Indian Journal of Experimental Biology Vol. 39, June 2001, pp. 520-525

/

Review Article

Pharmacological activities of Genistein, an isoflavone from soy (Glycine max): Part II-Anti-cholesterol activity, effects on osteoporosis &

menopausal symptoms

A C Suthar, M M Banavalikar* & M K Biyani

Ajanta Pharma Ltd., Research Centre, Ajanta House, 98, Gov!. Industrial Area, Charkop, Kandivali (West), Mumbai 400067, India

Tel : 8683945, 8683625, 8683718; Fax No: 8683930, 8682845, e-mail : ajanta@vsnl.com

Phytoestrogens represent a family of plant compounds such as isoflavones, flavones and lignans. A wide range of commonly consumed foods contains appreciable amounts of different phytoestrogens such as isoflavones and lignans. Soy and its products are particularly good sources of isoflavones and lignans. The evidence reviewed here represents the benefi­cial effects of most potential and promising isoflavone, Genistein in various types of diseases such as osteoporosis, cardio­vascular diseases, menopausal symptoms by accumulating evidence from molecular and cellular biology experiments, ani­mal studies, and, to a limited extent, human clinical trials. This review suggests that phytoestrogens may potentially confer health benefits related to various diseases such as cardiovascular disorder, menopausal symptoms, and osteoporosis.

Isoflavories of soy are important and fast-growing segments in the study of the relationship between health and nutrition. Of the isoflavones contained in soy (Glycine max), two-Genistein and Daidzein­seem to hold the most promise from the therapeutic point of view. Genistein and Daidzein are present in high concentrations (up to 3 mg/g) in soybeans 1. Soy (Glycine max) also contains Genistin and Daidzin, which are sugar containing isoflavones molecules. During digestion, intestinal bacteria cleave or cut off the sugar molecules, creating still more Genistein and Daidzein. Research suggests that soy isoflavones act in four distinct ways: as estrogens and antiestrogens, as cancer-enzyme inhibitors, as antioxidants and as immune enhancers. Accumulating evidence from molecular and cellular biology experiments, animal studies and human clinical trials suggest that isofla­vones may confer health benefits related to cardiovas­cular diseases, osteoporosis, menopausal symptoms and other diseases.

Genistein-Menopausal Symptoms During woman's post menopausal period in life,

the production of estrogen by the ovaries is reduced. The drop in blood-estrogen levels triggers common symptoms of menopause e.g. hot flashes; insomnia; heavy sweating (especially at night, which further contributes to insomnia); headaches; mood swings;

*Correspondent address

nervousness and irritability; depression, vaginal dry­ness and soreness. Based on life expectancy trends, women face the prospect of spending the last one­third to one-half of their lives in a state of hormonal imbalance. However, experiences vary from culture to culture around the world. For example, Asian women are typically one-third as likely as American women to report symptoms and there appears to be an actual difference in the severity and incidence of symptoms2

.4

• Hot flushes are uncommon in women from countries where the consumption of soy prod­ucts is high, such as in Asian countries5

•6

. In fact, there is no precise Japanese word to describe hot flushes. Japanese women reported much less night sweats and hot flashes than Canadian women did. Another study found that urinary excretion of iso­flavones in Japanese women was 100 to 1000 times higher than in American and Finnish women and that the high consumption of soy products may partly ex­plain why Japanese women report having fewer hot flushes7

•

Although women can reduce postmenopausal symptoms by taking exogenous estrogen, the compli­ance is very poor (about 10% of women older than fifty-five years). The development of SERMs (Selec­tive Estrogen Receptor Modulators) were designed to retain the benefits of estrogen without its unwanted effects. That is ideally, SERMs exert estrogenic ac­tions in tissues where that effect is thought to be bene­ficial, such as the bones and the heart, but would have

SUTHAR et at.: PHARMACOLOGICAL ACTIVITIES OF GENISTEIN: PART II 521

no effects or even an anti estrogenic effect in tissues such as the uterus and breast where estrogenic effects are undesirable. There have been much new recently about such SERM, Raloxifen, which has been shown to improve bone health without exerting an estrogenic effect on the endometrium and which may even exert an antiestrogenic effect on breast cancer. However, Raloxifen does not relieve hot flashes nor does it seem to offer protection against heart disease to the same extent as estrogens.

At the "Second International Symposium on the Role of Soy in Preventing and Treating Chronic Dis­ease", which was held in Brussels, Belgium, three studies found a significant decrease in the post­menopausal symptoms, especially hot flashes9

•11 . A

more recent study conducted in Italy using 104 post­menopausal women found a significant reduction (45% vs. 30% for the placebo) in the number of hot flushes after 12 weeks of consuming 60 g daily of soy protein containing 76 mg of isoflavones3. In another study, seventy-eight women with complaints about hot-flashes were given isoflavone-rich diet. After 12 week's of consuming the isoflavone-rich diet there was a significant increase in the women's serum lev­els of SHBG I2

.

Sex hormone-binding globulin (SHBG) is a plasma protein with high affinity-binding site for estrogens and androgens. An increase in SHGB decreases the relative amount of free testosterone and free estradiol and thereby lowers the steroids biological activity. There is some evidence that the binding of estrogen to SHGB is related inversely to breast cancer risk. The study indicates that prolonged elevation of SHBG by shifting to isoflavone-rich diet might offer some pro­tection against breast cancer. Also, there was a statis­tical significant reduction of hot flashes severity and vaginal dryness in the group of women receiving the isoflavone-rich diet.

In another study, fifty-one postmenopausal women were given isoflavone-rich supplements. The soy lessened menopausal symptoms, lowered blood pres­sure and resulted in a healthier blood lipoprotein pro­file. The supplements led to these benefits without the side effects associated with conventional hormone replacement therapyll. A recent review on common alternative remedies for treatment of symptoms at­tributed to menopause. The conclusion was "In avail­able controlled studies, the strongest data support phytoestrogens for their role in diminishing meno­pausal symptoms related to estrogen deficiency and

for possible protective effects on bones and cardio­vascular system,,13.

Genistein-Bone Disease (Osteoporosis) Osteoporosis is a reduction in bone tissue resulting

in brittle and fragile bones prone to fracture, usually occurring in postmenopausal women and elderly men. However, women are at greater risk for osteoporosis, because they have a lower peak bone mass (smaller skeletons), and they also lose bone more rapidly after menopause. Because differences in peak bone mass, women are an important determinant of the risk of developing osteoporosis later in life, osteoporosis is often considered to be a "pediatric disease with a geriatric outcome". The changes in bone mass and shape are due to hormonal changes, deficiency of cal­cium, deficiency of vitamin D, and low peak bone mass.

Osteoporosis is a problem of immense magnitude. Statistics from the National Osteoporosis Foundation indicate that one in three women over fifty years of age will suffer a fracture in her lifetime. Estrogen re­placement therapy (ERT) is one of the mainstays for the prevention and treatment of osteoporosis in post­menopausal women. However, ERT may have unde­sirable effects and risks, and compliance with ERT regimes tends to be low. Epidemiological studies have shown a lower incidence of osteoporosis among population consuming high soy diets compared to western population 14,15.

There are two types of cells in bone, one that makes bone (osteoblasts) and one that destroys bone (osteoclasts). Because of the drop in estrogen, the cell type that destroys bone starts to become predominant. Osteoclasts are usually dependent on the activity of the enzyme tyrosine kinase by inhibiting its activity; thus, tyrosine kinase inhibitors are candidates to pre­vent osteoporosis. Genistein is well known as tyrosine kinase inhibitor and as such can be used in preventing osteoporosis. A study presented at the "Second Inter­national Symposium on the Role of Soy in Preventing and Treating Chronic Disease" which was held in Brussels, Belgium, Sept 1996, indicates that Genistein suppresses osteoclastics function in vitro and in vivo at concentrations consistent with a tyrosine kinase mechanism, and has low toxicity 16. Another study presented at this symposium reviewed the effect of Genistein administration on bone loss in ovariecto­mized rats. The study showed that Genistein protected rats from bone loss associated with cessation of ovar-

522 INDIAN J EXP BIOL, JUNE 2001

ian function 17. Studies published later supported this effect1s. In a study conducted with postmenopausal women were given daily soy supplements. After six months, the women consuming soy isoflavones had significant increase in bone mineral content and den­sity in their lumbar spines, compared with the control group. Similar trends were noted for other skeletal areas 19.

Studies on various experimental animal models in­dicate that Genistein has a direct inhibitory effect on bone resorption (assimilation) in tissue culture2o. Other studies showed that Genistein and Genistin have an anabolic effect on bone metabolism in elderly rats and Genistein reduces both trabecular and com­pact bone loss and that this protective effect differs from that of estrogen, since it depends on stimulation of bone formation rather than on suppression of bone resorption21 . Given the promising results in animal models, one study investigated the effectiveness of soy diet rich in isoflavones in modulating the bone loss in estrogen-deficient, postmenopausal women. The results indicate significant increases in both bone mineral content and bone mineral density in the lum­bar spine22. Other studies showed that isoflavones are also estrogenic enough to promote bone formation. In fact, the European drug-Ipriflavone, used to treat osteoporosis, is a synthetic isoflavone; Daidzein is one of its metabolites23.

More recently, John J.B. Anderson published a re­view, which covered published reports of epidemiol­ogical and experimental studies of humans, animal models, isolated tissues and cells in structure, con­cluded that isoflavones (particularly Genistein and Daidzein), at optimal doses, result in improved bone mass24.

Genistein and Heart Disease Although progress has been made in prevention

and treatment of cardiovascular disease, it remains the number one killer of men and women in the United States, accounting for more than 40 percent of all deaths25. Of all the cardiovascular diseases heart dis­ease, primarily heart attack, has received the most attention. Atherosclerosis is a process in which mate­rial called plaque, builds up in an artery wall at the site of an injury, reducing blood flow. Complete blockage of this plaque-narrowed artery results in a heart attack. Atherosclerosis is present in most heart attacks. Susceptibility to atherosclerosis is determined by a combination of genetic and environmental fac-

tors, including diet, especially diet rich in cholesterol. Men are much more prone to atherosclerosis than women. Women are protected pre-menopausally at least by their estrogens and their death rate is 3.5-fold less. Lower incidence of heart disease has also been reported in populations consuming large amounts of soy products26. Lipoproteins are responsible for the transport of the major blood lipids and are classified as LDL cholesterol-low-density lipoproteins (main carrier of cholesterol), VLDL-very-Iow-density lipoproteins (the "bad" cholesterols), and HDL cho­lesterol-high density lipoproteins (the "good" cho­lesterol). The function of the LDL's is to carry most of the cholesterol in the body from the liver to the body's cells, where it has a vital role in providing cholesterol for incorporation into the membranes of new cells.

The role of HDL's is to return surplus cholesterol from the body's cells and also that in the arteries to the liver for disposal. This process protects the body from developing atherosclerosis by a process known as the reverse cholesterol transport process. Prevent­ing the oxidation of LDL-cholesterol is now viewed as an important process in the hypothesis known as the oxidative hypothesis of atherosclerosis. Only oxi­dized LDL-cholesterol is taken up by macrophages within the endothelial cells lining the arterial wall and ultimately contributing to atherosclerotic plaque for­mation. Although dietary soy protein is well recog­nized for its beneficial effects in the promotion of cardiovascular wellness27. Anthony and colleagues have conducted two studies with monkeys2s.29. In these studies soy protein rich in soy isoflavones fa­vorably affected serum lipid concentrations while soy protein from which the soy isoflavones had been ex­tracted had a minimal impact. These primate studies suggest that soy isoflavones may account for 60-70 percent of the effects of soy protein.

Isoflavones and Reduction in Cardiovascular Dis­ease via Several Distinct Mechanisms

Reduction of LDL cholesterol increasing HDL­cholesterol

In a recent study, sixty-six hypercholesterolemic postmenopausal women were given a diet with iso­lated soy protein containing high amount of isofla­vones. There was a significant reduction in LDL cho­lesterol and a significant increase in HDL cholesterol in the group receiving a protein diet with different

SUTHAR et at.: PHARMACOLOGICAL ACTIVITIES OF GENISTEIN: PART II 523

amounts of isoflavones compared with women con­suming pure isolated protein extract27

• During a 6-month intervention cross-over study, Anthony et al. fed peri pubertal monkeys a moderately athrogenic diet in which the protein was soy protein isolate either with the isoflavones Genistein and Daidzein intact or containing only the trace amounts still present after the isoflavones are removed29

•3°.When compared to

the soy protein diet, the soy protein with isoflavones significantly decreased LDL-cholesterol and VLDL­cholesterol by 30-40% in both males and females and significantly increased HDL-cholesterol in females. All sex hormones were normal, and at necropsy, it was found that the isoflavones had no adverse effects on either male or female reproductive systems. Crouse and his colleagues studied 156 patients with moderately elevated cholesterol levels who were ran­domly assigned to receive soy drink containing soy protein either with or without isoflavones. Another group of patients got beverage containing casein, the principal protein of cow's milk. Soy drinks containing isoflavones reduced both total cholesterol, and low­density lipoproteins. In patients who started with a high LDL cholesterol, the effects was even more dra­matic-a 10% reduction. Soy protein from which iso­flavones were removed by alcohol extraction had no such effece1

• Genistein may be able to protect LDL against oxidative damage. Studies examined the capa­bility of a range of isoflavones to enhance the resis­tance of LDL to oxidation. It was found that Genistein to be the most potent antioxidant both in the aqueous and in the lipophilic phases32.

Improving arterial elasticity 21 menopausal and premenopausal women were

given 80-mg daily isoflavones (45 mg Genistein) over lO-week periods33.Systemic arterial compliance (arte­rial elasticity), which declines with age in the women tested, improved significantly compared with the pla­cebo. It was concluded that the beneficial effect of the soy isoflavones on the arterial health of premeno­pausal and menopausal women improved to the "same extent as is achieved with conventional hormone re­placement therapy".

Beneficial effect on blood lipoproteins via estrogenic mechanism

Estrogens are known to protect against heart dis­ease in several ways including lowering the levels of LDL cholesterol and raising the level of HDL cho-

lesterol. The functions of estrogen help to explain the lower rate of heart disease in women before meno­pause. Genistein binds with the estrogen receptor (ER) beta, similar to the natural estrogen. The pre­dominance of ER beta in the cardiovascular system suggests that Genistein may be partly responsible for the lower incidence of heart disease in soy-consuming countries34

• A study showed that the isoflavones in soy improve cardiovascular disease risk factors with­out apparent deleterious effects on the reproductive system of peri pubertal monkeys35.

Inhibition of Cell Proliferation Genistein inhibits the tyrosine kinase enzymes,

which playa role in the arterial-wall changes that rep­resent the first step in the process of atherosclerosis. The effect of Genistein is more significant in hyper­lipdemic cells35

• Genistein can also inhibit cell adhe­sion, alter platelet activating factor and inhibits cell proliferation involved in atherosclerotic lesion forma­tion36

• Moreover, it also inhibits the activity of smooth muscle cell replication, which helps to prevent plaque formation on artery walls. A recent study in the Uni­versity of Washington, Seattle, found that Genistein selectively inhibited the growth of smooth muscle cells in arteries. Platelet-derived growth factors are believed to play an important part in the proliferation of smooth muscle cells in the atherosclerotic plaque37

•

Metabolism, Bioavailability and Toxicity of Genistein

As mentioned above, in soy foods, the aglycones Genistein and Daidzin are mainly present in the glu­coside forms Genistin and Daidzin, e.g. in soybean milk powder, only 4-5% of the isoflavones are in the form of aglycones. Absorption of soybean isoflavones may begin in the proximal small intestine. Lower down in the intestine, the gut bacterial de-conjugating enzymes hydrolyze the isoflavone glycosides to pro­duce Genistein and Daidzein. In addition, glucosi­dases of intestinal microflora in the lower bowel can liberate the aglycones, thereby promoting their ab­sorption. The intestinal microflora, however, also ex­tensively metabolize and degrade the isoflavones, thereb~ prohibiting their re-absorption from the lower bowel 8. After absorption, isoflavones are extensively transformed by phase II enzymes thus making their retention in the human body unlikell9

• Following transformation, isoflavones undergo urinary and bili­ary excretion38

• In a clinical study, Genistein was

524 INDIAN J EXP BIOL, JUNE 2001

shown to have relatively poor bioavailabilitl9.The considerable variation in Genistein bioavailability among seven female subjects in a similar study was attributed to the relative ability of gut microflora to degrade isoflavones4o• Despite the relatively low bio­availability of isoflavones, Genistein may still be ab­sorbed in sufficient quantities to exert potentially beneficial biological effects38

•39

• Since one-third of the world's population consume substantial amounts of soy, and hence Genistein, and have low rates of car­diovascular disease, it can be concluded that in hu­mans, Genistein is not toxic and may indeed be the cause of the lower risk of these diseases4o• The ad­ministration of upto 50 mg of Genistein in the form of one or more tablets is reported to have no significant toxicity and no or few side effects41

•

Conclusion It may be concluded that Genistein is potentially

beneficial in treating diseases such as menopause and osteoporosis as well as in the prevention of cardiovas­cular disease. In view of the multi-faceted benefits of this soy isoflavone, it may seem prudent to shift to­wards soy-rich diets. However, soy foods contain the b-glycoside, Genistin and conversion to the active aglycone form is dependent upon the activity of the gut microflora which in turn results in wide variations in the bioavailability of Genistein from soy products. Consequently, the administration of Genistein in the form of a standardized dietary supplement is a con­venient means of ensuring improved absorption with the resulting potential health benefits.

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