Phytoestrogens and the menopause

1. Phytoestrogens and the menopause G.B.Lockwood, School of Pharmacy & Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PL

2. Menopausal Symptoms • Vasomotor symptoms-mainly hot flushes, insomnia, heavy sweating, headaches, mood swings, irritability, depression • Vaginal dryness, soreness, loss of libido? • Osteoporosis • Breast cancer • Cardiovascular disease • Cognitive effects

3. Phytoestrogens • Defined as plant constituents which bind to estrogen receptors, ER and ER • Major classes include; Isoflavones, sources include soy, red clover and Phaseolus beans Lignans, sources include flax & grains Stilbenes, one example is resveratrol Coumestans, 3 and 4 methoxycoumesterol

4. Major phytoestrogens-widely available • Phytoestrogens are mainly used instead of HRT due to fears of links to breast cancer • Bind to estrogen receptors 0.001-100% activity of estradiol • Soy isoflavones-50mg/day based on presumed intake of Far Eastern populations • Soy products- soy milk/tofu mainly glycosides, miso/tempeh/soy sauce increasing aglycone composition • Red Clover isoflavones-  40mg • Flax Lignans- c40g flaxseed (20mg/day) • Resveratrol-15-200mg/day

5. Soy

6. Soy isoflavones

7. Soy for menopausal symptoms • Hot flushes are the major clinical symptom investigated • Epidemiological data-10-20% incidence of hot flushes in China/Japan, 70-80% in Western countries • Cause could be > Soy factor • < 1mg soy isoflavones in Western diets, 50-200mg in Japanese diets • A US survey revealed 7.4% of women used soy products for perimenopausal symptoms • 50-60% success of placebo in trials

8. Soy activity • Some phytoestrogens act as estrogen agonists, some antagonists-Concentration dependent? • Isoflavones have agonist effect in low estrogen environments, antagonist effect in high estrogen environment • Estrogen reduced at start of menopause, hence isoflavones have agonist effect • Antagonist effect due to competition with endogenous 17-estradiol

9. Rated success of Soy products in treating menopausal symptoms, mainly hot flushes

10. Variability in clinical trials of soy for menopausal symptoms • Different products and foods contain varying levels of isoflavones • Optimum dose is not known • Formulated products may not contain stated levels • Trial duration ranges from 1-4 months • Variability and deficiencies in reporting of outcomes

11. Vaginal symptoms • No beneficial effects on genital atrophy can be expected • Vaginal dryness variably improved • Little data from clinical trials

12. Osteoporosis • Reduction in bone density • Common in menopausal women • Reduction in estrogen at menopause causes increased osteoclastic bone resorption • Bone loss may continue for 5-10 years after menopause

13. Possible mechanism of action in osteoporosis • Prevention of calcium loss • Beneficial effects on osteoblasts • Influence on secretion of calcitonin which suppresses bone resorption • Genistein/daidzein suppress osteoblast activity in relation to bone turnover • May affect osteoblasts by mediating cytokine production in osteoblasts

14. Conventional treatments for osteoporosis • Women have higher incidence of osteoporotic fractures due to lower peak bone mass, and abrupt reduction in estrogen at menopause accelerates bone loss • HRT-not recommended for long term treatment • Inhibitors of bone turnover eg calcitonin, biphosphonates • Bone formation stimulating agents eg fluoride

15. Use of soy isoflavones for osteoporosis • Animal research consistently shows increase in bone mineral content (BMC) or bone mineral density (BMD) • Daidzein & genistein increase protein synthesis & alkaline phosphatase release by osteoblast cells in vitro • Epidemiological data show increased consumption of fermented products show lower osteoporotic bone fractures • Increased osteocalcin concentrations reported

16. Clinical data • Increased BMD at lumbar spine • Reduction in excretion of bone resorption markers eg pyridinoline • 54mg/day genistein reduce bone mineral loss at femoral neck and lumbar spine, as well as 1mg oestrogen • Lumbar spine BMD increases by 2.4% in equol producers (45% of postmenopausal women posses gut microflora capable of transformation of daidzein) • Calcium/Vitamin K2 present in soy products may act synergistically in osteoporosis • 50% reduction of osteoporotic fractures over 4.5 years in Chinese women (24,000 subjects) • Trials need to be 2-3 years as bone remodelling cycle can last 80 weeks

17. Significance of equol • Infants and germ-free animals do not produce it • Antibiotics inhibit equol production • 30-50% of population are equol producers • Equol is a non-steroidal estrogen • Equol binds to ER and ER similarly to genistein, greater than daidzein • Glycitein is not converted to daidzein, hence not to equol • Equol producers not identified in trials • S-enantiomer has affinity for ER

18. Breast cancer • HRT increases risk of breast cancer (1.3-2.4 times) over 5 years • Epidemiological evidence from Japan shows no link between isoflavone intake and breast cancer • In Australian women, increased urinary excretion of equol associated with reduced risk of breast cancer • Early and routine consumption is most beneficial • Soy isoflavones possibly stimulate breast cancers, particularly postmenopausally, correlations have been shown between oestrogenic effect, plasma prolactin levels, and breast cancer risk

19. Anti-cancer activity • Inhibition of DNA topoisomerase • Suppression of angiogenesis • Induction of differentiation in cancer cell lines • Induction of apoptosis • Genistein is a potent estrogen agonist and has cell growth inhibitory actions

20. Cardiovascular disease • Strong epidemiological evidence supports benefits, but diet may contribute • Soy protein reduces total cholesterol, LDL cholesterol and triglycerides • A meta analysis revealed that 34 out of 38 studies showed cholesterol reduction, but the roles of soy protein and isoflavones is not clear • Reduction in systolic blood pressure has been reported • Soy isoflavones have produced negative findings • Combinations of soy protein and isoflavones produce modest improvements • 45,694 Chinese women found systolic and diastolic BP reduced with 25g soy over 2-3 years

21. Cognitive function • Probably decreases due to decreased estrogen levels • Increased incidence of Alzheimer’s in postmenopausal women • One trial reported increase in verbal memory, but no effect in other indicators • 60mg/daily over 12 weeks was reported to increase memory, pattern recognition and mental flexibility • Significant improvements occur in males & young women taking 100mg/day over 10 weeks

22. Binding affinity of phytoestrogens for ER & ER Compound ER ER 17-estradiol 100 100 Genistein 4 87 Daidzein 0.1 0.5 Formononetin <0.01 <0.01 Biochanin A <0.01 <0.01 Ipriflavone <0.01 <0.01 Mode of action of soy isoflavones • Phytoestrogens require a flavonoid with 2-4 OH groups, methylation of these reduces oestrogenic activity. • Phytoestrogens bind to the oestrogen receptor (ER), predominantly ER and exert a weak oestrogenic effect, or anti-oestrogenic effect • Phytoestrogens may affect transcription of estrogen-regulated gene products • Phytoestrogens are antioxidants

23. Soy isoflavone distribution • Occur as glycosides, and hydrolysis, in the oral cavity and intestine, which allows absorption • >20 fold inter-individual variation in hydrolysis rate • 50mg isoflavone leads to 50-800 ng/ml in the plasma • Peak concentration 6-8 hours after 100mg dose • These levels are higher than normal plasma oestradiol levels • Isoflavones show less serum protein binding than oestradiol • Effects of the food matrix likely to be important

24. Ipriflavonesemi-synthetic isoflavone • Non-oestrogenic, mainly used in osteoporosis • Metabolised to daidzein and others • 200mg tds have been shown to produce statistically significant increases in BMD, and markers of bone metabolism • Increased calcium uptake in the duodenum • Some trials show no benefits • In one trial 13% developed subclinical lymphocytopenia

25. Red Clover

26. Red Clover • Lower isoflavone level than soy • 80mg isoflavone/day produced 44% reduction in hot flushes • Similar benefits to soy in osteoporosis • Reduced breast cancer, prostate cancer and ovarian cancer risks • Insignificant effects on lipoprotein levels

27. Red Clover isoflavones

28. Red Clover isoflavone distribution • Formononetin and biochanin A are efficiently demethylated to daidzein and genistein respectively • Peak concentrations of daidzein and genistein are 12 and 2 hours after a 40 mg dose of isoflavones • Formononetin is also converted to equol via demethylation to daidzein • Levels reported are c10% of those reported for equivalent doses of soy isoflavones

29. Flaxseed

30. Flaxseed • 40g flaxseed is as effective as HRT for mild menopausal symptoms (25g ineffective) • 25g Flax has a greater effect on estrogen metabolism than the same dose of soy • Urinary enterolactone positively correlated with BMD in Korean postmenopausal women • Urinary enterolactone was higher in Dutch women with greatest bone loss • Variable results in work on flaxseed supplementation on biochemical markers of bone metabolism

31. Flax lignans

32. Flaxseed • Reduction in LDL/HDL cholesterol levels • n-3-PUFAs, particularly -linolenic acid, in flax may have activity • 1.5g flax lignan/day gives a higher probability of intact cognitive function • Increased vascular compliance and induction of synaptogenesis in the hippocampus may be responsible

33. Flaxseed lignan metabolism • The importance of the metabolites, enterolactone and enterodiol have yet to be elucidated • No pharmacokinetic data available

34. Trans-Resveratrol

35. trans-Resveratrol • Physiological levels obtained from wine, up to 15mg/L • 10-200mg dosage forms available, no specified dosage • Antioxidant • Anticancer activity • Cardioprotective • Oestrogenic- binds to ER and ER receptors 1:7000 activity of estradiol • Increases in bone density in postmenopausal women have been reported

36. Quality • Soy isoflavones-10/15 failed, levels 30-99% • 300% variability between different soy food product types ie raw, soaked, cooked, drink, tofu • Soy infant formulas-up to 25% variability • Soy milk-70% variability • Tofu-50% variability • Phytoestrogens-28/32 failed, levels 0% and 383%, all products claiming genistein/daidxein failed • Red clover-Promensil passed • A number of products do not state levels of named constituents • No data on flax lignans or resveratrol

37. Adverse effects • Soy-gastrointestinal problems, supplement unpalatability (particularly soy drinks), nausea, allergy in one trial • In vitro genotoxicity with isoflavones • High level male tofu consumers suffered poor cognitive performance, lower brain weight! • High intake in animal studies suggests they may affect fertility • Adverse effects of HRT not reported in any of the trials (breast tenderness, vaginal bleeding) • Vaginal spotting • No reported adverse effects-Red clover/Flax

38. Conclusions • Soy, red clover, and flax are the main sources of dietary estrogens • Phytoestrogens may have oestrogenic or anti-oestrogenic activity depending on circulating levels of sex hormones, and may stimulate breast cancer • Generally safe-much epidemiological evidence • Other components may interfere with activity/bioavailability