.

Vasomotor symptoms per day, symptom intensity,

Wiklund Vasomotor Symptom Subscale score did

not differ between the herbal interventions and

placebo at 3, 6, or 12 mo or for the average over

all the follow-up time points (P > 0.05 for all

comparisons) with 1 exception: At 12 mo,

symptom intensity was significantly worse with

the multi botanical plus soy intervention than with

placebo (P > 0.016). The difference in

vasomotor symptoms per day between placebo

and any of the herbal treatments at any time

point was less than one symptom per day; for the

average over all the follow-up time points, the

difference was less than 0.55 symptom per day.

The difference for hormone therapy versus

placebo was −4.06 vasomotor symptoms per day

for the average over all the follow-up time points

(95% CI, −5.93 to −2.19 symptoms per day;

Randomized,

double-blind,

placebo-controlled

trial. Wiklund

Vasomotor

Symptom scale

Black Cohosh 67

Table 1 Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.) Year Extract/formulation/dosage Study length N Outcome measure/result Study design

P > 0.001). Differences between treatment

groups smaller than 1.5 vasomotor

symptoms per day cannot be ruled out.

Black cohosh containing therapies had no

demonstrable effects on lipids, glucose,

insulin, or fibrinogen (124)

Raus et al. (129) 2006 Dried aqueous/ethanolic

(58% vol/vol) extract CR

BNO 1055 of the rhizome of

Actaea or CR (black cohosh)

1 yr 400 Postmenopausal women with symptoms

related to estrogen deficiency were

recruited. The lack of endometrial

proliferation and improvement of climacteric

complaints as well as only a few gynecologic

organ-related adverse events are reported

for the first time after a treatment period of

1 yr

Prospective,

open-label,

multinational,

multicenter study.

Endovaginal

ultrasonography

Sammartino et al.

(130)

2006 Group A (n = 40) was

treated with 1 tablet/day

per os containing a

combination of isoflavones

[soy germ extracts, Glycine

max, no OGM-SoyLife:

150 mg, titrated in

isoflavones (40%) =

60 mg], lignans [flaxseed

extracts, Linum

usitatissimum, no

OGM-LinumLife: 100 mg,

titrated in lignans (20%) =

20 mg] and C. racemosa

[50 mg, titrated in

triterpene (2.5%) =

1.25 mg] (Euclim R

; Alfa

Wassermann, Italy); group B

(n = 40) was treated with

calcium supplements

(Metocal, Rottapharm,

Monza, Italy)

Three cycles

of 28 days

80 Healthy postmenopausal women were

recruited. At baseline no significant

difference was detected in KI between

groups A and B; however, after three cycles

of treatment, KI was significantly (P >

0.05) lower in group A compared with

baseline and with group B

Double-blind,

randomized,

placebo-controlled

trial, Kupperman

index

Gurley et al. (131) 2006 Milk thistle (300 mg, three

times daily, standardized to

contain 80% silymarin),

black cohosh extract

(20 mg, twice daily,

standardized to 2.5%

triterpene glycosides),

rifampin (300 mg, twice

daily), and clarithromycin

(500 mg, twice daily)

14 days 16 Young adults (8 females) (age, mean °æ

SD = 26 °æ 5 yr; weight, 75 °æ 13 kg)

compared with the effects of rifampin and

clarithromycin, the botanical supplements

milk thistle and black cohosh produced no

significant changes in the disposition of

digoxin, a clinically recognized P-gp

substrate with a narrow therapeutic index.

Accordingly, these two supplements appear

to pose no clinically significant risk for

P-gp-mediated herb–drug interactions

Randomized

controlled, clinical

pharmacokinetic

trial

Rebbeck et al. (132) 2007 Varied Case-control

design

949

breast

cancer

cases;

1524

controls

HRS varied significantly by race, with African

American women being more likely than

European American women to use any

herbal preparation (19.2% vs. 14.7%, P =

0.003) as well as specific preparations

including black cohosh (5.4% vs. 2.0%,

P > 0.003), ginseng (12.5% vs. 7.9%,

P < 0.001) and red clover (4.7% vs. 0.6%,

P < 0.001). Use of black cohosh had a

significant breast cancer protective effect

(adjusted odds ratio 0.39, 95% CI:

0.22–0.70). This association was similar

among women who reported use of either

black cohosh or Remifemin (a herbal

preparation derived from black cohosh;

adjusted odds ratio 0.47, 95% CI:

0.27–0.82)

Population-based

case–control study

(continued)

68 Fabricant et al.

Table 1 Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.) Year Extract/formulation/dosage Study length N Outcome measure/result Study design

Hirschberg et al.

(133)

2007 Remifemin (batch no.

229690), one tablet twice

daily. Each tablet contains

0.018–0.026 mL liquid

extract of black cohosh

rootstock (0.78–1.14:1)

corresponding to 20 mg

herbal drug [i.e., 2.5 mg dry

extract, extraction agent

isopropanol 40% (vol/vol)],

40 mg/day

6 mo 74 None of the women showed any increase

in mammographic breast density.

Furthermore, there was no increase in

breast cell proliferation. The mean change

°æ SD in proportion of Ki-67-positive cells

was 0.5% °æ 2.4% (median, 0.0; 95%

CI=−1.32–0.34) for paired samples.

The mean change in endometrial thickness

°æ SD was 0.0 °æ 0.9 mm (median, 0.0). A

modest number of adverse events were

possibly related to treatment, but none of

these were serious. Laboratory findings

and vital signs were normal

Prospective, open,

uncontrolled drug

safety study

Chung et al. (71) 2007 Gynoplus (264 mg tablet

with 0.0364 mL Cimicifuga

racemosa rhizome,

equivalent to 1 mg terpene

glycosides; 84 mg dried

Hypericum perforatum

extract, equivalent to 0.25

mg hypericin, with 80%

methanol)

12 wk 89 Kupperman index (KI) for climacteric

complaints. Vaginal maturation indices,

serum estradiol, FSH, LH, total cholesterol,

HDL-cholesterol, LDL-cholesterol, and

triglyceride levels. Significant

improvements in climacteric symptoms

and hot flashes, as well as an increase in

HDL (from 58.32 °æ 11.64 to 59.74 °æ

10.54) were observed in the Gynoplus

group by 4 wk and maintained after 12 wk,

compared with the placebo group. There

was no significant impact on superficial

cell proportion

Randomized,

double-blind,

placebo-controlled

trial

Ruhlen et al. (22) 2007 Remifemin R and CimiPure

(2.5% triterpenes; 40 mg

capsule contains 1 mg

23-epi-26-deoxyactein)

12 wk

followed by

12 wk

washout

61 Subjects experienced relief of menopausal

symptoms, with reversion to baseline after

washout. No effect on serum estrogenic

markers. No effect on pS2 or cell

morphology in nipple aspirate

Open study

Gurley et al. (134) 2008 Milk thistle (300 mg, three

times daily, standardized to

contain 80% silymarin),

black cohosh extract

(40 mg, twice daily,

standardized to 2.5%

triterpene glycosides),

rifampin (300 mg, twice

daily), and clarithromycin

(500 mg, twice daily)

14 days 19 Young adults [9 women; age (mean °æ SD)

= 28 °æ 6 yr; weight = 76.5 °æ 16.4 kg].

Milk thistle and black cohosh appear to

have no clinically relevant effect on CYP3A

activity in vivo. Neither spontaneous

reports from study participants nor their

responses to questions asked by study

nurses regarding supplement/medication

usage revealed any serious adverse events

Randomized

controlled, clinical

pharmacokinetic

trial

Amsterdam et al. (73) 2009 12 wk 28 (15

treatment/

13

placebo)

The primary outcome measure was

changed over time in total HAM-A scores.

Secondary outcomes included a change in

scores on the Beck Anxiety Inventory,

Green Climacteric Scale (GCS), and

Psychological General Well-Being Index

(PGWBI) and the proportion of patients

with a change of 50% or higher in baseline

HAM-A scores. There was neither a

significant group difference in change over

time in total HAM-A scores (P = 0.294)

nor a group difference in the proportion of

subjects with a reduction of 50% or higher

in baseline HAM-A scores at study end

point (P = 0.79). There was a significantly

greater reduction in the total GCS scores

during placebo (vs. black cohosh; P =

0.035) but no group difference in change

over time in the GCS subscale scores or in

the PGWBI (P = 0.140). One subject

(3.6%) taking black cohosh discontinued

treatment because of adverse events

Randomized,

double-blind,

placebo-controlled

trial

Black Cohosh 69

Table 1 Selected Black Cohosh Clinical Studies (Continued)

Author (reference no.) Year Extract/formulation/dosage Study length N Outcome measure/result Study design

Geller et al. (72) 2009 12 mo 89 Primary outcome measures were reduction

in vasomotor symptoms (hot flashes and

night sweats) by black cohosh and red

clover compared with placebo; secondary

outcomes included safety evaluation,

reduction of somatic symptoms, relief of

sexual dysfunction, and overall improvement

in quality of life. Reductions in number of

vasomotor symptoms after a 12-mo

intervention were as follows: black cohosh

(34%), red clover (57%), placebo (63%),

and CEE/MPA (94%), with only CEE/MPA

differing significantly from placebo. Black

cohosh and red clover did not significantly

reduce the frequency of vasomotor

symptoms as compared with placebo.

Secondary measures indicated that both

botanicals were safe as administered. In

general, there were no improvements in

other menopausal symptoms

Randomized,

double-blind,

placebo-controlled

trial

Studies listed by year of publication.

aAll with breast cancer history.

Abbreviations: CGI, Clinician’s Global Impression scale; HAM-A, Hamilton Anxiety scale; KMI, Kupperman Menopausal Index; MSS, unspecified menopausal index

using the Likert scale; Open, open-labeled; POMS, Profile of Mood States Scale; SDS, Self-Assessment Depression scale; VAS, Visual Analog Scale; VMI, Vaginal

Maturity Index.

of this study suggested the action was similar to that of the

SERM Raloxifene (86).Afollow-up study using BNO 1055

versus CE therapy showed beneficial effects of the extract

on bone metabolism in humans, specifically an increase in

bone-specific alkaline phosphatase in serum(64). While no

direct correlation between species has been established, it

is of note that studies of Asian Cimicifuga species have

demonstrated similar activity and may be of importance

for further investigation of this biological activity (87,88).

Uterine Weight/Estrous Induction

Uterine and ovarian weight increase, cell cornification,

and an increased duration of estrous are generally considered

evidence of endometrial estrogenic activity. However,

it has recently been proposed that uterine weight

is a poor marker for endometrial effects (89). Three studies

demonstrating that black cohosh extracts increased the

uterine weight of ovx rats have been reported (50,77,90)

with two of the studies using an undescribed root extract

(77,90). One study on immature mice reported similar

findings (50). By contrast, two studies on ovx rats

(79,91), as well as four studies on immature mice, reported

the converse (79,81,83,92). One of these studies

found that although there was no increase in uterine or

ovarian weight, the duration of estrous was significantly

increased by black cohosh (92). A subsequent study by the

authors and collaborators demonstrated no attenuation

in uterine weight at variable doses (4, 40, and 400 mg/

kg/day) of a 40% isopropanol extract in ovx rats (4).

Cell Proliferation

An unspecified black cohosh extract failed to significantly

induce growth of MCF-7 cells when compared with untreated

control cells (81). A study using isopropanolic and

ethanolic extracts also failed to induce growth of MCF-7

cells (93).

CNS Effects and Neurotransmitter Binding

A murine study using an unspecified extract (25–100

mg/kg, orally) measured effects on body temperature and

ketamine-induced sleep time using bromocriptine (D2-

agonist) as a positive control. Pretreatment with sulpiride

(D2 blocker) suggested a receptor-mediated dopaminergic

effect (84). An additional mouse study was carried

out to characterize neurotransmitter levels in the striatum

and hippocampus after pretreatment with the extract

for 21 days (94). Serotonin and dopamine metabolic levels

in the striatum were substantially lower in comparison

with the control group. These studies have led to

the hypothesis that dopaminergic, rather than estrogenic,

activity is responsible for the reported success of black cohosh

in reducing climacteric symptoms (95,96). A study

by the authors and collaborators has pointed to the effects

of black cohosh being mediated by serotonin (5-HT)

receptors (4). Three different extracts (100% methanol,

40% isopropanol, 75% ethanol) were found to bind to the

5-HT7-receptor subtype at IC50 ≤ 3.12 g/mL. The 40%

isopropanol extract inhibited (3H)-lysergic acid diethylamide

binding to the 5-HT7 receptor with greater potency

than (3H)-8-hydroxy-2(di-N-propylamino)tetralin to the

rat 5-HT1A. Analysis of ligand-binding data suggests that

the methanol extract functioned as a mixed competitive

ligand of the 5HT7 receptor. Further testing of the

methanol extract in 293T-5-HT7 transfected HEK cells

raised cAMP levels; these raised levels were reversed in

the presence of the 5-HT antagonist methiothepin, indicating

a receptor-mediated process and possible agonist

activity local to the receptor (4).

70 Fabricant et al.

Antioxidant

A black cohosh methanol extract protected S30 breast cancer

cells against menadione-inducedDNAdamage at variable

concentrations and scavenged DPPH free radicals at

a concentration of 99 M (38).

USE IN PREGNANT/LACTATING WOMEN

Despite an absence of mutagenic effects reported to date,

the use of black cohosh during pregnancy is contraindicated

according toWHOsuggestions (97). Data are inconclusive

regarding the effects on lactation.

DOSAGE (97,98)

Recommended doses for black cohosh are as follows:

1. Dried rhizome and root: 1 g up to three times daily.

2. Tincture (1:10): 0.4 mL daily (40–60% alcohol vol/vol).

3. Fluid extract (1:1): 20 drops twice daily (60% ethanol

vol/vol, equivalent to 40 mg dried herb).

4. Tablet equivalence: two tablets a day (equivalent to

40 mg dried extract).

The Commission E monograph also recommends

that usage not be extended for more than 6 months due to

a lack of long-term safety data. Experimental data are not

available to suggest this 6-month limit.

ADVERSE EFFECTS SAFETY

A majority of adverse event reports (AERs) for black

cohosh have been associated with Remifemin products,

probably due to its widespread use. Thus, the AER data

may speak more to the safety of this particular product

rather than black cohosh extracts in general. In clinical

trials, minor cases of nausea, vomiting, dizziness, and

headaches have been reported (61–73). An analysis of the

safety data from published clinical trials, case studies,

postmarketing surveillance studies, spontaneous report

programs, and phase I studies was carried out (99). The

data obtained from more than 20 studies, including more

than 2000 patients, suggest that adverse event occurrence

with black cohosh is rare, and that such events are mild

and reversible, the most common being gastrointestinal

upset and rashes. The same review investigated black cohosh

preparation and AERs and concluded that adverse

events are rare, mild, and reversible (99).

That said, black cohosh has garnered a great deal of

attention with respect to its safety over the past 5 years,

with the emergence of a few case reports citing acute hepatitis,

convulsions, cardiovascular, and circulatory insult

(100–104). It is important to note that in a number of

these reports, no effort was made to positively identify

the botanical associated with the event as black cohosh.

In one case, depositions taken during a legal proceeding

revealed that the lack of alcohol consumption and concomitant

medications reported in a published case report

(101) was inaccurate (105). Underreporting of adverse effects

may also be a common problem with botanical supplement

(100–104). However, these case reports have generated

much interest within the research community, so

much so that two workshops have been convened by the

National Institutes of Health (NIH) on the specific issue

of the safety of black cohosh preparations: one workshop

sponsored by the National Center for Complementary and

Alternative Medicine (NCCAM) and the Office of Dietary

Supplements (ODS) in November 2004 and a more recent

workshop sponsored by the ODS held in June 2007. The

report from the 2004 workshop indicated that there is “no

plausible mechanism of liver toxicity.” The 2007 workshop

offered no conclusions on safety to contradict those

of the 2004 meeting regarding hepatotoxicity of black cohosh

preparations. The 2007 workshop did recommend

that active steps be taken to monitor liver health in human

clinical trials of black cohosh (106).

It is also noteworthy that in the 2004 workshop, it

was agreed that “suspected hepatotoxicity should not be

broadcast when toxicity has not been demonstrated.” Despite

concerns by some scientists, a warning statement on

commercial black cohosh product labels was mandated

in Australia by the Therapeutic Goods Administration

(TGA), and the European Medicines Agency (EMEA) released

a press statement on July 18, 2006, urging patients

to stop taking black cohosh if they develop signs suggestive

of liver injury. It is noteworthy that it is not clear and

has never been fully disclosed as to how these agencies

reached their decision and what the scientific data were

that led to these warning statements.

While the notion of idiosyncratic hepatotoxicity was

raised in the June 2007 workshop by toxicologists from the

Food and Drug Administration (FDA), it was acknowledged

by these toxicologists that without data from a

mandatory adverse event reporting system, no real conclusion

on causality regarding idiosyncratic hepatotoxicity

can be drawn from case reports.

In the September–October 2007 edition of USP’s

Pharmacopeial Forum (100), the USP proposed the addition

of a cautionary statement for USP quality black

cohosh products with regard to liver toxicity. The American

Botanical Council (ABC) responded that given the

long history of safe black cohosh use and the lack of clear

scientific evidence for toxicity, there is not enough information

for such a warning. The ABC noted that of the 42

case reports of toxicity cited by the USP, only 18 met criteria

for assessment based on a standard-rating scale, and

of these, 3 met criteria for “possible” toxicity, and 2 for

“probable” toxicity. Many case reports were also said to

lack adequate documentation regarding the actual identity

of the black cohosh used and possible confounding

factors (107).

 

COMPENDIAL REGULATORY STATUS

Black cohosh products are regulated and marketed in the

United States as dietary supplements under the provisions

of the Dietary Supplement Health and Education

Act (DSHEA) of 1994 (U.S.C. § 321). Dried black cohosh

rhizome and roots, powdered black cohosh, black cohosh

fluid extract, powdered black cohosh extract, and black

cohosh tablets now have official standing in dietary supplement

monographs in the United States Pharmacopoeia-–

National Formulary (108). In the European Union nations,

Black Cohosh 71

black cohosh products are approved as nonprescription

phytomedicines when administered orally in compliance

with the German Commission E monographs (109).

 

CONCLUSIONS

With the elevated concern surrounding side effects

related to classical hormone/estrogen therapy for

menopause, modulation of certain climacteric symptoms

of menopause by both dopaminergic and serotonergic

drugs is becoming a more viable and frequent treatment

option. A review of the clinical trials associated with black

cohosh leads to the conclusion that women using hydroalcoholic

extracts of the rhizomes and roots of this plant may

gain relief from climacteric symptoms (i.e., hot flashes)

in comparison with placebo over the short term, whereas

longer studies have not shown the same degree of efficacy.

Further clouding the review of these clinical trials is the

wide variety and different types of extracts administered

in published studies. Early in vitro studies reported that

black cohosh extracts acted on ERs or had a sort direct

effect on ERs. Now it is becoming clear that the beneficial

effect of reducing hot flashes is related, at least in

part, to serotonergic or dopaminergic mechanisms that

regulate hypothalamic control and possibly mediate estrogenic

mechanisms. As mentioned earlier, the controversy

surrounding a purported direct estrogenic mechanism of

action may also be due to variance in the extracts assayed.

Overall, given variation in trial length, extract types, and

other potential confounders, the efficacy of black cohosh

as a treatment for menopausal symptoms is uncertain and

further rigorous trials seem warranted.

 

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Cyanobacteria

GlossarySuccess Chemistry Staff

In Asia, Africa, and parts of Central/South America,

naturally occurring green and blue-green algae have

been harvested and consumed for their nutritive properties

for centuries.

In western cultures, for approximately

30 years, certain freshwater blue green algae (cyanobacteria)

have been accepted as a source of food, in particular

Spirulina (Arthrospira) platensis and Spirulina maxima.

Beginning in the early 1980s, another blue-green species,

Aphanizomenon flos-aquae (AFA), was adopted for similar

uses. Both are rich in proteins, vitamins, essential amino

acids, minerals, and essential fatty acids. Consumers of

blue-green algae report a wide variety of putative effects,

such as mental clarity, increased energy, blood and colon

cleansing, increased focus, particularly in children with

attention deficit disorder, improved digestion, increased

eye health, healthier joints, and tissues. In the past 10

years, owing largely to the strong anecdotal consumer testimony

about them, studies have been conducted to verify

not only their nutritional efficacy but also their potential

pharmaceutical benefits as well.

BACKGROUND

Worldwide, algae, for thousands of years, have been a

food source and treatment for various physical ailments.

In coastal regions of the Far East, recorded use of macroalgae

(sea weed) as a food source began approximately

6000 BC, with evidence that many species were used for

food and medical treatment by around AD 900. The Spanish

recorded the use of microalgae as a food source when

they reported that the natives of Lake Texcoco collected

cyanobacteria from the waters of the lake to make sundried

cakes. In present day Africa, local tribes harvest

cyanobacteria in the Lake Chad region, primarily Spirulina,

and also use it to make hard cakes, called dihe.

In some regions of Chad, people consume from 9 to

13 g/meal, constituting 10% to 60% of the meal. However,

the longest recorded use of cyanobacteria as food

is the consumption of Nostoc flagelliforme in China, where

there are records of its use for some 2000 years and where

it is still harvested on a large scale. Use of microalgae

in the western culture began in the 1970s. Most commercial

producers of microalgae are located in the Asia-

Pacific rim, where approximately 110 commercial producers

of microalgae have an annual production capacity

from 3 to 500 tons. These cultivated microalgae include

Chlorella, Spirulina, Dunaliella, Nannochloropsis, Nitzschia,

Crypthecodinium, Tetraselmis, Skeletonema, Isochrysis, and

Chaetoceros.

Within the cyanobacteria, Spirulina (Arthrospira)

platensis and S. maxima have been commercially produced

as a human and animal food supplement and food coloring

for approximately 30 years. Spirulina is cultured in

constructed outdoor ponds in Africa, California, Hawaii,

Thailand, China, Taiwan, and India. World production in

1995 was approximately 2 °ø 106 kg.

The newest cyanobacterium to be used as a food

supplement is AFA, the production of which differs significantly

from Spirulina, because it is harvested from a

natural lake rather than constructed ponds. Since the early

1980s, this alga has been harvested from Upper Klamath

Lake, Oregon, and sold as a food and health food supplement.

The popularity of both Spirulina and AFA bluegreen

algae products over other seaweeds and green algae

may be attributable to the convenience of its packaging

and consumption, as well as to its highly directed marketing

to the health-conscious consumer. In 1998, the market

for AFA as a health food supplement was approximately

US $100 million with an annual production greater than

1 °ø 106 kg (dry weight) (1–18).

Chemistry and Preparation

Edible blue-green algae are nutrient dense food. The features

common to all blue-green algae include a high content

of bioavailable amino acids and minerals, such as zinc,

selenium, and magnesium. The nutrient profile is subject

to variation by habitat, harvest procedure, quality control

for contaminating species, proper processing to preserve

nutrients, and storage conditions, all of which influence

the vitamin content and antioxidant properties delivered

by the final product. However, the appeal of blue-green algae

is their raw, unprocessed nature and their abundance

of carotenoids, chlorophyll, phycocyanins, phytosterols,

glycolipids, -linolenic acid, and other bioactive components

(19–21).

Approximately, 40 cyanobacteria species and genera

produce potent toxins. Spirulina products have not been

associated with toxicity reports in humans, largely owing

to its being grown under cultured conditions (22). Natural

samples and cultured strains ofAFAhave been reported to

produce neurotoxins including paralytic shellfish poisons

(neosaxitoxin and saxitoxins) and anatoxin-a. Recent work

seems to indicate that a different Aphanizomenon species

is the toxin producer. A. flos-aquae has been reported to

be dominant or codominant in water blooms containing

Microcystis and Anabaena and is found in many eutrophic

water bodies. Species of Microcystis can produce a family

76 Carmichael et al.

of potent liver toxins called microcystins. Cylindrospermin

is a hepatotoxic and nephrotoxic compound produced

by several freshwater cyanobacteria, including Cylindrospermopsis

raciborskii and Anabaena spp (23). Several species

of marine and freshwater cyanobacteria (including a number

of Nostoc, Anabaena, and Microcystis species) produce

the neurotoxic amino acid BMAA (-N-methylamino-

L-alanine) (24). Although these toxic substances are probably

not naturally present in the target species discussed

below, the possibility that they might be present as contaminants

in commercial products highlights the need for

rigorous quality-control measures.

Blue-green algae products most often come in a

tablet form as algal material directly compressed. The

tablets can contain fillers such as sugars or starches called

binders, which give shape and stability to the tablets.

Algae supplements also come in a capsule form to neutralize

the taste and make the product easier to swallow,

or can be bought by the pound in powder form or in liquid

extract forms. Some companies combine the algae in

“green supplements” that contain other health-enhancing

ingredients such as alfalfa sprouts. Supplements come in

kosher or vegetarian forms, and can be combined with digestive

aids. Recommended dosages of blue-green algae

products vary widely, but can be as much as 20 g/day.

On the average, companies that produce algal products

for consumption as nutritional supplements recommend

500 mg to 1 g/day to start, with a build up over time

to several grams a day, often without an upper limit on

consumption (25,26).

 

Efficacy

Two types of blue-green algae form the major nutritional

supplement groups, Spirulina and AFA. As the traits of

each vary slightly, they are addressed separately below.

Spirulina

The blue-green alga Spirulina was sonamedfor its helically

coiled trichomes or rows of cells. Until recently, Spirulina

and Arthrospira were thought to belong to separate genera,

and the distinction was thought to be especially important

as only the strains of Arthrospira had been proven to be

safe for human consumption. These two are now referred

to as Arthrospira in scientific circles. Although the name

Spirulina has been persisted for commercial labeling, the

two are synonymous (27).

Spirulina is generally produced in large outdoor

ponds under controlled conditions. The safety of Spirulina

for human food has been established through long

use, and through various toxicological studies done under

the auspices of the United Nations Industrial Development

Organization (28). Spirulina is 60% to 70% protein

by weight and contains many vitamins, especially vitamin

B12 and -carotene, and minerals, especially iron and

-linolenic acid (Table 1). Recent reports suggest that a

number of therapeutic effects and pharmaceutical uses

are potential benefits of Spirulina as well (18).

Most studies of the effects of Spirulina on enhanced

body function have been performed on animals, and therapeutic

effects have been demonstrated in some cases.

Conclusive human studies are rare, but those that carry

substantive results are cited below.

Table 1 Nutritional Profile of a Commercial Spirulina Product

Composition Spirulina powder

  • Per 100 g

  • Macronutrientsa

  • Calories 382

  • Total fat 7.1 g

  • Total carbohydrate 15.5 g

  • Dietary fiber 6.8 g

  • Protein 55 g

  • Essential amino acids (mg)

  • Histidine 900

  • Isoleucine 3170

  • Leucine 5030

  • Lysine 2960

  • Methionine 1290

  • Phenylalanine 2510

  • Threonine 2770

  • Tryptophan 740

  • Valine 3500

  • Nonessential amino acids (mg)

  • Alanine 4110

  • Arginine 4130

  • Aspartic acid 5670

  • Cystine 580

  • Glutamic acid 9180

  • Glycine 2860

  • Proline 2170

  • Serine 2670

  • Tyrosine 2300

  • Vitaminsb

  • Vitamin A (as 100% -carotene) ≥200,000 IU

  • Vitamin K 548 g

  • Thiamine HCl (Vitamin B-1) 0.13 mg

  • Riboflavin (Vitamin B-2) 2.55 mg

  • Niacin (Vitamin, B-3) 14.3 mg

  • Vitamin B-6 (Pyridox.HCl) 0.77 mg

  • Vitamin B-12 93 g

  • Mineralsb

  • Calcium 446 mg

  • Iron 56 mg

  • Phosphorus 1010 mg

  • Iodine 39.1 g

  • Magnesium 305 mg

  • Zinc 1.27 mg

  • Selenium 19.6 g

  • Copper 0.32 mg

  • Manganese 3.0 mg

  • Chromium 91.7 g

  • Potassium 1620 mg

  • Sodium 815 mg

  • Phytonutrientsb

  • Phycocyanin 10 g

  • Chlorophyll 0.9 g

  • Superoxide dismutase (SOD) 531,000 IU

  • -linolenic acid (GLA) 1180 mg

  • Total carotenoids ≥370 mg

  • -Carotene ≥120 mg

  • Zeaxanthin ≥95 mg

  • Other carotenoids ∼155 mg

This is a natural product and nutrient data may vary from one lot to another.

One example of a nutrient profile for Earthrise R

Spirulina Powder, a

commercial Spirulina product, is shown in the above table.

a Macronutrient data are based on most recent proximate analysis.

bThe data indicate minimum values observed over a four-year period except

for sodium where the maximum observed value is used.

Blue-Green Algae (Cyanobacteria) 77

 

Nutritional Rehabilitation

A multicenter study of 182 malnourished children, aged

3 months to 3 years, reported that a 5 g/day dose of Spirulina

(Arthrospira) platensis had no added benefit over 90

days when compared to traditional renutrition (29).

Four groups of undernourished children under the

age of 5 (550 total) were provided with Misola (60% millet

flour, 20% soy, 10% peanut, 9% sugar, 1% salt), Misola

plus 5 g of S. platensis, traditional meals, or traditional

meals plus 5 g of Spirulina. All diets contained about the

same number of kilocalories/day. The authors concluded

that Misola, Spirulina plus Misola, and Spirulina plus traditional

diet are all good food supplements for undernourished

children, but that Misola plus Spirulina were

superior to the other combinations (30).

Cardiovascular

In ischemic heart disease patients, Spirulina supplementation

was shown to significantly lower blood cholesterol,

triglycerides, and LDL and very-low density lipoprotein

cholesterol, and raise HDL (the so-called “good”) cholesterol.

A 4 g/day supplementation showed a higher effect

in reducing total serum cholesterol and LDL levels than

did 2 g/day (31). In a small two-month study of the effects

of 1 g/day of Spirulina (species not specified) plus

medication versus medication alone on lipid parameters

in pediatric hyperlipidemic nephritic syndrome patients,

Samuels et al. (32) reported that supplementation of medication

with Spirulina helped reduce increased lipid levels

in these patients.

Several studies in healthy populations have shown

positive effects on cardiovascular endpoints. Consumption

of Spirulina was found to reduce total lipids, free fatty

acids, and triglyceride levels in a human study involving

diabetic patients. A reduction in LDL/HDL ratio was also

observed (33). A nonplacebo-controlled open label trial

of 36 healthy adults administered 4.5 g/day of S. maxima

for six weeks reported a hypolipidemic effect, especially

on triacylglycerols and LDL = cholesterol, systolic, and

diastolic blood pressure were also reduced (34). Effects

of 8 g/day Spirulina (species not given) versus placebo

on health-related endpoints in 78 healthy elderly Koreans

were determined in a 16-week double-blinded trial.

In the verum group, significant reductions in total plasma

cholesterol and interleukin (IL)-6 concentrations were observed,

along with increases in interleukin (IL)-2 concentrations

and total antioxidant status (35). Ju´arez-Oropeza

et al. (36) reported results of investigations of the effects

of S. maxima on vascular reactivity in rats and lipid status

and blood pressure in healthy humans. The authors

suggest that the results of the rat portion of the study

indicate that Spirulina induces a tone-related increase in

endothelial synthesis/release of nitric oxide and of a vasodilating

cyclooxygenase-dependent arachadonic acid

metabolite (or a decrease in synthesis/release of an endothelial

vasoconstricting eicosanoid). In the nonplacebocontrolled

study of the effects of 4.5 g/day Spirulina on

vascular and lipid parameters in 36 human volunteers, the

authors reported reductions in blood pressure and plasma

lipid concentrations (especially triacylglycerols and LDLcholesterol).

Immune System Function

Spirulina was found to have a positive effect on the immune

system. In a paper presented at a meeting of the

Japanese Society for Immunology, volunteers consuming

a Spirulina drink for two weeks experienced enhanced

immune system function, which continued for up to six

months after the extract administration was discontinued

(37). A follow-up study reported that administration of

50 mL of a hot water extract of S. platensis augmented

interferon production and natural killer cell (NK) cytotoxicity

in more than 50% of 12 healthy human volunteers

(38). Results of a study on immunoglobulin-A in

human saliva showed a significant correlation between

the immunoglobulin-A level in saliva and the amount of

Spirulina consumed (39).

Much attention has been focused on the potential

mitigation of allergies through Spirulina intake. A group

of Russian researchers are pursuing a patent on their success

with the normalization of immunoglobulin-E in children

living in radioactive environments (40). In a more

recent study of allergic rhinitis patients, the production

of cytokines, critical in regulating immunoglobulin-E–

mediated allergy, was measured. In a randomized doubleblind

crossover study versus placebo, allergic individuals

were fed daily with either placebo or Spirulina at 1000 or

2000 mg for 12 weeks. Although Spirulina seemed to be

ineffective at modulating the secretion of Th-1 cytokines

(one type of the so-called “killer” cells), the study reported

that at 2000 mg/day, Spirulina significantly reduced IL-4

levels by 32% (41). A six-month double-blind placebo controlled

trial of the effects of 2 g/day of S. platensis on

allergic rhinitis in 150 otherwise healthy individuals aged

19 to 49 reported that the cyanobacterium treatment significantly

improved symptoms and physical findings including

nasal discharge, sneezing, nasal congestion, and

itching (42).

Cancer

The sole human cancer intervention study involving

Spirulina intake was done in India on a group of tobacco

chewers afflicted with oral leukoplakia. In a study involving

44 subjects in the intervention group and 43 in

a placebo group, it was found that supplementation with

1 g of Spirulina per day for one year resulted in complete

regression of lesions in 45% of the intervention group

and in only 7% of the control group. As supplementation

with Spirulina did not result in an increase in retinal

-carotene, the authors concluded that other components

in Spirulinamaybe responsible for the regression of lesions

observed (43).

Other Endpoints

A series of four N-of-1 double-blind randomized trials

were performed on four individuals who complained of

idiopathic chronic fatigue. Each patient was his own control

and received three pairs of treatments comprising four

weeks of S. platensis and four weeks of placebo in doses

of 3 g/day. Outcome measures were severity of fatigue

measured on a 10-point scale. The score of fatigue was

not significantly different between Spirulina and placebo

(44). A small study compared the effects of S. platensis

plus a normal diet against soy protein plus normal diet

in the prevention of skeletal muscle damage in untrained

78 Carmichael et al.

student volunteers. Sixteen subjects were divided into two

equal groups (7.5 g/day S. platensis or soy protein). They

were administered the Bruce incremental treadmill exercise

prior to treatment, took the intervention for three

weeks, and were then readministered the treadmill exercise.

Results suggested that ingestion of Spirulina (but not

soy protein) protected against skeletal muscle damage and

may have led to postponement of the time to exhaustion

during all-out exercise (45).

Most of the research on Spirulina’s efficacy forhuman

nutrition and pharmaceutical use has been concerned with

the areas of vitamin and mineral enrichment, immune system

function, antioxidant effects, and anticancer and antiviral

effects. Although the number of studies referenced

by Amha Belay for his Spirulina research review article in

1993 contained 41 references, 18 his review in 2002 contained

98, 17 and this chapter has added additional information,

few of the human studies in almost any area can

be said to be conclusive. Studies are small or very small,

and most are open label nonplacebo-controlled studies. A

number of the publications do not provide adequate information

(many fail to identify the test cyanobacteria to

the species level). Interesting results in both the human

studies and in vitro and animal studies show that further

research is merited.

 

Adverse Effects

As previously noted, Spirulina products have not been

associated with toxicity reports in humans, probably because

commercial production is via large-scale culture

rather than wild harvest (22).

AFA (Aphanizomenon flos-aquae)

In western cultures, certain cyanobacteria have been an accepted

source of microalgal biomass for food for approximately

30 years, in particular, as discussed earlier, Spirulina

(Arthrospira) platensis and S. maxima. Beginning in the

early 1980s, another species, AFA, was adopted for similar

uses. Members of this genus are free floating (planktonic)

and occur either singly or form feathery or spindle-shaped

bundles, are cylindrical in shape, much longer than they

are wide, and contain abundant gas vesicles. They occur

in temperate climates and are most abundant in summer

and fall (46). The only known commercial harvesting of

AFA is from Upper Klamath Lake, the largest freshwater

lake system in Oregon. In 1998, the annual commercial

production of AFA was approximately 1 °ø 106 kg. As this

species is not cultured like Spirulina in outdoor ponds or

raceways, it requires very different procedures for harvesting

and processing. Other procedures, such as those for

removal of detritus and mineral materials, and those for

monitoring and reducing the amounts of certain contaminant

cyanobacteria, which can produce cyanotoxins, have

also become important in quality control and marketing

(47). The nutrient profile for AFA is very similar to that

for Spirulina (Table 2). Consumers of AFA nutritional supplements

report a variety of benefits from enhanced energy

to boosted immune system function. Cited below are

the peer-reviewed human studies extant in the literature

confirming certain of these nutritive and pharmaceutical

attributes.

Nutritional Profile of a Commercial AFA Product

Nutrient Units Amount

General composition

  • Protein % 55.1

  • Carbohydrate % 29.1

  • Calories % 3.7

  • Minerals (ash) % 6.8

  • Fat calories cal/g 0.3

  • Cholesterol mg/g 0.3

  • Total dietary fiber % 5.7

Sugar profile

  • Dextrose (glucose) mg/g 19.4

  • Fructose mg/g 0.5

  • Maltose mg/g 5.6

  • Sucrose mg/g 0.8

  • Total sugars mg/g 26.2

  • Minerals and trace metals

  • Calcium mg/g 8.5

  • Chloride mg/g 2.0

  • Chromium g/g 1.2

  • Copper g/g 10.5

  • Iron mg/g 0.7

  • Magnesium mg/g 1.8

  • Manganese g/g 31.2

  • Molybdenum g/g 4.7

  • Phosphorus mg/g 4.7

  • Potassium mg/g 10.6

  • Selenium g/g 0.4

  • Sodium mg/g 2.5

  • Zinc g/g 12.1

  • Vitamins

  • Vitamin A (-carotene) IU/g 1523

  • Thiamin (B1) g/g 19.0

  • Riboflavin (B2) g/g 44.9

  • Pyridoxine (B6) g/g 14.6

  • Cobalamin (B12) g/g 3.7

  • Ascorbic acid (C) mg/g 0.4

  • Niacin mg/g 0.4

  • Folic acid g/g 0.6

  • Choline mg/g 1.3

  • Pantothenic acid g/g 3.1

  • Biotin g/g 0.2

  • Vitamin D IU/g 0.4

  • Vitamin E IU/g 0.1

  • Vitamin K g/g 47.7

  • Amino acids

  • Arginine mg/g 29

  • Histidine mg/g 9

  • Isoleucine mg/g 25

  • Leucine mg/g 43

  • Lysine mg/g 29

  • Methionine mg/g 9

  • Phenylalanine mg/g 21

  • Threonine mg/g 29

  • Tryptophan mg/g 6

  • Valine mg/g 29

  • Asparagine mg/g 49

  • Alanine mg/g 39

  • Glutamine mg/g 78

  • Cystine mg/g 3

  • Glycine mg/g 23

  • Proline mg/g 20

  • Serine mg/g 25

  • Tyrosine mg/g 16

  • Aspartic acid mg/g 46

  • Glutamic acid mg/g 49

  • Total amino acids mg/g 579

  • Blue-Green Algae (Cyanobacteria) 79

  • Table 2 Nutritional Profile of a Commercial AFA Product (Continued)

  • Nutrient Units Amount

  • Lipid analysis

  • Total lipid (fat) content 4.4% 44 mg/g

  • Total saturated fat 43% 19 mg/g

  • Total unsaturated fat 57% 25 mg/g

  • Total essential fatty acids 45% 20 mg/g

  • Total Omega-3 essential fatty acids 38% 17 mg/g

  • -Linolenic acid (ALA) 37% 16 mg/g

  • Eicosapentanoic acid (EPA) 0.4% 0.2 mg/g

  • Total Omega-6 essential fatty acids 8% 3 mg/g

  • Linoleic acid (LA) 8% 3 mg/g

  • Arachidonic acid (AA) 0.1% 0.04 mg/g

One example of a nutrient profile for Cell Tech Super Blue-Green Algae, a

commercial AFA product, as of 4-22-05, is shown in the above table.

Circulation and Immune Function

In a study examining the short-term effects of consumption

of moderate amounts (1.5 g/day) of AFA on the

immune system, it was discovered that AFA resulted

in increased blood cell counts when compared to subjects

taking a placebo. When the volunteers were grouped

into long-term AFA consumers and na¨ıve volunteers, the

na¨ıve volunteers exhibited a minor reduction in natural

killer cells, and the long-term consumers exhibited a pronounced

reduction. It was further determined that AFA

does not activate lymphocytes directly, but that it does increase

immune surveillance without directly stimulating

the immune system. The authors of this study conclude

that AFA has a mild but consistent effect on the immune

system and could function as a positive nutritional support

for preventing viral infections. They also recommend

further research intoAFA’s potential role in cancer prevention

(48). In a later report, an aqueous extract of AFAfound

to contain a novel ligand for CD62L (L-selectin). Consumption

of the extract by 12 healthy subjects in a doubleblind

randomized crossover study caused mobilization of

human CD34+CD133+ and CD34+CD133− stem cells (49).

Eye Disease—Blepharospasm and Meige Syndrome

A study to determine whether blue-green algae could be

helpful in improving the eyelid spasms associated with

essential blepharospasm and Meige syndrome was undertaken

by a group of physicians. Although a few patients

exhibited a positive effect, for most patients, neither the

severity nor the frequency of facial spasms was significantly

reduced (50).

 

Adverse Effects

No cases of human intoxication by AFA were found in

the literature. As noted, around 40 cyanobacterial species

have been reported to produce potent natural toxins. Certain

A. flos-aquae strains were long thought to produce neurotoxins

including the paralytic shellfish poisons neosaxitoxin

and saxitoxin and anatoxin-a. A reevaluation of the

species using gene sequencing data led to the conclusion

that a different Aphanizomenon species was the actual toxin

producer (51). While it now seems clear that AFA is not

a toxin-producing species, the toxigenic Aphanizomenon

species seems to be distinguishable from AFA only by the

presence or absence of toxins and by genetic sequencing.

No evidence exists to suggest that AFA is a toxin producing

strain (51), but water blooms of AFA may also

contain Microcystis and Anabaena. Species of Microcystis

can produce a family of potent liver toxins called microcystins,

while Anabaena species can produce antitoxins and

BMAA. The microcystins, especially, are of concern, since

hepatic damage caused by this toxin is cumulative. This

has led the State of Oregon to set a safe level of microcystin

in AFA product from Klamath Lake. Currently this

level is set at 1 g/g dry weight of product, and was set

to correspond to an average daily adult intake for AFA of

2 g. Recently, the cyanobacterial toxin anatoxin-a was reported

in 3 of 39 cyanobacterial dietary supplement product

samples at concentrations of 2.5 to 33 g/g (52). Exposure

guidelines have been summarized by Burch (23). This

points to the need for rigorous quality-control measures in

production of products. These measures may range from

existing practices such as modifying the harvesting equipment

to exclude Microcystis, harvesting the water bloom

only at times when Microcystis is at a minimum, or developing

and using toxin-detecting methods in an integrated

testing scheme (47,53,54).

CONCLUSIONS

As a natural source of many vitamins and minerals, proteins,

and chlorophyll, it is not surprising that blue-green

algae have attracted attention among those interested in

natural sources of nutrition. Thousands of people consume

blue-green algae in its most popular forms (Spirulina

and AFA), and as a result, a large body of anecdotal material

has existed for many years concerning the positive

health benefits of blue-green algae consumption. The volume

of the testimony has contributed to a growing interest

in recent years in verifying these benefits through scientific

research. Beginning with animal and in vitro studies,

and moving toward human studies, scientists have only

recently begun to investigate some of the positive health

effects attested to by long-term consumers of blue-green

algae. These include cholesterol reduction, weight loss,

enhanced immune system function, regression of cancer related

lesions, and enhancement of blood circulation, as

well as many vitamin and mineral benefits. It must be

stated, however, that there is an overall paucity of well designed,

controlled human trials using blue-green algal

products as interventions.

As the industry relies on self-regulation, it is important

to be aware of the quality-control issues involved in

harvesting and packaging blue-green algae for consumption,

particularly in the case of AFA, which is harvested

from the wild. The World Health Organization has determined

through current knowledge of microcystin toxins

and what it calls a tolerable daily intake (TDI), an estimate

of the intake over a lifetime that does not constitute an appreciable

health risk. This TDI is derived through existing

knowledge of toxin tolerance in mice combined with principles

used in defining the health risks of other chemicals.

It carries with it a degree of uncertainty owing to the lack

of long-term data for the effect of microcystin on humans

(55). To be sure that their risk has been minimized as much

as possible, consumers of blue-green algae supplements

would be wise to check to see that the product has been

80 Carmichael et al.

tested for toxins, and it has been found to be below the

WHO/Oregon Department of Health Regulatory Level of

1 g/g of microcystin (56).

ACKNOWLEDGMENTS

The authors would like to thank Jerry Anderson (CellTech,

Inc.) and Diana Kaylor (Wright State University) for supplying

a number of references used in this text.

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Boron

Glossary, elementsSuccess Chemistry Staff

boron is essential for all higher plants in

phylogenetic kingdom Viridiplantae

 

(1) and at least some organisms in the phylogenetic kingdoms Eubacteria (2),

Stramenopila (3), and Animalia (4,5). Specific species in

the kingdom Fungi have a demonstrated physiological

response to boron, an important finding because Fungi

species are thought to share a common ancestor with animals

exclusive of plants (6). Physiologic concentrations of

the element are needed to support metabolic processes in

several species in Animalia. For example, embryological

development in fish and frogs does not proceed normally

in the absence of boron. There is evidence that higher vertebrates,

that is, chicks, rats, and pigs require physiological

amounts of boron to assist normal biologic processes including

immune function, bone development, and insulin

regulation. In humans, boron is under apparent homeostatic

control and is beneficial for immune function and

calcium and steroid metabolism.

 

COMMON CHEMICAL FORMS

Boron is the fifth element in the periodic table with a

molecular weight of 10.81 and is the only nonmetal in

Group III. Organoboron compounds are those organic

compounds that contain B–O bonds, and they also include

B–N compounds, because B–N is isoelectronic with

C–C (7). Organoboron compounds are apparently important

in biological systems and are the result of interaction

with OH or amine groups. Organoboron complexes occur

in plants and are produced in vitro with biomolecules

isolated from animal tissues (8).

 

BORON SPECIATION

Environmental Forms

Boron does not naturally occur free nor bind directly to

any element other than oxygen except for trivial exceptions,

for example, NaBF4 (ferrucite) and (K,Cs)BF4 (avogadrite)

(7). Its average concentration in the oceans is

4.6mg/Land is the 10th most abundant element in oceanic

salts (9). Weathering of clay-rich sedimentary rock is the

major source of total boron mobilized into the aquatic environment

(10). Undissociated boric acid (orthoboric acid)

is the predominant species of boron in most natural freshwater

systems (10) where most concentrations are below

0.4 mg/L and not lowered by typical treatments for drinking

water. The most common commercial compounds

of boron are anhydrous, pentahydrate, and decahydrate

(common name: tincal) forms of disodium tetraborate

(borax, Na2B4O7), colemanite (2CaO°§3B2O3°§5H2O), ulexite

(Na2O°§2CaO°§5B2O3°§16H2O), boric acid (H3BO3), and

monohydrate and tetrahydrate forms of sodium perborate

(NaBO3) (11).

Inorganic boron, within the concentration range

expected for human blood (2–61 M B; 22–659 ng

B/g wet blood) (12), is essentially present only as the

monomeric species orthoboric acid (common name: boric

acid) B(OH)3 and borate, that is, B(OH)4

− (13). Boric acid

is an exclusively monobasic acid and is not a proton donor,

but rather accepts a hydroxyl ion (a Lewis acid) and leaves

an excess of protons to form the tetrahedral anion B(OH)4

(14):

B (OH)3

+ 2H2O ⇔ H3O+ + B (OH)−

4 pKa = 9.25(25◦C)

Within the normal pH range of the gut and kidney,

B(OH)3 would prevail as the dominant species (pH 1:

∼100% B(OH)3; pH 9.3: 50%; pH 11: ∼0%) (15).

 

Biochemical Forms

Many biomolecules contain one or more hydroxy groups

and those with suitable molecular structures can react with

boron oxo compounds to form boosters, an important

class of biologically relevant boron species. Several types

of boron esters exist. Boric acid reacts with suitable dihydroxy

compounds to form corresponding boric acid monoesters

(“partial” esterification) (Fig. 1) that retain the

trigonal-planar configuration and no charge.

In turn, a boric acid monoester can form a complex

with a ligand containing a suitable hydroxyl to create

a borate monoester (“partial” esterification; monocyclic)

(Fig. 2), but with a tetrahedral configuration and a negative

charge. A compound of similar configuration and

charge is also formed when borate forms a complex

with a suitable dihydroxy compound. The two types

of boro monoesters can react with another suitable dihydroxy

compound to give a corresponding spirocyclic

borodiester (“complete” esterification) that is a chelate

Figure 1 Boric acid may complex with a suitable dihydroxy ligand to form

a boric acid monoester (“partial” esterification) that retains a trigonal-planar

configuration and no charge.  Borate may complex with a suitable dihydroxy ligand to form

a borate monoester (“partial” esterification; monocyclic) with a tetrahedral

configuration and a negative charge.

complex with a tetrahedral configuration and negative

charge (16)

Boric acid and boric acid–like structures, instead of

borate, are most likely the reactive species with biological

ligands, because it is probably easier for a diol to substitute

for a relatively loosely bound water molecule associated

with boric acid or a boric acid-like structure than it is for

the diol to substitute for a charged hydroxyl ion in borate

or a borate-like structure (16).

Procaryotes

Boron is an integral component of several biomolecules

in which it is thermodynamically stabilized in a covalent

bond (17–20) or by forming a boroester (21). Its presence in

these molecules is essential; in its absence, they no longer

perform their normal physiologic functions. Of great interest

is a boron-containing biomolecule produced by a bacterium

that is not an antibiotic but rather a cell-to-cell communication

signal (20). Communication between bacteria

is accomplished through the exchange of extracellular signaling

molecules called autoinducers (AIs). This process,

termed “quorum sensing,” allows bacterial populations

to coordinate gene expression for community cooperative

processes such as antibiotic production and virulence factor

expression. AI-2 is produced by a large number of bacterial

species and contains one boron atom per molecule.

Not surprisingly, it is derived from the ribose moiety of

biomolecule, (S)-adenosylmethionine (SAM). The gliding

bioluminescent marine bacterium, Vibrio harveyi (phylum

Proteobacteria), produces and also binds AI-2. In V. harveyi,

the primary receptor and sensor for AI-2 is the protein

LuxP, which consists of two similar domains connected by

a three-stranded hinge. The AI-2 ligand binds in the deep

cleft between the two domains to form a furanosyl borate

diester complex (Fig. 4) (20).

Boron is a structural component of certain antibiotics

produced by certain myxobacteria, a distinct and unusual

group of bacteria. For example, tartrolon B (Fig. 5)

is characterized by a single boron atom in the center of the

Figure 3 Boric acid monoesters or borate monoesters can combine with a

suitable dihydroxy compound to form a corresponding spirocyclic borodiester

[“complete” (add the close parenthesis) esterification] that is a chelate

complex with a tetrahedral configuration and negative charge.

Figure 4 The autoinducer, AI-2, with its integral boron atom is stabilized

by a hydrogen network in the binding site of the receptor. The O–O or O–N

distances for potential hydrogen bonds are shown in angstroms. Source: From

Ref. 20.

molecule (18). Another related antibiotic, boromycin, was

discovered to be potent against human immunodeficiency

virus (HIV) (22). It strongly inhibits the replication

of the clinically isolated HIV-1 strain and apparently, by

unknown mechanisms, blocks release of infectious HIV

particles from cells chronically infected with HIV-1.

Animal and Human Tissues

Only meager information is available on the speciation of

boron in animal or human tissues. However, animal and

human biocompounds with vicinal cis-diol moieties bind

Figure 5 Tartrolon B, an example of certain antibiotics produced by certain

myxobacteria that require the presence of a single atom of boron for

functionality.

Experimental data indicate that biochemical species with vicinal cis-diols bind strongly to boron: (S)-adenosylmethionine (SAM) ≡ diadenosine

hexophosphate (Ap6A) ≡ Ap5A > Ap4A > Ap3A ≡ NAD+ > Ap2A > NADH ≡ 5ATP > 5ADP > 5AMP > adenosine (ADS). Species without these moieties

do not bind boron well: 3AMP ≡ 2AMP ≡ cAMP ≡ adenine (ADN).

boron; those without these moieties typically do not. Of

these animal or human biocompounds examined, SAM

has the highest known affinity for boron (8). It is the predominant

methyl donor in biological methylations and is

therefore a versatile cofactor in various physiologic processes.

NAD+, an essential cofactor for five sub-subclasses

of oxidoreductase enzymes, also has a strong affinity

for boron (23). The di-adenosine-phosphates (ApnA) are

structurally similar to NAD+. Boron binding by Ap4A,

Ap5A, and Ap6A is greatly enhanced compared with

NAD+ but is still less than that of SAM (8). The ApnA

molecules are present in all cells with active protein synthesis

and reportedly regulate cell proliferation, stress response,

and DNA repair (24). At physiologic pH, the adenine

moieties ofApnAare driven together by hydrophobic

forces and stack interfacially (25). Stacking of the terminal

adenine moieties brings their adjacent ribose moieties

into close proximity, a phenomenon that apparently potentiates

cooperative boron binding between the opposing

riboses.

Plant-Based Foods

All higher plants require boron and contain organoboron

complexes. There may have been considerable evolutionary

pressure exerted to select for carbohydrate energy

sources that do not interact with boron. Sugars often form

intramolecular hemiacetals: those with five-membered

rings are called furanoses and those with six-membered

rings are called pyranoses. In cases where either five- or

six-membered rings are possible, the six-membered ring

usually predominates for unknown reasons (26). In general,

compounds in a configuration in which there are

cis-diols on a furanoid ring (e.g., ribose, apiose, and erythritan)

form stronger complexes with boron than do those

configured to have cis-diols predominately on a pyranoid

ring (e.g., the pyranoid form of -D-glucose). D-Glucose

reacts with boric acid (27) but the near absence (<0.5%)

of an -furanose form of D-glucose in aqueous solutions

(26) suggests that glucose was selected as the aldose for

general energy metabolism because of its lower reactivity

with boric acid. On the other hand, ribose may have

been selected as part of the chemistry of nucleic acid and

nucleotide function and apiose for, rather than against, its

extraordinary borate-complexing capability.

Recent evidence suggests that the predominant

place of boron function in plants is in the primary cell

walls where it cross-links rhamnogalacturonan II (RG-II)

(Fig. 7), a small, structurally complex polysaccharide of the

pectic fraction. RG-IIs have an atom of boron that crosslinks

two RG-II dimers at the site of the apiose residues

to form a borodiester (28). However, this function is not

adequate to explain all boron deficiency signs in plants.

Twenty-six boron-binding membrane-associated proteins

were identified recently in the higher plant, Arabidopsis

thaliana (29), and boron oxo compounds also form stable

ionic complexes with the polyol ligands mannitol, sorbitol,

and fructose in liquid samples of celery phloem sap

and vascular exudate and phloem-fed nectaries of peach

(30).

Dietary Supplements

Boron speciation in dietary supplements varies widely

(31) as does the relevant information provided by various

dietary supplement manufacturers. It is sometimes listed

only in a general manner (e.g., “borates” or “boron”),

and occasionally in a more specific manner (e.g., “sodium

Boron

Schematic representation of two monomers of the pectic polysaccharide

rhamnogalacturonan-II cross-linked by an atom of boron at the site

of the apiose residues to form a borodiester. Multiple cross-links form a

supramolecular network. Source: From Ref. 75.

borate” or “sodium tetraborate decahydrate”). Several

commercially available forms (e.g., “boron amino acid

chelate,” “boron ascorbate,” “boron aspartate,” “boron

chelate,” “boron citrate,” “boron gluconate,” “boron glycerborate,”

“boron glycinate,” “boron picolinate,” “boron

proteinate,” “boron bonded with niacin,” and “calcium

fructoborate”) are not well characterized in the scientific

literature. Most often, dietary boron supplements are provided

in conjunction with other nutrient supplements.

 

BIOAVAILABILITY, EXCRETION, AND HOMEOSTASIS

If plant and animal boron absorption mechanisms are

analogous, the organic forms of the element per se are

probably unavailable (32). However, the strong association

between boron and polyhydroxyl ligands (described

later) is easily and rapidly reversed by dialysis, change in

pH, heat, or the excess addition of another low-molecular

polyhydroxyl ligand (27). Thus, within the intestinal tract,

most ingested boron is probably converted to orthoboric

acid (common name: boric acid), B(OH)3, the normal end

product of hydrolysis of most boron compounds (7). Gastrointestinal

absorption of inorganic boron and subsequent

urinary excretion (33) is near 100%.

Several lines of evidence suggest regulation of boron

in humans. For example, lack of boron accumulation and

relatively small changes in blood boron values during a

substantial increase in dietary boron support the concept

of boron homeostasis (33). Boron contents in human milks

0

10

20

30

40

50

60

Weeks After Birth

Mean Concentration and Model

of Boron (ug/kg milk)

B FT Means

B FT Model

B PRT Means

B PRT Model

1 2 3 4 5 6 7 8 9 10 11 12

Figure 8 Model and mean (°æSE) concentrations of boron in breast milk

from mothers of full-term (FT) and premature (PRT) infants; n = 9 per group

over 12 weeks after birth. During the first 12 weeks of lactation, prematurity

affected the rate of change in concentrations (P = 0.01).

were similar and stable throughout lactation of full-term

infants in two cohorts of women living in either Houston,

TX (34), or St. John’s, Newfoundland (Fig. 8) (35), have

been interpreted as suggestive of regulatory mechanisms

for the elements, which remain undefined. Evidence for

the homeostatic control of boron is enhanced further by a

report (36) of a specific borate transporter, NaBC1, in mammalian

cell lines, a finding that has yet to be confirmed by

another laboratory.

 

DIETARY BORON SOURCES AND INTAKES

Dietary Recommendations

The tolerable upper intake level (UIL) for boron varies by

life stage (Table 1) (37).No Estimated average requirement,

recommended dietary allowance, or adequate intake has

been established for boron for any age–sex group.

Dietary Supplements

For adults, the amount of boron commonly provided in

a single dietary boron supplement is 0.15 mg (31). However,

the reported amount of boron available per serving

varies considerably among commercially available products

as indicated in the relevant information provided by

Table 1 Upper Limits for Boron Set by the 2001 Food and Nutrition

Board of the National Academy of Sciences (37)

Life stage Age (yr) Upper limit (mg/day)

Children 1–3 3

4–8 6

9–13 11

Adolescents 14–18 17

Adults 19–70 20

70 20

Pregnancy ≤18 17

19–50 20

Lactation ≤18 17

19–50 20

86 Hunt

various dietary supplement manufacturers. Some manufacturers

publish reported values of 6.0mgboron per serving

of dietary supplement (38). The mean of usual intake

of boron (mg/day) from dietary supplements for children

(1–8 years), adolescents (9–15 years), males (19+ years),

females (19+ years), and pregnant/lactating women is

0.269, 0.160, 0.174, 0.178, and 0.148, respectively. The median

boron intake from supplements in the U.S. population

is approximately 0.135 mg/day (37).

Nonfood Personal Care Products

Boron is a notable contaminant or ingredient of many

nonfood personal care products. For example, an antacid

was reported to have a high concentration of boron

(34.7 g/g) (39) such that the maximum recommended

daily dose would provide 2.0 mg B/day, two times the

estimated daily boron consumption for the overall adult

U.S. population.

Dietary Sources and Intakes

Ten representative foods with the highest boron concentrations

are distributed among several food categories

(40): raw avocado (14.3 g/g), creamy peanut butter,

(5.87 g/g); salted dry roasted peanuts (5.83 g/g),

dry roasted pecans (2.64 g/g), bottled prune juice

(5.64 g/g), canned grape juice (3.42 g/g), sweetened

chocolate powder (4.29 g/g), table wine (12.2% alcohol)

(3.64 g/g), prunes with tapioca (3.59 g/g), and granola

with raisins (3.55 g/g). Several fruit, bean, pea, and nut

products contained more than 2 g B/g. Foods derived

from meat, poultry, or fish have relatively low concentrations

of boron.

Infant foods supply 47% of boron (B) intake to infants.

For toddlers, consumption from fruits and fruit

juices, combined, is twice that from milk/cheese (38% vs.

19%). For adolescents, milk/cheese foods are the single

largest source of boron (18–20%), and for adults and senior

citizens, it is beverages (mainly represented by instant regular

coffee) (21–26%). For all groups (except infants), 7%

to 21% of boron intake is contributed by each of the vegetable,

fruit, and fruit drink products. Infants, toddlers,

adolescent girls and boys, adult women and men, and

senior women and men are estimated to consume the following

amounts of boron: 0.55, 0.54, 0.59, 0.85, 0.70, 0.91,

0.73, and 0.86 mg/day, respectively.

 

INDICATIONS AND USAGE

Boron and Calcium Metabolism and Bone Structure

There are several lines of evidence that dietary boron is

important for normal bone growth and function. Boron

deprivation induced abnormal limb development in frogs

(41) and retarded maturation of the growth plate in chicks

(42). Dietary boron deprivation decreases bone strength

in pigs (43) and rats (44). The trabecular microarchitecture

of vertebral bone was impaired in rats deprived of the

element (44). Similarly, in mice, modeling and remodeling

of alveolar bone (45), as well as alveolar bone healing

after experimental tooth extraction (46), was impaired by

dietary boron deprivation.

Findings from human studies suggest that boron

influences calcium metabolism. For example, in postmenopausal

women, boron supplementation (3 mg/day)

of a low-boron diet (0.36 mg B/day) resulted in a 5%

increase in urinary calcium excretion (33). A similar

phenomenon occurred in either free-living sedentary or

athletic premenopausal women consuming self-selected

typical Western diets: boron supplementation increased

urinary calcium loss (47). These findings may reflect an

increase in intestinal calcium absorption because increase

in dietary calcium often result in increased urinary calcium

excretion.

Dietary boron also alleviates the signs of marginal

vitamin D deficiency relevant to bone structure and function.

Marginal vitamin D deficiency impairs bone structure,

elevates plasma alkaline phosphatase concentrations,

and reduces body weight. In the growing rachitic

chick, dietary boron substantially alleviated the perturbed

histomorphometric indices of bone growth cartilage

(42,48), reduced elevated serum concentrations of

alkaline phosphatase (49,50), and improved body weight

(50,51).

Boron and Insulin and Energy Substrate Metabolism

Circulating insulin concentrations respond to dietary

boron in a manner that suggests the element may function

to reduce the amount of insulin needed to maintain

glucose levels. For example, in the rat model (with

overnight fasting), boron deprivation increased plasma

insulin with no concurrent change in glucose concentrations

(52). In the chick model, boron deprivation increased

in situ peak pancreatic insulin release (52). In

older volunteers (men and women) fed a low-magnesium,

marginal copper diet, dietary boron deprivation induced

a modest but significant increase in fasting serum glucose

concentrations (53).

Findings from several studies indicate that dietary

boron may attenuate the deleterious effects of marginal

vitamin D deficiency on insulin and energy substrate

metabolism. In vitamin D–deprived rats, hyperinsulinemia

was decreased by dietary boron (54). It has been

demonstrated repeatedly in the chick model that physiological

amounts of dietary boron can attenuate the rise

in plasma glucose concentration induced by vitamin D

deficiency (42,48,55). In addition, boron decreases the abnormally

elevated plasma concentrations of pyruvate, ßhydroxybutyrate,

and triglycerides that are typically associated

with this inadequacy (42). It is not understood how

boron deprivation perturbs energy substrate metabolism

in humans and animal models, particularly when other

nutrients are provided in suboptimal amounts.

Boron and Immune Function

Dietary boron may have a role in control of the normal

inflammatory response, especially as it relates to production

of various cytokines. Cytokines, including interferongamma

(IFN-), tumor necrosis factor- (TNF-), and

interleukin-6 (IL-6), are produced and secreted by immune

cells that regulate immune responses. Production of IFN-

and TNF- was increased in peripheral blood monocytes

cultured in the presence of lipopolysaccharide (an inflammatory

agent) after isolation from pigs fed supplemental

dietary boron. In the same animals, boron caused a reduction

in localized inflammation following a challenge with

Boron

the antigen phytohemagglutinin (PHA) (56). In cell culture

studies with human fibroblasts (57) and chick embryo cartilage

(58), the addition of boric acid also increased TNF-

release by the respective cells. Certain boron-containing

RG-II s from Panax ginseng leaves enhanced the expression

of IL-6–producing activity of mouse macrophages (59).

Finally, perimenopausal women who excreted <1.0 mg

B/day during the placebo period exhibited an increased

percentage of polymorphonuclear leukocytes during the

boron (as sodium borate) supplementation period (60). Dietary

boron may serve as a signal suppressor that downregulates

specific enzymatic activities typically elevated

during inflammation at the inflammation site. Suppression,

but not elimination, of these enzyme activities by

boron is hypothesized to reduce the incidence and severity

of inflammatory disease.

Boron and Steroid Metabolism

There is a clear evidence that dietary boron affects steroid

metabolism. In particular, circulating concentrations of

vitamin D metabolites are sensitive to boron nutriture.

Findings from animal models indicate that dietary boron

enhances the efficacy of vitamin D but cannot substitute

for the vitamin. In volunteers (men, and women

on or not on estrogen therapy), boron supplementation

after consumption of a low-boron diet increased serum

25-hydroxycholecalciferol concentrations (62.4 °æ 7.5 vs.

44.9 °æ 2.5 mmol/L; mean °æ SEM) (61,62), an effect that

may be especially important during the winter months

when these concentrations normally range between

35 and 105 mmol/L (63).

The circulating concentrations of 17-estradiol also

respond to boron nutriture. Perimenopausal women who

excreted <1.0 mg B/day during the placebo period exhibited

increased serum concentrations of estradiol after

boron supplementation (2.5 mg B/day) of self-selected diets

(60). In a separate study, postmenopausal women on

estrogen therapy, but neither men nor postmenopausal

women not ingesting estrogen, also exhibited increased

serum concentrations of estradiol after boron supplementation

(3 mg B/day) of a low-boron diet (0.25 mg B/2000

kcal) (62). However, plasma estradiol, but not testosterone,

concentrations increased in young male volunteers when

their self-selected diets were supplemented with ample

amounts of boron (10 mg/day) (64).

 

Boron and Cancer

Indirect evidence from several epidemiological and cell

culture studies indicate that dietary boron intake may affect

cancer risk. For example, observations from epidemiologic

human studies suggest that increased intakes of

boron are associated with decreased risk of prostate (65)

and lung (66) cancers and abnormal cervical cytopathology

(67). In cultures of human prostatic epithelial cells

(not tested for proliferative activity), physiological levels

of boron reduced Ca2+ release from ryanodine receptor sensitive

stores in a dose-dependent manner, without affecting

cytoplasmic Ca2+ concentrations (68). In immunocompromised

mice fed physiological amounts of dietary

boron, the element reduced the growth of transplanted

human prostate adenocarcinoma tumors (69).

 

OVERDOSAGE

As with all other elements, boron produces toxicity in

all tested biological organisms when excessive amounts

are absorbed. The toxicity signs associated with boric acid

when used as an antiseptic in lieu of antibiotics on abraded

epithelium (i.e., surgical wounds and diaper rash) were

overlooked for many years even though signs of poisoning

were reported soon after its introduction into clinical use.

Boron is more bacteriostatic than does bactericidal and,

thus, may suppress bacterial growth.

Deaths can occur at doses between 5 and 20 g of boric

acid for adults and below 5 g total for infants (70,71). Potential

lethal doses are usually cited as 3 to 6 g total for infants

and 15 to 20 g total for adults. However, an independent

examination of 784 cases of boric acid ingestion found

minimal or no toxicity at these intake levels or higher (72).

Signs of acute boron toxicity, regardless of route of administration,

include nausea, vomiting, headache, diarrhea,

erythema, hypothermia, restlessness, weariness, desquamation,

renal injury, and death from circulatory collapse

and shock. Autopsy may reveal congestion and edema of

brain, myocardium, lungs, and other organs, with fatty

infiltration of the liver. Chronic heavy borax dust occupational

exposure (average air concentration: 4.1 mg/m3;

range: 1.2–8.5 mg/m3) may manifest as eye irritation,

nosebleeds, chest tightness, sore throat, dry mouth, and

productive cough (71). Chronic boron toxicity symptoms

include poor appetite; nausea; weight loss; decreased sexual

activity, seminal volume, sperm count, and motility

and increased seminal fructose. At present, death from

boron poisoning is exceptionally rare probably because of

the emphasis placed on maintaining electrolytic balance

and supporting kidney function during the worst part of

the illness. Depending upon boron blood levels, treatment

ranges from observation to gastric lavage to dialysis.

 

CONCLUSIONS

Boron is ubiquitous in the environment and daily dietary

boron intakes of adult American males, for example, are

slightly less than 1.0 mg. The evidence to date suggests

that higher animals (43,73) and humans (33,62,74) probably

require boron to support normal biological functions.

Despite the progress made in studies of boron essentiality

for plants, animals, and man, the biochemical mechanisms

responsible for the beneficial physiologic effects of boron

across the phylogenetic spectrum are poorly understood.

However, the unique nature of boron biochemistry suggests

specific lines of investigation. In particular, further

characterization of the various cell signaling molecules

that form complexes with boron under physiological conditions

should provide insights into the specific biochemical

function(s) of boron in humans.

 

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of the normal inflammatory response: A review and

current research progress. J Trace Elem Exp Med 1999; 12:

221–233.

74. Travers RL, Rennie GC, Newnham RE. Boron and arthritis:

The results of a double-blind pilot study. J Nutr Med 1990;

1:127–132.

75. Power PP, Woods WG. The chemistry of boron and its speciation

in plants. Plant Soil 1997; 193:1–13.

Caffeine

GlossarySuccess Chemistry Staff

Caffeine is undoubtedly one of the most widely consumed

and studied dietary supplements.

It is found in many products, including numerous foods and

drugs. Approximately 50% of the U.S. adult population

regularly uses one or more dietary supplements, but

80% or more regularly consumes caffeine. Thousands

of studies have investigated this substance, and a comprehensive

discussion of all aspects of the literature on

caffeine would require hundreds of pages of text. Substantial

literature on caffeine can be found in multiple

scientific fields including pharmacology, exercise and cardiovascular

physiology, psychology, psychiatry, and epidemiolog.

Caffeine occurs naturally in beverages and foods,

including coffee, tea, and chocolate.

Additional caffeine is added to beverages,

including colas, which naturally contain caffeine, because

manufacturers of these products have determined

that optimal levels of caffeine should be greater than

their naturally occurring concentration. Caffeine is behaviorally

active in the doses present in foods (3) and

is the most widely consumed psychoactive substance in

the world. Caffeine is recognized in scientific and regulatory

domains as both a naturally occurring food and

a drug, a distinction that few, if any, other substances hold.

HISTORY OF CAFFEINE USE

The first written mention of a caffeine-containing food or

beverage, tea, is in a Chinese dictionary from about AD 350

(4). However, it is likely that tea was in use long before

then. Coffee was cultivated in Ethiopia as early as the sixth

century AD, where it originated. Coffee beans were probably

first eaten whole or mixed with food (4). Coffee came

into use as a hot beverage around AD 1000 in the Middle

East but did not spread to Europe until the 17th century.

Coffee is currently second only to water as the beverage

of choice around the world, with an estimated 400 billion

cups of coffee consumed each year (5). The two most

common species of the coffee plant are Coffea arabica and

Coffea canephora Pierre ex. A. Froehner (commonly known

as robusta). Approximately two-thirds of the world’s coffee

comes from arabica plants, whereas one-third comes

from robusta (5). Arabica coffee has a smoother and superior

taste but requires extensive care in growing. Arabica

beans contain approximately 1.5% or less of caffeine by

dry weight (5). Robusta beans, grown in regions such as

Brazil, have a higher caffeine content, 2.4% to 2.8%, which

may explain their less-preferred flavor, because caffeine

itself is quite bitter (5). At least 60 species of plants contain

caffeine. The reason so many plants contain caffeine

is not known, but caffeine protects plants from certain

insects (6).

Tea [Camellia sinensis (L.) Kuntze] is the caffeinated

beverage of choice in a large part of the world, although it

contains less caffeine than coffee (Table 1). It appears that

caffeine-containing beverages originated independently

in at least four different locations throughout the world.

In both North and South America, caffeine-containing

beverages were made by the native inhabitants prior to

contact with Europe. The sources of caffeine in North

America were the cassina or Christmas berry tree (Ilex

vomitoria Ait.) and in South America guarana (Paullinia

cupana Kunth) and yoco (4). Caffeine is present in cocoa

beans (Theobroma cacao L.), native to Central and South

America. A compound chemically similar to caffeine,

theobromine, is also found in cocoa but in substantially

greater amounts than caffeine. Although the popularity of

caffeine is widely recognized, the rationale for its unique

status in the diets of humans is not known, but many have

speculated its mild stimulant properties account for its

popularity.

PHARMACOLOGY OF CAFFEINE

Caffeine is a methylated xanthine, 1,3,7-trimethylxanthine;

theophylline and theobromine are two other methylated

xanthines found in foods and/or drugs (Fig. 1).

Theobromine, 3,7-dimethylxanthine, is not behaviorally

active in doses found in foods (7). Theophylline, 1,3-

dimethylxanthine, used to treat asthma, is not present in

coffee but found in small quantities in tea (C. sinensis).

The parent compound of these methylated compounds

is xanthine, a dioxypurine structurally similar to uric

acid (8).

When ingested, caffeine is rapidly absorbed into the

systemic circulation and reaches peak levels in 45 minutes

or less (9). Caffeine is distributed to all tissues and

readily crosses the blood–brain barrier, which explains

its behavioral activity. Caffeine is initially absorbed by the

buccal membranes (in the mouth) and, when consumed in

chewing gum, enters the circulatory system more rapidly

than when ingested in pill form (10).

Caffeine  Estimated Caffeine Content of Selected Beverages, Foods, and Dietary Supplements

Caffeine content

Item (mg/serving)

  • Coffee (5 oz)

  • Drip method 90–150

  • Instant 40–108

  • Decaffeinated 2–5

  • Tea, loose or bags

  • 1-minute brew (5 oz) 9–33

  • 5-minute brew (5 oz) 20–50

  • Iced tea (12 oz) 22–36

  • Chocolate products

  • Hot cocoa (6 oz) 2–8

  • Chocolate milk (8 oz) 2–7

  • Milk chocolate (1 oz) 1–15

  • Baking chocolate (1 oz) 35

  • Cola beverages (12 oz)

  • Coca-Cola R  Classic 35 Diet Coke R 47

  • Pepsi R 38

  • Diet Pepsi R 36

Other Soft drinks (12 oz)

  • Dr Pepper R

  • Mountain Dew R 55

  • Pibb Xtra R 41

  • Barq’s Root Beer 23

Energy drinks and shots

  • AMPTM (16 oz) 142

  • Monster EnergyTM (16 oz) 160

  • Monster EnergyTM Lo-Carb (16 oz) 135

  • Red Bull R (8.3 oz) 80

  • Rockstar R (16 oz) 160

  • 5-Hour Energy R Shot (2 oz) 138

  • DynaPepTM Micro Shot (4 mL) 80

  • Extreme EnergyTM 6-Hour Shot 220

  • Endurance Shot 200

Dietary supplements

  • Thermogenic HydroxycutTM Advanced (2 pills) 200a

  • Zantrex R 3 (2 pills) 320

  • Stacker 2 R Ephedra Free (1 pill) 200

  • MetaboliftTM (2 pills) 176

  • SlenderiteTM (2 pills) 75

  • Skinny Fast R (3 pills) 0

  • Nature’s Plus R Fat Busters (2 pills) 0

CAFFEINE METABOLISM

The period of time caffeine remains in the circulatory system,

measured as half-life, varies dramatically. Half-life of

caffeine in a healthy adult is approximately four to five

hours, but in women taking oral contraceptives, it can increase

substantially. In cigarette smokers, caffeine is metabolized

more rapidly and has a half-life of about three

hours (11,12).

Caffeine is metabolized in the liver by a complex series

of processes. The principal metabolic pathway, which

accounts for approximately 95% of initial breakdown of

caffeine, is catalyzed by the cytochrome P450 enzyme

CYP1A2 (13).

 Chemical structures of caffeine, its demethylated derivatives

(theobromine, theophylline, paraxanthine), its parent compound (xanthine),

and uric acid.

group to form paraxanthine and, to a lesser extent, theobromine

and theophylline.

Various factors alter CYP1A2 activity. For example,

both pregnancy and severe liver disease result in

decreased caffeine clearance (13,14). Conversely, smoking

induces CYP1A2 activity, thereby decreasing half-life

of caffeine (14). Many pharmacological substances also affect

this enzyme: oral contraceptives and cimetidine inhibit

the enzyme and slow caffeine clearance (14), whereas

other drugs (e.g., phenytoin, carbamazepine) induce the

enzyme, accelerating caffeine metabolism.

Dietary practices influence CYP1A2 activity. Caffeine

intake itself induces this enzyme, so heavy consumers

metabolize caffeine more rapidly (15), explaining,

in part, why they are less sensitive to its behavioral and

physiological effects. Cruciferous vegetables (e.g., broccoli,

kale, turnip) increase CYP1A2 activity, whereas apiaceous

vegetables (e.g., cilantro, parsnip, celery) inhibit

it (16).

Genetic Differences in Caffeine Metabolism

Genetic variation is partly responsible for different phenotypes

of caffeine metabolism (17). Several CYP1A2 single

nucleotide polymorphisms (SNPs) have been characterized

(18). One, CYP1A2∗1F, is a substitution (A → C) at

position 734 on the CYP1A2 gene. Approximately 10%

to 16% of individuals have the CYP1A2 C/C (homozygous)

genotype, whereas about half possess two A alleles

(18). Individuals with the CYP1A2∗1F A → C polymorphism

are slower to metabolize caffeine and less likely

to increase CYP1A2 activity following exposure to inducers.

There may be physiological consequences of genetic

differences in caffeine metabolism. For example, a recent

study linked the slow-metabolizing CYP1A2∗1F C/C genotype,

with increased risk of heart disease associated with

coffee consumption (19).

Metabolic pathways of caffeine and its derivatives. Only pathways that begin with N-demethylation are shown, which account for almost 95% of

initial breakdown. Less than 3% of caffeine is excreted unchanged in the urine; the remainder is metabolized to 1,3,7-trimethyl uric acid (not shown). Source:

From Ref. 17.

MECHANISM OF ACTION

Caffeine’s behavioral, as well as ergogenic effects can be attributed

to central adenosine receptors (20,21). Adenosine

is an inhibitory neuromodulator in the central nervous

system that has sedative-like properties. Under normal

physiological conditions, caffeine is a nonselective competitive

antagonist at these receptors. Four subtypes, A1,

A2a, A2b, and A3, of G-protein–coupled adenosine receptors

have been identified, each with a unique tissue distribution,

signaling pathway, and pharmacological profile

(22,23). Through the respective activation of Gi and Gs

proteins, adenosine decreases adenylate cyclase activity,

and hence, cAMP levels, when bound to A1 or A3 receptors,

and increases activity when bound to A2a or A2b

receptors (22,23).

Prior to discovery of caffeine’s action on adenosine

receptors, effects of caffeine were attributed to inhibition

of phosphodiesterase (PDE) (24). However, the concentration

of caffeine required to inhibit PDE substantially exceeds

that achieved from consumption of caffeine in foods

or dietary supplements. While caffeine blocks A1 and A2a

receptors at concentrations in the low micromolar range

(5–30 M), approximately 20 times as much caffeine is required

to inhibit PDE, well above physiological levels as

illustrated in Figure 3, which also presents the approximate

concentration of caffeine from consumption of a cup

of coffee (25,26).

All four adenosine receptor subtypes are expressed

to various extents in the brain and periphery (23). Adenosine

A1 receptors are widely distributed in the periphery,

spinal cord, and brain, with high levels found in

hippocampus, cortex, cerebellum, and hypothalamic nuclei;

lower levels of the A1 subtype are found in the basal Blockade of:

  • GABAA

  • receptors

  • Ca2+-release

  • Inhibition of

  • phosphodiesterase

  • A1-receptors

  • A2a-receptors

Concentration–effect curves for caffeine at various potential sites

of action. Caffeine markedly affects A1 and A2a receptors at low micromolar

concentrations. To inhibit phosphodiesterase (PDE), concentrations as large

as 20-times are required. Approximate caffeine concentration resulting from

a single cup of coffee and toxic doses of caffeine is indicated.
Ref. 26.

ganglia (23). The A2a, A2b, and A3 receptors are mainly expressed

in the periphery; however, there is marked expression

of the A2a receptors in regions heavily innervated by

dopamine-containing fibers, including the striatum, nucleus

accumbens, and olfactory tubercle, where they are

coexpressed with dopamine D2 receptors (27).

Caffeine binds with highest affinity at A2a receptor

and has slightly lower affinity at the A1 and the A2b receptors;

the A3 subtype has little to no affinity (24). At

standard physiological concentrations (i.e., low micromolar),

effects of caffeine are due to blockade at A1 and A2a

receptors, with binding at A2b and A3 receptors having a

minor role, if any (22,27).

Both adenosine A1 and A2a receptors may be responsible

for behavioral effects of caffeine, but the contribution

of each is uncertain. The A1 receptors are located

predominantly on presynaptic nerve terminals and mediate

release of several neurotransmitters, including glutamate,

dopamine, and acetylcholine. Caffeine is thought

to enhance arousal, vigilance, and attention by blocking

inhibition by adenosine at these receptors, particularly

those in the striatum (22). Caffeine may stimulate arousal

via A1 receptors by preventing inhibition of mesopontine

cholinergic neurons that regulate cortical activity and

arousal (22).

Unlike dopaminergic stimulants, such as cocaine

and amphetamine, which facilitate dopamine D2 receptor

transmission, caffeine does not alter dopamine release

in ventral striatum (22). This may explain why caffeine

does not have the abuse potential of these stimulants.

Genetic Differences in Adenosine Receptors

Recently, it was shown in humans that a particular polymorphism

(a T→C substitution at position 1976; also

known as SNP rs5751876 or 1976T→C, and formerly

known as 1083T→C) of the A2a2a receptor gene (ADORA2A)

is associated with effects of caffeine on sleep (28). It appears

that 16% of individuals are homozygous for the T allele

and roughly 35% are homozygous for the C allele (29).

While inhibition of A1 receptors may be partly responsible

for wakefulness promoted by caffeine, most evidence

suggests caffeine-induced arousal is due to blockade at

A2a receptors (23).

Individuals with the ADORA2A C/C genotype

are more likely to report disturbed sleep following

caffeine consumption compared with individuals with

the T/T genotype (28). Consistent with these genetic

differences, associations were observed between self reported

caffeine-sensitivity, assessed by questionnaire,

and ADORA2A genotype.Higher proportion of sensitive

subjects had the C/C genotype, whereas the T/T genotype

was more frequent in insensitive subjects (28). In a

sleep deprivation study conducted in a subset of the survey

population, caffeine-sensitive men reported greater

stimulant-like effects of caffeine compared with those who

were caffeine-insensitive. In addition, ratings of caffeine

sensitivity were positively correlated with psychomotor

vigilance after sleep loss (28). Also, the C allele is associated

with caffeine-induced insomnia, and the T allele

appears to be related to caffeine-induced anxiety. Infrequent

caffeine users consuming less than 300 mg/wk,who

possess the ADORA2A 1976T/T genotype, experienced

greater anxiety following 150 mg caffeine compared with

those who possessed at least one C allele (29). Individuals

with the T/T genotype are significantly more likely

to limit caffeine intake (i.e., consume <100 mg/day) than

those who possess at least one C allele, with the probability

of having the T/T genotype decreasing as caffeine

intake increases (30).

CAFFEINE CONTENT OF VARIOUS PRODUCTS

In the United States, most of caffeine (approximately

80%) is consumed in coffee

(31). Caffeine is also found in

soft drinks, especially colas, energy drinks, tea, chocolate,

over-the-counter (OTC) drugs, and dietary supplements

(Table 1) (32). Some non-cola beverages also contain caffeine

such as Mountain Dew R , Dr Pepper R , and Pibb

Xtra R . There is tremendous variation in the caffeine in a

cup of coffee. Instant coffee (5 oz) can have as little as 40

mg of caffeine, whereas drip-method brewed coffee can

have as much as 150 mg (Table 1). There is considerable

variation in coffee prepared using the same method, due

to differences in caffeine content of different types of coffee

beans, especially arabica versus robusta, and variations in

brewing technique.

 

CAFFEINE INTAKE

Recent information on caffeine consumption in the United

States is not available. The most current data are based on

information collected between 1994–1998 (33) and 1999

(34). Using data from the nationally representative U.S.

Department of Agriculture Continuing Survey of Food

Intakes by Individuals (n = 18,081), Frary et al. reported,

in 1994–1998, that 87% of the population was caffeine consumers,

with an average caffeine intake of 193 mg/day

in users. Adult males consumed more caffeine than females

(268 mg/day vs. 192 mg/day). Coffee was the major

source of caffeine for consumers of all ages (68 mg/day),

followed by 15 mg/day from soft drinks and 12 mg/day

from tea (33). However, Ahuja et al. (35) concluded that

Frary et al. overestimated caffeine intake and revised their

estimates downward by about 25%. According to Ahuja

et al., average daily intake of caffeine in the U.S. population

is 131 mg/day, with males and females (20+ years)

consuming 193 and 149 mg/day, respectively (35). Knight

et al. (34) estimated caffeine intake in caffeine consumers

(n = 10,712) from beverages on the basis of data from

the 1999 U.S. Share of Intake Panel as 141 mg/day for

adults. There are other reports of higher caffeine consumption

in the United States. Barone and Roberts (31)

reported that U.S. caffeine intake for all consumers is about

210 mg/day.

Since these data were collected, changes in availability

of caffeine-containing products have occurred and

consumer preferences have changed (36). Energy drinks,

introduced in the United States in 1997, contain caffeine

(Table 1) and are a popular component of the diet, especially

among young adults (36). Another new product

containing high levels of caffeine (Table 1) termed “energy

shots” is rapidly gaining in popularity.

94 Lieberman et al.

High levels of caffeine are present in many dietary

supplements (Table 1), particularly those intended to promote

weight loss, for example, Zantrex 3 R and new Hydroxycut

AdvancedTM (37–39). Unfortunately, manufacturers

of such products are not required to disclose their

caffeine content, so this information can be difficult to

obtain. These products have not been shown to increase

weight loss, and recently some of the HydroxycutTM family

of products was withdrawn from the market after the

FDA issued a warning (40).

 

REGULATORY STATUS OF CAFFEINE

In the United States, complex regulations govern use of

caffeine. Once caffeine is ingested and enters circulation,

its source is of little physiological or health significance,

but U.S. government agencies, in practice, regulate it on

the basis of the medium in which it is consumed. Caffeine

consumed in dietary supplements, occurring naturally in

foods, caffeine added to foods, and caffeine in OTC and

prescription drugs are all regulated differently. Multiple

sources of caffeine ingestion and regulation can lead to

peculiar consequences. For example, a large cup of coffee

purchased at a coffee shop can contain more caffeine than

the recommended dose of an OTC stimulant. The recent

popularity of energy drinks has lead to calls for additional

regulation of such products, because it has been argued

that these products are abused (36).

 

BEHAVIORAL EFFECTS OF CAFFEINE

It is likely that caffeine is the most widely studied behaviorally

active compound not only in dietary supplements,

but also in any exogenously administered compound. Caffeine’s

behavioral effects have been examined in a large

number of laboratories and in well-controlled studies conducted

with males, females, young, and older volunteers

(41–43). Effects observed on specific aspects of cognitive

function andmoodstate are usually consistent with the lay

perception of caffeine as a mild stimulant when consumed

in moderate doses, just as Pietro della Valle recognized

400 years ago (44).

However, it can be difficult to detect effects of caffeine

if insensitive behavioral tests that assess parameters

not affected by caffeine are employed or doses that are

too low or high administered. In addition, it is essential to

control intake of caffeine before testing and monitor and

control for habitual patterns of caffeine consumption, as

these factors can have substantial effects on study findings.

Controlling for tobacco use is also essential, because

smoking significantly decreases caffeine’s half-life. Furthermore,

well-designed studies typically employ a range

of doses, since caffeine’s behavioral effects are dose dependent

and nonmonotonic.

 

Effects on Cognitive Performance and Mood

In rested volunteers, caffeine consistently improves both

auditory and visual vigilance (3,41,45–48). When a dose

of 200 mg of caffeine is given, effects on vigilance are

seen for several hours and are so robust that they can be

detected on a minute-by-minute basis (45) (Fig. 4). Such

effects are present with doses equivalent to a single serving

of a cola beverage, about 40 mg, up to multiple cups of

coffee (3,43,46). However, when higher doses are administered

(approximately 400–500 mg or above), cognitive

performance begins to deteriorate, so optimal dose appears

to be in the range found in foods (49,50). Caffeine

also improves simple and choice reaction time in rested

individuals (51). In general, it appears that sustained tests

of vigilance or tasks with substantial embedded vigilance

components are the most sensitive to behavioral effects of

caffeine in rested individuals.

Mood state is also altered by doses of caffeine equivalent

to those found in single and multiple servings of

dietary supplements, foods, and drugs. Aspects of mood

affected by caffeine are consistent with its effects on cognitive

functions such as vigilance and reaction time.

 

Effect of a 200-mg caffeine dose administered at

time “0” on visual vigilance reaction time assessed continuously

and plotted in 10 minute time blocks. Slower reaction

time (higher number) indicates worse performance (p < 0.002;

caffeine vs. placebo). Caffeine consistently improved cognitive

performance for two hours. Source: From Ref. 45.

Effects of 64 to 256 mg of caffeine compared to placebo (mean

°æ SEM) on the fatigue and vigor subscales of the Profile of Mood States

(POMS). Difference scores were computed by subtracting the baseline from

posttreatment values. Higher numbers on the vigor subscale indicate increased

vigor; lower numbers on the fatigue subscale indicate lowered fatigue.

∗p < 0.05 caffeine vs. placebo Source: From Ref. 32.

questionnaire, are altered by caffeine in a dose-dependent

manner (Fig. 5). Caffeine typically increases vigor and reduces

fatigue. At higher doses, these beneficial effects may

be reduced or disappear. An analog scale mood questionnaire

designed to assess effects of caffeine consistently detects

effects on moods such as tired/energetic, listless/full

of go, and efficient/inefficient (41).

 

Fine Motor Performance

Caffeine consumption has been associated, at least anecdotally,

with impaired fine motor performance. When administered

in a dose of 160 mg, it disrupted hand steadiness

in nonusers but not in users (53). In a study with low

and moderate consumers, doses of 32 to 256 mg of caffeine

had no effect on tests of complex motor function (54). A

recent study examined effect of caffeine on handwriting

in caffeine consumers (54). In this study, subjects were administered

caffeine in doses of 0, 1.5, 3.0, or 4.5 mg/kg and

performed a writing exercise on a digitized tablet. Compared

to placebo, high doses of caffeine improved aspects

of handwriting, such as fluidity of movement (54). Caffeine

has also been reported to improve marksmanship, a

task that requires fine motor performance (55).

 

Caffeine and Anxiety

It appears that caffeine increases anxiety when administered

in single bolus doses of 300 mg or higher, a dose

not ordinarily found in single servings of beverages, although

there are some exceptions. Generally, large servings

of beverages, such as 16 oz of coffee, that contain high

amounts of caffeine, are consumed slowly over time. Some

products, for example certain brands of energy drinks

or energy shots, do contain such high levels of caffeine

and may be consumed quickly, especially energy shots

(Table 1). The effects of caffeine on anxiety at lower doses

are unclear, with both positive and adverse effects reported,

perhaps due to differences in the testing environment

(50,56).

Several papers suggest caffeine consumption can

adversely affect individuals suffering from anxiety disorders.

Also, consumption of more than 600 mg of caffeine

per day may induce, in normal individuals, a syndrome

known as “caffeinism,” characterized by anxiety,

disturbed sleep, and psychophysiological complaints (57).

Effects on Cognitive Performance and Mood During

Sleep-Deprivation and Stress

Caffeine has substantial beneficial effects on cognitive performance

and mood when individuals are sleep deprived

or exposed to multiple stressors (50,58,59). Under such

conditions, caffeine positively affects various behavioral

parameters, including vigilance and mood state. For example,

during a night of sleep deprivation, 200 mg of caffeine

administered every two hours maintained vigilance

performance (60). Other behavioral parameters, such as

learning, memory, and reasoning, not altered when caffeine

is administered to rested volunteers, are affected

when individuals are sleep-deprived (50). When individuals

were sleep-deprived for 30 hours but not exposed

to additional stressors, 300 mg of caffeine per 70 kg of

body mass improved working memory, logical reasoning,

mathematical processing, pursuit tracking, and visual vigilance

(61). In a study conducted with U.S. Navy SEAL

trainees exposed to multiple stressors including cold, intense

physical challenges, 72 hours of sleep deprivation,

and severe psychological stress, caffeine in doses of 200

and 300 mg improved visual vigilance, choice reaction

time, and self-reported alertness (50).

Simulator and Applied Behavioral Studies

Based, in part, on studies demonstrating caffeine has

beneficial effects in laboratory studies of cognitive performance

and mood, studies have been conducted to determine

whether caffeine will have beneficial effects in

simulated or real work environments. For example,

Regina et al. (6) tested rested males in a realistic simulation

96 Lieberman et al.

U.S. Navy SEAL trainees are exposed to multiple stressors during

a segment of training known colloquially as “Hell Week.” This provided a

unique opportunity to test the behavioral effects of caffeine during sustained

exposure to severe stress (50). Cold stress serves as a key physiological

stressor during most of Hell Week. As instructors look on, trainees are required

to walk into the cold ocean water. Source: Photo courtesy of H.R. Lieberman.

of highway driving. Caffeine (200 mg) improved several

aspects of driving performance including response time

to accelerations and decelerations of a lead car. Philip

et al. (63) examined effects of approximately 200 mg of

caffeine administered in coffee on rested, young male volunteers

driving a distance of 200 km late at night on a

highway. Caffeine improved ability to maintain control

of the vehicle as measured by deviation from the traffic

lane. In a study simulating sentry duty, Johnson and

Merullo (64) evaluated the effects of 200 mg of caffeine on

marksmanship for three hours following caffeine administration.

Soldiers in the study responded to infrequent

appearance of a target by picking up a rifle, aiming, and

firing as rapidly, and accurately, as possible. Caffeine decreased

detection time but did not increase the number of

targets hit.

Recently, a series of studies have been conducted

by Kamimori and colleagues using a caffeine-containing

gum to determine whether caffeine would enhance performance

under conditions simulating combat, including

intermittent or continuous sleep deprivation and extensive

physical activity. These studies uniformly demonstrate

that in a wide variety of circumstances, various aspects

of cognitive, operational, and aerobic performance

are enhanced by caffeine (58,59).

In aggregate, these behavioral studies have important

practical implications. Use of caffeine in moderate

doses can improve the performance of individuals who

must drive automobiles or stand sentry duty for long periods

of time during the day or night. These beneficial

effects increase in situations where vigilance is reduced

due to sleep loss, jet lag, or circadian variations in arousal.

Recently, at the request of the U.S. Defense Department,

an independent panel conducted a comprehensive review

of the scientific literature and concluded that caffeine, in

doses of 100 to 600 mg, could be used to maintain cognitive

performance of military personnel. As a consequence,

caffeine is currently available in certain field rations (2,65).

 

CAFFEINE AND MENTAL ENERGY

Considerable attention has focused on dietary supplements

and foods that may increase “mental energy,” and

most of these contain caffeine. As noted above, new product

categories of “energy drinks” and “energy shots” have

emerged as popular products among the population (36).

Scientific literature on mental energy is quite limited (66–

68), although related factors, such as fatigue and alertness,

have been extensively examined. Scientists have typically

used the term “energy” to describe the concept of physical

energy measured in calories or joules. Mental energy,

however, cannot be easily defined or measured, but a distinction

between physical and mental energy clearly exists

(32,68–70).

In the United States, surveys have observed a high

prevalence of feelings of low energy. For the lay public,

mental energy is perceived as critical for the conduct

of daily activities and quality of life. On health-related

Internet sites, “fatigue,” “tiredness,” or “absence of energy”

are among the largest concerns for which remedies

are sought. To address consumer demand for such

products, dietary supplements and foods containing caffeine

have been marketed asserting they enhance energy

(36,69). Some have been evaluated by use of cognitive

tests, and the results are usually consistent with the claims

made (68).

 

Caffeine is one of the few constituents found in dietary

supplements or foods that clearly increase mental

energy

As noted above, low and moderate doses

improve aspects of cognitive performance and mood associated

with the perception of mental energy such as

vigilance, reaction time, vigor, and fatigue (68,71). Beneficial

effects of caffeine that appear related to mental

energy are observed in simulations of real-world activities

(58,59,62,64,72). Caffeine (100 mg) increases alertness

and self-reported attention in college students attending a

lecture (56). Epidemiological studies of large populations

indicate caffeine consumption has positive effects on factors

related to mental energy in large populations (73). In

a sample of over 7000 British adults, a significant dose–

response relationship between increased overall caffeine

intake and improved cognitive performance was observed

(73). Inclusion of caffeine in products intended to increase

perception of mental energy therefore appears warranted.

Caffeine may be the only active ingredient in such products,

if mental as opposed to physical energy is the implied

benefit (32,68).

 

EFFECTS OF CAFFEINE ON SLEEP

It is not surprising that caffeine may interfere with sleep,

because it improves ability to sustain vigilance and increases

alertness. Many individuals abstain from caffeine

consumption in the afternoon and evening because they

believe caffeine will disrupt nighttime sleep. Others report

they consume caffeine-containing beverages before

bedtime with no adverse impact on sleep (74). Genetic

differences in sensitivity to caffeine, as discussed above,

and acquired tolerance by individuals who consume caffeine,

probably contribute to these differences. Consumers

of 3 to 6 cups of coffee per day are less likely to report sleep

disturbances than individuals who consume 0 to 1 cups

per day (74).

Anecdotal reports that caffeine interferes with sleep

are supported by the scientific literature. In both high and

low consumers, a high dose of caffeine (4.0 mg/kg) at bedtime

reduced sleep tendency, as measured by the Multiple

Sleep Latency test (75). A recent study examined effects

of moderate doses of caffeine before bedtime on various

sleep parameters assessed with polysomnography. Subjects

received 100 mg of caffeine (or placebo) three hours

and then one hour before sleeping in the laboratory. Caffeine

lengthened sleep latency, increased stage 1 sleep

(light sleep) and reduced slow-wave and stage 2 sleep

(deeper sleep) (76).

 

BEHAVIORAL EFFECTS OF CAFFEINE: NONUSERS

AND WITHDRAWAL

One of the most controversial issues regarding the behavioral

effects of caffeine is whether it affects performance

and mood independent of withdrawal symptoms. It has

been suggested that behavioral effects of caffeine can only

be observed in habituated individuals experiencing effects

of caffeine withdrawal on performance and mood when

treated with placebo (77). If this hypothesis is correct then

caffeine should have no effects on individuals who are

not regular users. Several studies that fail to find behavioral

effects of caffeine in individuals who are not habitual

users support this hypothesis (77,78). However, there are

several problems with this hypothesis and the experimental

evidence supporting it. No plausible mechanism has

been advanced to explain how a substance could have

no acute effects on cognitive function, yet have effects

when it is withdrawn. Many substances produce tolerance

when administered for sustained periods. However, these

substances have acute behavioral effects consistent with

behavioral consequences of their withdrawal. Examples

include drugs of abuse, such as heroin, and therapeutic

compounds such as the benzodiazepines. To test the hypothesis

advanced by Rogers and colleagues that caffeine

only has behavioral effects on habitual users, a number of

laboratories have conducted studies. These demonstrate

caffeine has behavioral effects on nonusers and affects

users who continue with their typical patterns of caffeine

consumption (47,48,51).

 

CAFFEINE AND PHYSICAL PERFORMANCE

Just as caffeine improves specific aspects of cognitive performance

and mood, it also has positive effects on some

aspects of physical performance; however, it not clear that

these effects occur in doses found in most dietary supplements

or foods. Evidence that caffeine enhances aerobic

performance is convincing. A review of the literature

concluded that “caffeine effectively increases athletic performances

in endurance events” (79). For example, when

4 mg/kg of caffeine was administered to eight male volunteers,

time to run to exhaustion increased (80). In another

study, 3 and 6 mg/kg of caffeine enhanced endurance but

a higher dose, 9 mg/kg, did not (81). Beneficial effects of

caffeine assessed with a bicycle ergometer were observed

at doses 5 and 9 mg/kg, a higher dose (13 mg/kg) was no

more effective than lower doses (82).

These dose-dependent findings are similar to behavioral

studies with caffeine, in which higher doses can have

adverse effects on mood and do not enhance performance

to the same extent as moderate doses (49,50). It appears

that ergogenic effects of caffeine, like caffeine’s behavioral

effects, are attributable to its effects on central adenosine

receptors (21).

 

CAFFEINE WITHDRAWAL

Sudden withdrawal of caffeine from the diet, if regularly

consumed in substantial doses, can have adverse

effects in approximately 50% of users. Most notable is

headache, which is relieved by consumption of caffeine.

Onset of symptoms typically occurs in 12 to 24 hours

and may last for several days. Other symptoms can include

fatigue, lower energy, and difficulty concentrating.

Caffeine-withdrawal headaches are relieved by OTC analgesics

(83,84). Individuals who wish to reduce or eliminate

caffeine from their diet should probably do so gradually.

 

ABUSE POTENTIAL OF CAFFEINE

Although controversial, it has been suggested that caffeine

should be considered an addictive compound and is similar

to drugs that have substantial abuse potential, such

as nicotine and cocaine (85). Evidence for this association

includes adverse physical effects of caffeine withdrawal in

animals and human studies of caffeine self-administration

(84). Hirsh (83) has noted that addiction can best be defined

as compulsion to use a drug, and specifically, involvement

with the abused substances to the exclusion of

other interests. The use of methylxanthines in foods and

beverages would not appear to qualify as such behavior

(83). Most individuals who are regular users of caffeine

can readily halt its use and not feel compelled to continue

consuming caffeine-containing products. It is much more

difficult to stop using drugs of abuse despite the fact that

these substances are known to be extremely harmful. Caffeine

clearly has low abuse potential compared to more

widely recognized drugs of abuse (86).

 

CONCLUSION

When caffeine is consumed in doses found in foods and

dietary supplements, it improves ability to perform tasks

requiring sustained vigilance, including real and simulated

automobile driving, and activities that require maintenance

of vigilance (50,58,59). In addition, caffeine increases

self-reported alertness and decreases sleepiness.

Caffeine positively affects a wide range of cognitive functions

in sleep-deprived individuals, including learning,

memory, and reasoning. Caffeine can be found in many dietary

supplements, which are marketed to increase weight

loss, but evidence to support this implied claim is lacking.

Adverse behavioral effects of caffeine occur when

it is consumed in excessive doses or by individuals who

are more sensitive to the substance. Genetic factors and an

98 Lieberman et al.

individual history of caffeine consumption may be the key

factors explaining individual differences. In high doses,

caffeine can increase anxiety but its effects on fine motor

performance vary with improvement and impairment reported.

It also interferes with sleep when consumed by certain

individuals at bedtime. Like many other drugs, regular

caffeine consumption appears to produce tolerance to

its behavioral effects. Sudden withdrawal of caffeine from

the diet will lead to adverse symptoms, such as headache

and undesirable changes in mood state, in approximately

50% of individuals. Some scientists believe that caffeine

has properties that are similar to those exhibited by drugs

of abuse; others strongly disagree with this hypothesis.

An evidence-based determination of the risk-tobenefit

ratio of caffeine consumption is not possible. Positive

behavioral consequences of caffeine are well documented.

These beneficial effects generalize to highway

driving, various military duties, and presumably other

transportation and industrial operations. Use of caffeine

in such circumstances could potentially prevent accidents

attributable to lapses of vigilance such as “falling asleep at

the wheel.” Such accidents are a significant cause of motor

vehicle accidents. However, adverse effects of caffeine on

sleep quality have been observed, and some scientists believe

that caffeine has characteristics of an addictive drug.

It must also be noted that a large and complex literature

on possible beneficial and adverse effects of caffeine

on the incidence of various diseases exists. There are many

methodological concerns with both positive and negative

studies. The difficulty in accurately assessing caffeine intake

is a critical issue in such studies as is the lack of

double-blind, placebo-controlled clinical trials. Therefore,

both positive and negative findings regarding possible

health risks and benefits of caffeine should be regarded

with skepticism.

 

DISCLAIMER

Portions of this chapter are based on previous reviews by

the first author (32,67,68). This work was supported by

the U.S. Army Medical Research and Materiel Command

(USAMRMC). The views, opinions, and/or findings in

this report are those of the authors and should not be

construed as an official Department of the Army position,

policy, or decision, unless so designated by other official

documentation. Citation of commercial organization and

trade names in this report do not constitute an official

Department of the Army endorsement or approval of the

products or services of these organizations.

 

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Calcium

GlossarySuccess Chemistry Staff

Calcium is an alkaline earth, divalent, cationic element,

abundant in the biosphere, and widely distributed in nature.

 

It exhibits intermediate solubility. As a solid, calcium

forms crystalline minerals with various anions. These salts

make up the bulk of limestone, marble, gypsum, coral,

pearls, seashells, bones, and antlers. In solution, the calcium

ionic radius (0.99 ˚A units) allows the ion to fit snugly

into the folds of protein molecules.

ACTIONS AND PHARMACOLOGY PHYSIOLOGY

Calcium is unusual—perhaps unique—among the nutrients

in that its intake (whether from foods or supplements)

is not related to its primary intracellular, metabolic

function. Rather, calcium nutrition is centered almost

exclusively on the secondary functions of the nutrient.

Accordingly, the primary functions are described here for

completeness, but only briefly. More information can be

found in standard textbooks of cell physiology or in reviews

of calcium signaling (1).

 

Primary Metabolic Functions

Calcium acts as a second messenger within cells, linking

external stimuli acting on cells to the specific, internal

responses a cell is able to make (e.g., nerve signals and

muscle contraction). By forming up to 8 to 12 coordination

bonds with oxygen atoms in amino acid side chains,

calcium stabilizes the tertiary structure of numerous catalytic

and structural proteins. Cytosolic calcium ion levels

are normally maintained at very low concentrations

[3–4 orders of magnitude below extracellular fluid (ECF)

levels]. The second messenger response occurs when calcium

ions flood into critical cytosolic compartments in

response to first message stimuli.

Additionally, dissolved calcium in the circulating

blood and ECF of all vertebrates supports such diverse

functions as blood clotting and neuromuscular signal

transmission. Calcium is not consumed in the exercise of

these metabolic functions.

ECF [Ca2+] is tightly maintained at approximately

4.4 to 5.2 mg/dL (1.1–1.3 mmol/L). The regulatory apparatus

behind this constancy consists of parathyroid

hormone (PTH), calcitonin, and 1,25-dihydroxyvitamin D

[1,25(OH)2D], acting jointly through control of intestinal

calcium absorption efficiency, bone resorption, and the renal

excretory threshold for calcium.

Secondary Functions

Effects on the Size and Strength of the Nutrient Reserve

(Bone Mass)

Calcium is lost continuously from the body through shed

skin, hair, nails, sweat, and excreta. For this reason, landliving

vertebrates, needing a continuous supply of calcium,

have evolved an internal reserve, in the form of

bone. Because bone also serves structural/mechanical

functions, the reserve has become far larger than would be

needed solely to protect calcium’s primary functions. It is

for this reason that the primary functions themselves are

not threatened by deficient calcium intake, or enhanced

by calcium repletion.

The bony reserves are accessed by a process termed

“bone remodeling.” Bony tissue is continuously renewed

by first resorbing preexisting volumes of bone and then

subsequently replacing them with new bone. Mineralization

of the new bone occurs at a rate that is the integral of

the prior several days of osteoblast activity, and for that

reason tends to be relatively constant over the short term.

By contrast, osteoclastic bone resorption is controllable

minute by minute. Thus, by modulating bone resorption,

the body can, in effect, withdraw calcium from, or cause

it to be taken up by, bone whenever ECF [Ca2+] departs

from optimal levels.

When daily absorbed calcium intake is less than that

needed to offset daily calcium losses, bone resorption exceeds

bone formation and the bony reserves are depleted.

This occurs by net destruction of microscopic volumes

of bony tissue and scavenging of the calcium released in

the process. Such decrease in skeletal mass results in a

corresponding reduction in strength. Additionally, bone

remodeling itself directly contributes to bony structural

weakness (2), insofar as the remodeling locus is, for the

several months of its life cycle, depleted of its normal

complement of bony material, thereby greatly weakening

the involved microscopic bony elements.

The principal purpose of calcium intake during

growth is to support the accumulation of the skeletal

mass called for in the genetic program, that is, the building

of a large calcium reserve. During the adult years,

intake serves to (i) offset daily losses, thus preventing

unbalanced withdrawals from the skeletal reserves, with

their inevitable, associated reduction in bony strength; and

(ii) reduce the level of bone remodeling to the minimum

needed for optimum structural maintenance (2). These

two effects are the basis for the protective effect of calcium

with respect to osteoporosis.

Intraluminal Effects of Unabsorbed Dietary Calcium

Net absorption efficiency for ingested calcium is of the order

of 10% to 15% (see later). Accordingly, up to 90% of dietary

and supplemental calcium remains in the intestinal

lumen and is excreted as a component of the feces. At high

calcium intakes, unabsorbed calcium amounts to 1000 mg

(25 mmol) per day or more. This unabsorbed calcium complexes

with other constituents of the digestive residue,

blocking their absorption or neutralizing their luminal actions

(3). This occurs, for example, with oxalic acid, which

may be either present in ingested plant foods or produced

by bacterial degradation of unabsorbed food fatty acids.

The formation of calcium oxalate in the gut lumen reduces

oxalate absorption and hence the renal oxalate load. It

thereby reduces the risk of kidney stones. Similarly, the

calcium ion complexes directly with free fatty acids and

bile acids in the digestate substances, which, in their free

form, act as mucosal irritants. In colon cancer–prone individuals,

these irritants would otherwise serve as cancer

promoters. These intraluminal actions are the basis for the

protective effects of high calcium intakes on risk of renal

stone disease and colon cancer.

Additionally, calcium complexes with dietary phosphorus,

blocking its absorption to some extent. This is the

basis for the use of calcium salts as a part of the control of

hyperphosphatemia in patients with end-stage renal disease

(ESRD). Every 500 mg of ingested calcium (whether

from foods or supplements) binds ≈166 mg of coingested

phosphorus, preventing its absorption (4).

“Off-Loop” Effects of Alterations in Calcium Homeostasis

When calcium intake is low, PTH is secreted to improve

renal calcium conservation and intestinal absorption efficiency,

the latter through 1--hydroxylation of 25(OH)D

to 1,25(OH)2D in the kidney. The calcium-conserving effects

of these hormones are part of a classical negative

feedback loop, in the sense that 1,25(OH)2D, by increasing

calcium absorptive extraction from food, counteracts

to some extent the original stimulus to PTH secretion and

1,25(OH)2D synthesis.

In addition to these functions within the feedback

control loop, 1,25(OH)2D binds to membrane receptors

in many tissues not directly involved in calcium regulation

(3). These include vascular smooth muscle cells and

adipocytes. These effects are termed “off-loop,” because

they occur as a result of reduced ECF [Ca2+] but do not

act to change that level. Hence, they do not influence the

signals that caused them in the first place, that is, they

are not a part of the regulatory feedback loop. The cell

membrane receptors are linked to calcium channels that

open and let calcium ions into the cytosol, where they

may trigger their usual second messenger function (but

without the normal first messenger). The presence of high

cytosolic calcium levels, when dietary calcium is low, has

given rise to the term “calcium paradox disease.” In individuals

with limited control of cytosolic [Ca2+], this rise

in cytosolic calcium triggers inappropriate, tissue-specific

cell activity, for example, smooth muscle contraction in

arterioles and adipogenesis in fat cells. These relationships

are the basis for the protective effects of high calcium intake

against hypertension and obesity, and probably for

premenstrual syndrome and polycystic ovary syndrome

as well (3).

 

The Internal Calcium Economy

The adult human body contains approximately 1000 to

1300 g (25,000–32,500 mmol) of calcium, with more than

99% being locked up in bones and teeth. Low hydration

of bone, together with the insolubility of hydroxyapatite

(the principal form of calcium phosphate in mineralized

tissues), means that most body calcium is effectively exterior

to the ECF and accessible only by cellular action (e.g.,

osteoclastic bone resorption).

The ECF, which is the locus of all body calcium traffic,

contains about 1 g (25 mmol) calcium (i.e., ≈0.1% of

total body calcium). Soft tissues contain another 7 to 8 g

(175–200 mmol) of calcium, mostly locked up in intracellular

vesicles, which store calcium for its critical, second

messenger function. The calcium homeostatic regulatory

apparatus functions solely to maintain the constancy of

the concentration of the ≈1 g of calcium in the ECF. In

healthy midlife adults, ECF calcium turns over at a rate

of approximately 650 mg/day (≈10 mg/kg/day), with

bone mineralization and resorption accounting for half to

two-thirds of that traffic.

 

The calcium economy of a middle-aged woman. In considering

the magnitudes of these transfers, it is important to

recognize that they do not vary independent of one another.

An increase in absorption, for example, produces

an immediate decrease in bone resorption. This linkage

is mediated by the PTH–calcitonin–vitamin D regulatory

apparatus.

Absorption

Calcium is absorbed mainly from the small intestine by a

combination of active, transcellular transport and passive,

paracellular diffusion.

The active transport component

is mediated by a vitamin D–dependent calcium-binding

protein (“calbindin”) that shuttles calcium ions from

the luminal brush border to basolateral portions of the

cell membrane, where calcium is released into the ECF.

Calbindin activity is highest in the duodenum and drops

along the length of the remaining bowel (including the

colon). Accordingly active transport capacity is greatest

in the duodenum. However, the residence time of the

digestate in the duodenum is short, and most of the actual

mass transport occurs in the jejunum and ileum, where

residence time is longer.

The partition of absorption between the active and

the passive mechanisms is not well studied, but data

from various sources suggest that, at nutritionally relevant

intake loads (i.e., 7.5+ mmol/meal), passive absorption

amounts to approximately 15% of intake. Fractional

absorption above that value thus reflects the vitamin D–

mediated active transport component. The latter is highly

variable, both because it is physiologically regulated in

response to body need for calcium, and, in part, because it

is often limited by vitamin D availability. The interactions

between intake load of calcium and its active absorption

are complex and are summarized in Figure 2. As measured

fractional absorption is typically on the order of

0.30 to 0.32; it follows that the active transport component

amounts to approximately 0.16 (16%). As is shown

in Figure 2, the 16% isogram intersects the dashed line for

5 mmol (200 mg) net absorption at an intake of approximately

1200 mg (30 mmol), and thus designates the oral

intake needed to maintain total body equilibrium. When

vitamin D status is less than optimal, the body is generally

unable to maintain active absorption at a 16% level

and absorption occurs along lower and lower isograms

until, at severe vitamin D deficiency, active absorption is

zero. As is shown in Figure 2, intake at such absorption

values would need to be in the range of 3000 mg/day to

ensure absorption of sufficient calcium to offset obligatory

losses.

the calcemic rise above baseline in healthy adults for a 500-mg calcium supplement source ingested as part of a low-calcium breakfast. It illustrates

a number of features of calcium absorption: (i) a delay of

approximately 30 minutes before serum calcium begins to

rise, reflecting gastric residence time; (ii) peak calcemia at

3 to 5 hours after ingestion, indicating continuing absorptive

input throughout that period of time; (iii) a degree of

calcemia approximating a 1% rise for every 100 mg calcium

ingested, that is, a perturbation that is within the

usual normal range for serum calcium and hence effectively

undetectable outside of a research context; and (iv)

gradual return to baseline by 9 to 10 hours. Tracer studies

show that calcium absorption is effectively completed by

five hours after ingestion (5), and the slow fall to baseline

after the peak reflects offsetting declines in other inputs

into the ECF.

It is commonly considered that calcium salts must

be dissociated to be absorbed, and hence that solubility

predicts absorbability. However, this is probably incorrect.

The pH of the digestate in the small intestine is close

to neutral, and it is likely that most of the digestate calcium

is complexed with prevailing anions in the digestate.

 

Relationship of vitamin D–mediated, active calcium

absorption, calcium intake, and net calcium gain across the gut.

Each of the contours represents a different level of active absorption

above a baseline passive absorption of 12.5%. (The values

along each contour represent the sum total of passive and variable

active absorption.) The horizontal dashed lines indicate 0

and 5 mmol/day net absorption, respectively. The former is the

value at which the gut switches from a net excretory to a net absorptive

mode, and the latter is the value needed to offset typical

urinary and cutaneous losses in mature adults.

 

Time course of the rise in serum calcium following a single oral

dose of a commercial calcium carbonate preparation (containing 500 mg

calcium) taken as part of a light, low-calcium breakfast. Error bars are 1 SEM.

Source: Copyright Robert P. Heaney, 2001, 2004; used with permission.

Aqueous solubility of calcium salts spanning 4 to 5 orders

of magnitude has been shown to have little or no effect on

absorbability if the calcium source is coingested with food

(6). Double-tracer studies have demonstrated absorption

of insoluble calcium complexes without prior dissociation

(7). Thorough dispersion of calcium salts among food particulates

is probably more important than actual solubilization.

Additionally, continuous slow release of calcium

from the stomach, exposing the duodenal mucosa to only

small amounts of calcium at a time, substantially improves

absorption.

 

Calcium leaves the body through unabsorbed digestive

secretions, through sweat and shed skin, hair, and nails,

and through urine

In non exercising adult humans

with typical calcium intakes, digestive calcium losses

amount to approximately 120 mg/day, cutaneous losses

amount to approximately 60 mg/day, and urinary

losses amount to approximately 120 mg/day, with

great individual variability around these figures. Only

the urinary loss is physiologically regulated by the

system controlling calcium homeostasis, and much of

even the urinary calcium represents obligatory loss,

that is, excretion determined by forces outside of the

calcium regulatory system (9), such as salt intake and

net endogenous acid production (as, for example, from

metabolism of S-containing amino acids). On average,

urine calcium rises by approximately 45 mg (1.1 mmol)

for every 1000 mg (25 mmol) increase in calcium intake.

This increase is a reflection of the small absorptive

calcemia (Fig. 3), which produces a corresponding rise in

the filtered load of calcium.

In adults, the primary purpose served by ingested

calcium is the offsetting of obligatory excretory losses,

thus protecting the skeletal reserves and thereby preserving

their structural integrity. Ingested calcium, thus, does

not so much “go” to bone as prevents net removal of calcium

from bone.

 

INDICATIONS AND USAGE

Calcium is a nutrient and would normally be ingested as

a component of food. However, except for dairy foods,

modern diets, especially seed-based plant foods (which

are the basis of most contemporary diets), are calciumpoor

diets. Hence, for many individuals, achieving an adequate

calcium intake may be difficult without recourse

to supplements or calcium-fortified foods. (The latter are

effectively equivalent to taking a supplement along with

the otherwise unfortified food.)

 

Supplementation to Achieve Recommended

Intake Levels

Diets free of dairy foods typically contain no more than

200 to 300 mg calcium, far below currently recommended

intakes (Table 1). Supplementation (or fortification) will

often be required to meet optimal intake objectives.

Table 1 Estimated Average Requirements (EARs) for Calcium and the

Corresponding RDAs (mg/day)

Age range EAR RDAa

Infants, 7–12 mo 270 350

Children

1–3 yr 500 600

4–8 yr 800 1000

Boys and girls, 9–18 yr 1300 1550

Men and women

19–50 yr 1000 1200

>50 yr 1200 1450

aUsing an estimated 10% coefficient of variation of individual requirements

around the population mean.

Source: From Ref. 10.

Calcium 105

Since absorption efficiency is inversely proportional

to the logarithm of the ingested load (11), absorption is

maximized by a divided dose regimen (e.g., 3°ø per day;

Fig. 4). Also, because delivery of calcium to the absorptive

sites in the upper small intestine is optimized under meal

conditions, it is best to take calcium supplements with

meals. (N.B.: Fortified foods tend, automatically, to meet

both objectives.)

Preparations

The nutritional preparations of calcium include mainly

salts with such anions as carbonate, citrate, phosphate,

lactate, acetate, fumarate, and citrate-malate (CCM). In

addition, salts with gluconic acid may occasionally be

found, and calcium chelates with amino acids are also

marketed. The calcium content (i.e., “elemental” calcium)

varies from 40% for the carbonate salt to ≈13% for CCM.

For phosphate binding in ESRD, the acetate salt is more

commonly used. In the United States, most preparations

come in the form of swallowable or chewable tablets, with

calcium contents ranging from 200 to 600 mg per tablet.

Bioavailability is approximately the same for all the

leading salts, although CCM and the chelates tend toward

the high end of the range and the gluconic acid salts toward

the low end. Absorbability of the salt is only very

weakly related to solubility, and gastric acid is not necessary

for calcium absorption if the supplement is taken

(as recommended) with meals. The most extensive, sideby-

side comparisons have involved the carbonate and citrate

salts, and the bulk of the evidence for such comparisons

indicates approximately equal absorbability for the

two sources, with perhaps a slight edge for the carbonate

salt (12). Poor pharmaceutical formulation will impede

disintegration and hence impair absorption, a problem

encountered with many generic calcium supplement

products sold in the 1980s and early 1990s (13). For this

reason, preference should be given to supplements that

meet United States Pharmacopeia (USP) disintegration

standards, and, even better, to those that have demonstrated

bioavailability.

A growing variety of fortified foods has been available

since 1999. As noted, fortification tends to improve

the nutritional value of low-calcium foods and, to some

extent, it can be thought of as equivalent to taking supplements

with meals. However, interactions between added

calcium and various food constituents during food processing

and storage may alter the absorbability characteristics

of the former. For example, it was noted during the

early days of juice supplementation that CCM was well

absorbed from orange and grapefruit juices, and even better

from apple juice, but poorly from lemon juice. These

differences could not have been predicted from what was

known of food chemistry. Hence, with fortified foods as

with supplements, actual bioavailability of the product

reaching the consumer should be demonstrated.

When calcium is added to beverages (such as orange

juice or soy beverage), an additional problem arises. Solubility

of the principal calcium salts is relatively low, and

serving size portions of such beverages would not sustain

in solution more than a small fraction of the calcium

content of, say, a comparable serving of milk. Hence, such

fortification almost always requires physical suspension

of a particulate. In some beverages, this suspension is so

poor that the calcium settles as a dense sludge at the bottom

of the beverage container and may, accordingly, not

be ingested at all (14).

Supportive Therapy as a Part of Anti Osteoporosis

Pharmacotherapy

Current anti osteoporosis pharmacotherapy includes bisphosphonates,

selective estrogen receptor modulators

(SERMs), estrogen, and anabolic agents such as the fluoride

salts, PTH derivatives such as teriparatide, and

RANK-ligand antibodies and cathepsin-K inhibitors. All

have at least stabilization of bone mass as their goals.

Some of them, such as the bisphosphonates, can lead

to slow, steady-state bone gain (0.5–1.0% per year), and

the anabolic agents can produce as much as 8% to 10%

bone gain per year. To support this increase, especially for

the anabolic agents, calcium intake from diet must usually

be augmented by supplements. Optimal doses for calcium

during pharmacotherapy have not been established.However,

all the bisphosphonates and SERMs have been tested

only with 500 to 1000 mg supplemental calcium, whereas

fluoride has been shown to produce bone hunger calling

for as much as 2500 mg of calcium per day. Only estrogen

has been studied with and without supplemental calcium,

and here the evidence is very clear: bony effects of estrogen

are augmented two- to threefold, and estrogen dose

can be reduced by half if calcium intake is above 1000

mg/day (15,16). With the more potent anabolic agents, a

calcium phosphate preparation may be preferable, so as

to ensure an adequate intake of both of the components of

bone mineral and to compensate for the intestinal binding

of diet phosphorus by high-dose calcium supplementation.

The high phosphorus loads of the phosphate salts

produce no adverse metabolic consequences, and calcium

phosphate supports bone anabolism fully as well as the

carbonate salts.

 

Ancillary Therapy for Prevention or Treatment of Miscellaneous Disorders

Hypertension, pre-eclampsia, colon cancer, renolithiasis,

premenstrual syndrome, polycystic ovary syndrome,

and obesity—all multifactorial disorders—have each been

shown to have a calcium-related component (3), and

for several of them, calcium supplementation has been

shown in randomized-controlled trials to reduce incidence

and/or severity. Optimal calcium intake for this protection

has not been established for any of the disorders

concerned, but several threads of evidence indicate that

total intakes of 1200 to 1800 mg of calcium per day may

be sufficient. The role of calcium in these disorders has

been described earlier (see sections “intraluminal effects of

unabsorbed dietary calcium” and “off-loop effects of alterations

in calcium homeostasis”). Specific pharmacotherapy

of any of the disorders concerned should always be

accompanied by an adequate calcium intake, using supplements

if necessary.

 

CONTRAINDICATIONS

There are few, if any, true contraindications to calcium

supplementation. In general, supplementation moves

106 Heaney

contemporary intakes into the range that would have been

the Paleolithic standard, and hence helps to normalize

modern diets. However, patients receiving calcitriol therapy

or suffering from disorders such as sarcoidosis, in

which calcium absorption may be high, should not take

supplements except under medical supervision.

 

PRECAUTIONS AND ADVERSE REACTIONS

Calcium supplements may bind with tetracycline antibiotics

and hence reduce their absorbability. The element has

also been reported to interfere slightly with thyroxin absorption.

Hence, a person requiring both calcium and thyroid

replacements should take them at different times of

the day or have plasma thyroxin and thyroid-stimulating

hormone (TSH) levels checked to ensure that the thyroid

dose produces the desired therapeutic effect. Both calcium

salts and high-calcium foods reduce absorption of nonheme

iron ingested at the same meal in unprepared subjects.

However, chronic supplementation studies show no

long-term deterioration in iron status in adults and no

interference with augmenting iron status during growth

(17). The single-meal tests that are used to demonstrate

this interference could not have detected physiological

upregulation of iron absorption.

Adverse reactions tend to be extremely rare and

mostly idiosyncratic. Although constipation is often said

to be a consequence of taking calcium carbonate, the evidence

is scant (18), and in several randomized-controlled

trials, the difference in degree of constipation between the

calcium- and placebo-treated groups has generally been

small and usually not statistically significant.

 

OVERDOSAGE

The Food and Nutrition Board of the Institute of Medicine,

in its 1997 recommendations, set a tolerable upper intake

level for calcium to be 2500 mg/day (9). However, it is important

to note that there has never been a reported case of

overdose of calcium from food sources, even at continuing

intakes over 6000 mg/day. Supplement intakes above

2500 mg/day are occasionally associated with a syndrome

similar to the milk alkali syndrome. The pathogenesis of

the hypercalcemia seen in this condition is complex, but

there is usually hypoperfusion of both the kidneys and the

skeleton, the two most important internal regulatory organs

for calcium. The condition can usually be managed

by giving attention to adequate hydration and maintenance

of blood flow to these critical organs. Except as support

for the most potent osteoporosis pharmacotherapy, or

in management of the hyperphosphatemia of ESRD, there

is no known reason to use supplements at a dose above

2500 mg of calcium per day.

 

REGULATORY STATUS

Calcium supplements are regulated as foods in the United

States. Bioavailability is not a regulated characteristic of

marketed supplement products. Nevertheless, because of

pharmaceutical formulation and food matrix effects on

absorbability, bioavailability of different preparations of

the same salt (e.g., calcium carbonate) may vary over a

twofold range.

 

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