Phytoestrogens and the menopause

G.B.Lockwood,School of Pharmacy &

Pharmaceutical Sciences,University of Manchester,

Manchester, M13 9PL

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

Phytoestrogens

• Defined as plant constituents which bind to estrogen receptors, ER and ER

• Major classes include;Isoflavones, sources include soy, red clover and Phaseolus beansLignans, sources include flax & grainsStilbenes, one example is resveratrol Coumestans, 3 and 4 methoxycoumesterol

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

Soy

Soy isoflavones

OOH

HO O

OHgenistein

O

HO O

OHdaidzein

O

HO O

OHglycitein

H3CO

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

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

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

Trials included

No. +ve No. -ve

Reference

10 4 6 Huntley A. L; Ernst E. Soy for the treatment of perimenopausal symptoms--a systematic review. Maturitas (2004), 47, 1-9.

8 on soy foods,34-134mg/day

1 7 Krebs, E. E, Ensrud, K. E, MacDonald, R, Wilt, T. J. Phytoestrogens for treatment of menopausal symptoms: a systematic review. Obstetrics & Gynecology (2004), 104, 824-836. 

5 on soy extract, 50-150mg/day

2 2

8 3 5 Kronenberg F, Fugh-Berman A. Complementary and alternative medicine for menopausal symptoms: a review of randomized, controlled trials. Annals of Internal Medicine (2002) , 137, 805-13.

13 Overall benefit

Messina, Mark; Hughes, Claude. Efficacy of soyfoods and soybean isoflavone supplements for alleviating menopausal symptoms is positively related to initial hot flush frequency. Journal of Medicinal Food (2003), 6(1), 1-11.

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

Vaginal symptoms

• No beneficial effects on genital atrophy can be expected

• Vaginal dryness variably improved• Little data from clinical trials

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

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

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

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

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

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

HO O

OHequol

O

HO O

OHdaidzein

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

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

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

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

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

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

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

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

O

O O

Ipriflavone

O

HO O

OHdaidzein

Red Clover

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

Red Clover isoflavones

O

HO O

OMeformononetin

O

HO O

OMeBiochanin

OH

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

O

HO O

OMeformononetin

O

HO O

OMeBiochanin

OH

OOH

HO O

OHgenistein

O

HO O

OHdaidzein

Flaxseed

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

Flax lignans

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

Flaxseed lignan metabolism

• The importance of the metabolites, enterolactone and enterodiol have yet to be elucidated

• No pharmacokinetic data available

Trans-Resveratrol

OH

HO

HO

Trans-resveratrol

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

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

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

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