(Food and nutritional components in focus)

In the past three decades there have been major advances in our understanding of the chemistry and function of nutritional components. This has been enhanced by rapid developments in analytical techniques and instrumentation. Chemists, food scientists and nutritionists are, however, separated by divergent skills, and professional disciplines. Hitherto this transdisciplinary divide has been difficult to bridge. The series Food and Nutritional Components in Focus aims to cover in a single volume the chemistry, analysis, function and effects of single components in the diet or its food matrix. Its aim is to embrace scientific disciplines so that information becomes more meaningful and applicable to health in general. The series Food and Nutritional Components in Focus covers the latest knowledge base and has a structured format. Isoflavones has four major sections, namely:

Food and Nutritional Components in Focus

IsoflavonesChemistry, Analysis, Function and Effects Edited by Victor R Preedy

Chemistry, Analysis, Function and Effects

Food and Nutritional Components in Focus

Series Editor:

Professor Victor R Preedy, School of Medicine, King’s College London, UK

Titles in the Series:

1: Vitamin A and Carotenoids: Chemistry, Analysis, Function and Effects2: Caffeine: Chemistry, Analysis, Function and Effects

3: Dietary Sugars: Chemistry, Analysis, Function and Effects

4: B Vitamins and Folate: Chemistry, Analysis, Function and Effects5: Isoflavones: Chemistry, Analysis, Function and Effects

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Food and Nutritional Components in Focus No 5

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In the past three decades there have been major advances in our standing of the chemistry and function of nutritional components This hasbeen enhanced by rapid developments in analytical techniques and instru-mentation Chemists, food scientists and nutritionists are, however, separated

under-by divergent skills, and professional disciplines Hitherto this plinary divide has been difficult to bridge

transdisci-The series Food and Nutritional Components in Focus aims to cover in a singlevolume the chemistry, analysis, function and effects of single components in thediet or its food matrix Its aim is to embrace scientific disciplines so thatinformation becomes more meaningful and applicable to health in general.The series Food and Nutritional Components in Focus covers the latestknowledge base and has a structured format

Isoflavones has four major sections, namely:

Isoflavones in Context

Chemistry and Biochemistry

Analysis

Function and Effects

The first section covers phytoestrogens in health and plants, then material onisoflavones in foods and the diet The chemistry and biochemistry sectioncovers structures, bioconversion and biotransformation, the human estrogenreceptor, homonuclear NMR spectroscopy, genistein, daidzein, xenoestrogens,methylated derivatives and non-natural isoflavonoids The section on analysisincludes foods, beverages, nuts, traditional medicines, herbs, pharmacologicallyactive isoflavones, plasma and urine Methodology encompasses microwave-extraction, HPLC, LC-MS/MS and LC-UV/PDA and many other techniques.Finally, the section on function and effects covers ingestion of isoflavones by

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Edited by Victor R Preedy

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v

different populations, isoflavones in beverages, inherited metabolic diseases,mucopolysaccharidoses, clinical trials, obesity, inflammation in adipose tissue,menopausal vasomotor syndrome, estrogenic activity, testicular function,thyroid function, gastric cancer, learning and memory, prenatal exposure, cellproliferation, bone, NMDA and GABA receptors, and insulin secretion.Individual isoflavones are also described, such as daidzein, genistein and gly-citein, as well as their derivatives, such as equol and tetrahydroxyisoflavone Asisoflavones occur within a complex plant matrix, other estrogenic and bioactivecompounds in isoflavones-rich foods, e.g coumestrol, are described for com-parative reference.

Each chapter transcends the intellectual divide with a novel cohort of tures namely by containing:

fea- Abstract

 Summary Points

 Key Facts (areas of focus explained for the lay person)

 Definitions of Words and Terms

It is designed for chemists, food scientist and nutritionists, as well ashealthcare workers and research scientists Contributions are from leadingnational and international experts, including contributions from world-renowned institutions

Professor Victor R PreedyKing’s College London

Isoflavones in ContextChapter 1 Phytoestrogens in Health: The Role of Isoflavones 3

Rodney J Baber

1.2 Absorption, Metabolism and Excretion of Isoflavones 4

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2.3 Chemical Features of Plant Isoflavones 17

2.6 Biological Function of Isoflavones in Plants 222.6.1 Interaction of Isoflavones with

Micro-organisms of the Rhizosphere 222.6.2 Isoflavones as Phytoalexins and Phytoanticipins 22

Chapter 3 Isoflavones in Foods and Ingestion in the Diet 28

Baskaran Stephen Inbaraj and Bing Huei Chen

3.4 Growth, Variety, Environmental and Post-harvest

Storage Conditions Affecting Isoflavone Composition

Kashif Ghafoor, Fahad Y Al-Juhaimi and Jiyong Park

Chapter 5 Chemistry and Synthesis of Daidzein and its Methylated

Derivatives: Formononetin, Isoformononetin, and

Namita Bhan and Mattheos Koffas

6.2 Phenylpropanoid and Isoflavonoid Pathways 83

ixContents

6.4 Non-natural Isoflavonoids 856.4.1 Semi-synthesis of Non-natural Isoflavonoids 856.4.2 Chemical Synthesis of Non-natural Flavanones 86

Chapter 7 The Structure of Isoflavones by 1D and 2D Homonuclear

Kristiina Wa¨ha¨la¨, Somdatta Deb and Tapio Hase

Chapter 8 Biotransformation and Transfer of Genistein: a Comparison

with Xenoestrogens and a Focus on the Human Placenta 115Hsiu-Wen Chan, Greg E Rice and Murray D Mitchell

8.1 Biotransformation of Genistein Compounds in the

9.1 Genistein and its Biological Activities 131

Francesco Squadrito and Alessandra Bitto

Chapter 11 Isoflavones and Human Estrogen Receptor: When Plants

Patricia de Cremoux and Yves Jacquot

11.2 From Plant Biosynthesis to Mammalian Biosynthesis 158

xiContents

11.2.1 Biosynthesis of Isoflavonoids in Leguminous

11.2.2 Biosynthesis of Isoflavonoids in ERs 16311.3 Interaction of Isoflavonoids with ERs 16311.4 Mammalian Metabolism and Bioavailability 16511.5 Biochemical and Physiological Functions of

Dorin Boldor and Cristina Mirela Sabliov

12.3 Microwave-assisted Extraction Process 18312.4 Oil Separation and Isoflavone Purification 183

12.5.2 Influence of Time and Temperature

on the Oil and Isoflavones Extraction

Chapter 13 Isoflavones: High-performance Liquid Chromatographic

Analysis of Glucuronic Acid- and Sulfuric Acid-conjugated

Metabolites of Daidzein and Genistein in Human Plasma

13.2.1 Selective Enzymatic Hydrolysis 19813.2.2 Fractionation by Ion-exchange

Chapter 14 High-throughput Quantification of Pharmacologically Active

Wahajuddin and Sumit Arora

Chapter 15 Methods for Isoflavones: A Focus on Beverage Analysis 244

Rita C Alves and M Beatriz P P Oliveira

Definitions of Words and Terms 260

Chapter 17 Isoflavones: LC-MS/MS Profiling of Isoflavone Glycosides

Piotr Kachlicki and Maciej Stobiecki

17.5 Differentiation of Isoflavone and Flavone

Glycoconjugates with Instrumental Methods 28517.6 Differentiation of C-Glycosides and O-Glycosides of

List of Abbreviations 292

Lei Wan and Chia-Hung Lin

Chapter 19 Pattern Profiling and Quantitative Determination of

Isoflavones in Herbal Chemotypes using Liquid

Lakshmi Manickavasagam, Smriti Mishra and

Girish Kumar Jain

19.2 Herbal Preparations for Osteoporosis 31719.2.1 Pattern Profiling and Quantitative Analysis

19.2.2 Osteogenic Herbal Fractions and

19.3 LC-MS/MS Method for Qualitative and

Quantitative Analysis of Herbal Fractions 319

19.3.2 Mass Spectrometric Conditions 32119.3.3 Validation of the LC-MS/MS Method 32219.3.4 Pattern Profiling and Quantitative Analysis

xvContents

20.3.3 Isolation and Purification of a Novel

Isoflavone from Groundnut Extract 33520.3.4 Content of a Novel Isoflavone in Groundnuts 335

20.4.1 Isoflavone Profile of Groundnuts 33620.4.2 Influence of the Extraction Temperature on

the Isoflavone Composition of Groundnut

20.4.3 Purification of a Novel Isoflavone (Peak-1)

20.4.4 Structural Analysis of a Novel Isoflavone

20.4.5 Content of a Novel Isoflavone from the

21.2 Isoflavone Ingestion by Multiethnic Populations 350

21.2.4 China and Hong Kong 354

Rita C Alves and M Beatriz P P Oliveira

23.4.3 Isoflavone Action on GAG Synthesis and

Chapter 24 Optimizing Isoflavone-rich Food Delivery Systems for

Jennifer Ahn-Jarvis, Steven Schwartz and Yael Vodovotz

24.2.1 Preparation of Fermented and Thermally

24.2.2 Soy-almond Bread (SAB) Preparation 403

24.2.5 Characterization of Organoleptic Properties 406

24.3.1 Process of Selecting the Optimal

Chapter 25 Isoflavones and Thyroid Function: An Overview 423

Francesco Squadrito and Alessandra Bitto

25.1.1 Goitrogenic Effects of Flavonoids 425

25.2.1 Genistein and Thyroid Function 426

Chapter 26 Isoflavones against Gastric Cancer: Function and Effects 438

Sue K Park and Kwang-Pil Ko

27.2 Pre-clinical Studies of the Deleterious,

Endocrine-disrupting Effects of Dietary Isoflavones

27.3 Pre-clinical Studies of the Beneficial Effects of

Dietary Isoflavones in Rodent Models of

27.4 Pre-clinical Studies of Beneficial Effects of Dietary

27.5 Human Clinical Studies of Beneficial Effects of

Dietary Isoflavones on Learning and Memory 457

List of Abbreviations 461

Brian R Stephens and Joshua A Bomser

28.6 Glycitein as an Estrogen Receptor (ER)

29.3 Equol: Biosynthesis (Plants and Mammals),

Antioxidant Activity, and Safety Data in Postnatal

Chapter 30 Genistein: GABAAand NMDA Receptors 500

Renqi Huang and Glenn H Dillon

30.2 GABAAReceptor Function and Structure 501

30.3 Genistein Modulation of GABA Receptors 50330.3.1 Genistein as an Inhibitor of Protein Tyrosine

30.3.2 Direct Inhibition of GABAAReceptors 50430.4 NMDA Receptor Function, Structure and

30.5 Genistein Modulates NMDA Receptor Modulation 50830.5.1 Genistein as an Inhibitor of Protein Tyrosine

Chapter 31 Estrogenic Activity and Molecular Mechanisms

Kenneth Ndebele, Barbara Graham and Paul Tchounwou

Chapter 32 Genistein and Insulin Secretory Function 529

Dongmin Liu

32.2 Genistein may have Anti-diabetic Effects 53032.3 Genistein at Physiological Concentrations AugmentsGlucose-stimulated Insulin Secretion (GSIS) in

Chapter 33 Prevention and Management of Obesity by Isoflavones 541

Barbara B Doonan, Erxi Wu and Joseph M Wu

33.2 Evidence that Dietary Factors and Soy Isoflavones

33.2.1 Introduction to Soy Phytochemicals and

33.3 Identification and Mechanism of Soy Isoflavones

33.3.1 Modulation of Fat Deposition and

33.3.2 A Facile Strategy for the Identification of

Soy Isoflavones with Potential for

33.3.3 Discovery of Proteins Targeted by Soy

Isoflavones with a Regulatory Role in

33.3.4 Candidate Molecular Targets and

Mechanisms of Anti-obesity Isoflavones:

Interplay Between NQO1 and p53 in

33.3.5 Role of PPARa, PPARg, C/EBPa and Other

Candidate Molecular Sensors/Effectors of

Definitions of Words and Terms 556

34.3 Hormonal Regulation of Reproductive Tract

34.5 Isoflavone Action in Testicular Cells 566

34.5.3 Effects on Developing versus Mature Stages

34.7 Isoflavone Action in the Human Testis 572

Zhong Li, Caiyun Zhong and Chunyan Hu

35.2 Equol and Hormone-positive Cell Proliferation 58135.2.1 Equol Binds to ERs with a Greater Affinity

35.2.2 Estrogenic Activity and Breast Cancer 581

35.2.4 Equol and the Reproductive System 586

35.3 Equol and Other Cancer Cell Proliferation 586

xxiiiContents

35.9 Equol and Drug Metabolism 590

Marina Komrakova, Ewa Klara Stuermer,

Klaus Michael Stuermer and Stephan Sehmisch

36.4 Bone and Purified Genistein, Daidzein and Equol 600

Chapter 37 Isoflavones and Inflammation in Adipose Tissue and

Maria Teresa Blay, Montserrat Pinent and Anna Arde´vol

37.1 An Introduction to Inflammation in Adipose Tissue 61137.2 An Overview of Isoflavones as Anti-inflammatory

37.2.1 Isoflavones can Modulate Proinflamatory

Factors in Macrophages and Adipocytesand Down-regulate Gene Expression of

37.2.2 Isoflavones can Modulate Proinflamatory

Factors in Adipose Tissue by a Decrease inAdipose Tissue Mass and/or Number 61737.3 What We Know from Animal Models and

Isoflavone Effects on Adipose Tissue Inflammation

37.3.1 The Effects of Isoflavones in Animal

Models of Obesity: Effect of Isoflavones inRats with High-fat Diet (HFD)-inducedObesity/Insulin Resistance and the Analysis

37.4 What We Know from Human Studies and the Effects

of Isoflavone on Adipose Tissue Inflammation 62137.4.1 Studies in Obese Postmenopausal Women 62137.4.2 Studies with Genistein (Gen) in Human Cell

Chapter 38 Isoflavones for Menopausal Vasomotor Syndrome 627

Rafael Bolan˜os Diaz and Juan Carlos Zavala Gonzales

Isoflavones in Context

et al.2007) These compounds have a steric structure similar to that of steroidalestrogens, allowing them to bind to the human estrogen receptor (ER) and theyare therefore capable of exerting various estrogenic or anti-estrogenic effects(Duncan et al 2003) Isoflavones bind with a greater affinity to ERb than toERa (Kuiper et al 1996) The binding affinity of isoflavones for ERs has beenestimated to be between 10–2and 10–4 of that of 17b-estradiol (Collins et al.1997; Miksicek 1994), however, these substances can be present in the blood atlevels up to 10 000 times that of steroidal estrogens (Adlercreutz et al 1991).Over 10 000 peer-reviewed papers have been published on the role of iso-flavones in health Amongst commonly consumed foods, isoflavones are found

in physiologically relevant amounts in soybeans and foods derived from soy

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(Adlercreutz and Mazur 1997) The greater reliance on vegetables andparticularly legumes such as soy for dietary protein in Asian and CentralAmerican communities means that those communities typically havesubstantially higher dietary isoflavone intake than those found in Westerncountries (Goldin et al 1986).

Soybeans contain three primary isoflavones in their glycoside form: genistin,daidzin and glycitin Digestion leads to the cleavage of the sugar moiety and theformation of the respective aglycones: genistein, daidzein and glycitein Redclover has also been used to manufacture supplements for human use Redclover contains four isoflavones: formononetin, biochanin, daidzein and gen-istein At physiological concentrations, formononetin does not bind to the ERbut is metabolized to daidzein and then by intestinal bacteria to equol, both ofwhich have been shown to have estrogenic properties (Baber 2010)

Interest in these compounds began over 20 years ago with research by the USNational Cancer Institute exploring possible chemo-preventive properties forphytoestrogens, especially in regard to breast cancer Ongoing research led tofurther investigation of these compounds in relation to cardiovascular andskeletal health, cognitive function and the alleviation of menopausal symptoms.Despite continuing research, the role of isoflavones in health remainscontroversial, with concerns expressed that the estrogen-like effects of thesecompounds may pose a risk to certain individuals, especially women withbreast cancer or at high risk of that disease Clinical trials, although numerous,have typically been small, short, of variable quality, have tested different (andoften impure) substances and have chosen different endpoints to measureusefulness, efficacy and safety

There are substantial inconsistencies between results from Asian logic studies, which are generally viewed as supportive of health benefits, andWestern clinical trials, which are much less so The epidemiologic data arebased on the intake of traditional soy foods, such as tofu, miso and soymilk,whereas the intervention trials have usually utilized isolated soy protein and soy

epidemio-or isoflavone supplements The outcome differences may be due to differencesbetween traditional isoflavone-rich foods and prepared isoflavone supplements,

to individual differences in absorption of isoflavones, the timing and duration

of exposure to isoflavones and ethnic differences or to a healthy user effect

of Isoflavones

Isoflavone aglycones are absorbed in the upper small intestine by passive fusion, peaking in the blood within 1 h of being ingested (Sfakianos et al 1997)

dif-In contrast, the b-glucosides are not passively absorbed They are hydrolyzed

by b-glucosidases from intestinal bacteria or an intestinal enzyme, phlorizin hydrolase Isoflavone aglycones are converted into their b-glucur-onides by UDP-glucuronyltransferases in gut mucosal cells (King et al 1996)and to a lesser extent to sulfate esters catalyzed by 30-phosphoadenosine

50-phosphosulfate (PAPS)-sulfotransferases (Ronis et al 2006) tion and sulfation also occur in the liver These phase II metabolites areexcreted in the bile and are deconjugated in the lower bowel, allowing them to

Glucuronida-be reabsorGlucuronida-bed again, creating an enterohepatic circulation (Sfakianos et al.1997) Daidzein is metabolized to dihydrodaidzein, which is further metabo-lized to equol and O-desmethylangolensin (O-DMA) Genistein is transformed

to dihydrogenistein and then metabolized to 6-hydroxy-O-DMA Humanurinary excretion of these metabolites is variable, and only approx 30–40% ofsubjects excrete significant quantities of equol after isoflavone consumption(Kurzer et al 1997) The same is not true of animals in which equol excretionappears quite consistent Based on many studies, the consensus is that only25–30% of the adult population of Western countries excrete equol when fedsoy foods This is significantly lower than the reported 50–60% frequency ofequol-producers in adults from Japan, Korea or China, or in Western adultvegetarians This regional or ethnic difference in equol production has led tothe hypothesis that equol production is necessary for an individual to derivethe predicted health benefits from isoflavone consumption (Setchell andClerici 2010)

The ability of Isoflavones to bind to to mammalian ERs has been known forover 40 years, although, compared with 17b-estradiol, isoflavones have approx

100 times weaker affinity (Kuiper et al 1997)

A greater understanding of estrogen action began in 1996 with the discovery

of ERb (Kuiper et al 1996) Although related to ERa, which is located onchromosome 6, ERb is located on chromosome 14 The ligand-binding sites arehighly homologous between ERa and ERb However, the few amino aciddifferences result in isoflavones exerting preferential binding affinity to ERb.ERa and ERb are expressed at various concentrations in different organs anddifferent cell types This has led to the development of compounds, known asselective estrogen receptor modulators (SERMs), which selectively targetreceptors and which may be antagonistic in some and agonistic in others Somehave suggested that isoflavone binding to ERb is an antagonistic process, not

an agonistic one, and that hence isoflavones may be ‘natural SERMs’ thews and Gustafsson 2003) The mechanism of action of isoflavones may thus

(Mat-be estrogenic or anti-estrogenic in different tissues depending upon the centrations of ERa and ERb, the concentration of the isoflavone and theconcentration of endogenous sex hormones Isoflavones also have non-genomicactivity Genestein, in vitro, will inhibit the activity of tyrosine protein kinase(Akiyama et al 1987) Genistein also affects genes involved in the control of cellgrowth via effects on natural killer cell function, as well as enzyme inhibitionand the peroxisome proliferator regulator (Sarkar and Li 2003) Overall, evi-dence suggests that many pathways, not just estrogen-dependent events,mediate the biological effects of isoflavones and their metabolites Future

con-5Phytoestrogens in Health: The Role of Isoflavones

studies may utilize data that have been obtained in DNA microarray ments Lastly, studies have suggested that some of the benefits of dietary iso-flavones observed in other populations may depend on early life exposure(Korde et al 2009), which may involve their impact on gene expression at anepigenetic level.

1.4.1 Cardiovascular Health

Isoflavones may exert an effect on the cardiovascular system by three majormechanisms:

(1) directly through ER-mediated effects;

(2) through ER-independent effects directly on cardiovascular risk factorsand putative atherogenic risk factors;

(3) indirectly through the displacement of animal protein intake

Isoflavones exert estrogenic and anti-estrogenic effects (Kuiper et al 1998) Inanimal models, isoflavones have been shown to require the presence of the ER toexert anti-atherogenic efffects The vascular endothelium is a rich site of ERbexpression and the preferential binding of isoflavones for ERb suggests they mayexert anti-atherogenic effects in vascular tissue (Makela et al 1999) However,binding affinities for the ER do not explain the functional complexity of iso-flavones For example, although genistein and equol have a 20-fold greater affinityfor ERb than ERa, the transcriptional expression is greatest for equol relative toall other isoflavones The contribution of the protein portion of soy versus theisoflavone portion to the cardiovascular effects reported also remains unclear

At present any cardiovascular effects of isoflavones are thought to be related

to their effects on lipid metabolism Results are awaited from trials examiningthe effects of isoflavones on progression of atherosclerosis and whether thecapacity to convert daidzein into equol confers greater cardiovascular benefits

No randomized clinical trials examining clinical endpoints of cardiovascularhealth are available Therefore, although there is some evidence of a beneficialeffect of isoflavones on lipids and vascular function, and while a healthy dietshould be encouraged as a general health measure, it is not appropriate torecommend phytoestrogen supplements or diets as a primary cardiovasculardisease preventive intervention

1.4.2 The Brain

Both ERa and ERb are abundantly expressed in brain and exhibit a pattern ofdistribution consistent with their roles in reproductive and cognitive function(Spencer et al 2008) As expected, ERa occurs in brain regions involved inregulation of reproduction but both occur, particularly ERb, in brain regionsinvolved in cognition The expression and localization of ERs are dynamic and

can vary depending upon brain region, cell type, hormonal status and logical condition The effects of isoflavones in the brain may be due to genomic

neuro-or non-genomic actions Some clinical trials also point to different effects oncognition in women depending on the age since the menopause, a situationanalogous to the ‘critical window’ hypothesis for the effects of postmenopausalestrogens on the cardiovascular and cognitive health of women

1.4.3 Bone Health

A number of genomic and non-genomic mechanisms exist by which trogens may affect bone metabolism Isoflavones increase the synthesis ofvitamin D in a number of non-renal cell types, have been shown to stimulatecalcium uptake in bone and to increase bone cell proliferation and differ-entiation in animal studies These effects may lead to improved bone healthconsistent with data on postmenopausal estrogen therapy and bone health(Writing Group for the Women’s Health Initiative Investigators 2002) Morethan 25 studies have examined the effects of isoflavones on bone mineraldensity (BMD) in postmenopausal women most, as usual, being small andshort A 2-year trial in postmenopausal osteopenic women given 54 mg day–1genistein found that spinal and hip BMD increased significantly compared withplacebo (Marini et al 2008) However, two long-term trials (Brink et al 2008;Vupadhyayula et al 2009) showed no significant effect Possible explanationsfor this inconsistency include chronological differences in exposure (Asian adultsoy intake assessed in the epidemiologic studies may reflect lifelong intake) andthe ability to convert daidzein into equol There may be a critical dose ofisoflavones required to achieve an effect on BMD, and there may be differences

phytoes-in bioavailability between various supplements, foods and purified compounds.Consequently, it remains unclear whether isoflavones have a beneficial effect onBMD and, importantly, no randomized trials have demonstrated efficacy ofphytoestrogens on fracture prevention (Baber 2010)

1.4.4 Breast Cancer

Isoflavones have been identified as putative chemopreventives (Messina 2010).Most research interest has focused on breast and prostate cancer because of thedifference in incidence of these cancers in Western and Asian communities.Isoflavones may alter the metabolism of endogenous estrogens, potentiallyproducing indirect effects on estrogenic pathways Other mechanisms by whichisoflavones may be cancer protective include antiproliferative effects, tyrosinekinase inhibition, modulation of steroid hormone-metabolizing enzyme activ-ity, induction of apoptosis and inhibition of angiogenesis Dietary isoflavonesalso reduce circulating and intra-breast estradiol concentrations in monkeys,with a corresponding decrease in uterine and breast tissue proliferation(NAMS 2011)

A recent meta-analysis (Wu et al 2008) found that high soy intake wasassociated with an odds ratio (OR) of 0.71 [95% confidence interval

7Phytoestrogens in Health: The Role of Isoflavones

(CI)¼ 0.60–0.85] for breast cancer However, there is an intriguing body ofevidence suggesting that to derive protection against breast cancer, soy con-sumption must occur during childhood and/or adolescence (Messina andHilakivi-Clarke 2009) The current operating hypothesis for this observation isthat isoflavone exposure stimulates differentiation of breast tissue leading to areduction in the anatomical structures that give rise to cancer cells.

1.4.5 Prostate Cancer

A meta-analysis (Yan and Spitznagel 2009) reported a combined relative risk/OR

of 0.74 (P¼ 0.01) for prostate cancer when comparing high with low soy intake.Animal studies generally show isoflavone-containing diets retard the develop-ment of prostate cancer (Pollard and Suckow 2006) There is also evidence thatisoflavones inhibit prostate tumour spread via the non-genomic mechanisms

1.4.6 Menopausal Symptoms

Adlercreutz et al (1992) suggested that the low prevalence of hot flushes inJapanese postmenopausal women might be partially due to their highconsumption of soy foods It was speculated that the estrogen-like effects ofisoflavones might mitigate the drop in estrogen levels This hypothesis encour-aged the development of a vast complementary therapy industry marketingvarious mixtures of isoflavones, sometimes combined with other botanicals, astreatments for menopausal vasomotor symptoms More than 50 clinical trialsevaluating the efficacy of isoflavone-containing products have been conducted.Most trials have failed to show a significant benefit (Baber 2010; Lethaby et al.2007) and there is increasing evidence that women who lack the capacity toconvert daidzein into equol may derive little benefit Placebo response rates inclinical trials of interventions to alleviate vasomotor symptoms typically show a40–50% response (Baber 2010) and if only 30% of isoflavone users are able toconvert them into the more estrogenic equol, it is not surprising that results havebeen disappointing To test whether conversion into equol is critical, a trial wasconducted on 96 healthy postmenopausal women (Jou et al 2008) Volunteerswere randomized to the isoflavone or placebo group The isoflavone group wasfurther divided into equol producers (n¼ 34) and non-producers (n ¼ 32) based

on urinary equol levels after consuming 135 mg of isoflavones day–1for a week.Equol producers showed significantly greater reduction in some categories ofKupperman menopausal symptom scores than the placebo group

Most concern regarding high level isoflavone consumption has focused on theestrogenicity of isoflavones and potential harm when used by breast cancerpatients and women at high risk of developing breast cancer Although iso-flavones in vitro may stimulate breast cancer cells, observational studies havefound that isoflavones do not adversely affect markers of breast cancer risk,

including breast tissue density, breast cell proliferation or circulating estrogenlevels, and epidemiologic studies report the consumption of traditional soyfoods after a diagnosis of breast cancer has no effect on or improves prognosis(Baber 2010; Guha et al 2009; Shu et al 2009) Isoflavones may affect thyroidfunction in patients receiving thyroid replacement therapy, but this effect is due

to an impact on drug absorption rather than a direct effect on thyroid function(Villar et al 2007)

A Cochrane Review found no adverse effects of phytoestrogens on metrial pathology for up to 1 year (Lethaby et al 2007) However, a 5-yearplacebo-controlled study of the effects of soy isoflavone (150 mg daily) onendometrial tissue in 376 women found isoflavone use increased the incidence

endo-of endometrial hyperplasia (Unfer et al 2004) The increased incidence wassmall and there were no cases of endometrial cancer identified These findingshave not been confirmed by other studies and hence the long-term endometrialsafety of high doses of phytoestrogen supplements is not fully established

Summary Points

 Isoflavones are diphenolic plant chemicals structurally similar to estradiol, which bind selectively but weakly to mammalian ERs with apreference for ERb

17b- Isoflavones are capable of exerting receptor-mediated estrogenic and estrogenic effects, as well as non-genomic effects

anti- The effects on human health predicted from epidemiological studies ofAsian communities consuming substantial dietary isoflavones have notbeen born out in clinical trials using dietary supplements containingequivalent amounts of isoflavones on Western populations

 The discord seen between these results may be due to a number of factors:

 Lifelong, or at least pre-pubertal, exposure may be required to manifestsome biological effects

 It may be that biological effects achieved from whole food sources ofisoflavones are different to those achieved with the use of extracts orsupplements

 The effects attributed to isoflavones may be due to some as yet unidentifiedsubstance found in popular dietary sources of isoflavones

 Biological effects may only be seen in those people who are capable ofconverting isoflavones into equol

Key Facts

Key Facts for Isoflavones

 Isoflavones are diphenolic compounds of plant origin, which bind weaklybut selectively to mammalian ERS Isoflavones exert biological effects viagenomic and non-genomic mechanisms

9Phytoestrogens in Health: The Role of Isoflavones

 Epidemiological studies in Asian communities, which consume substantialquantities of dietary isoflavones, suggest a beneficial effect of isoflavones

on cardiovascular health, menopausal vasomotor symptoms and somehormone-dependent cancers

 The promise of these benefits has not been born out in clinical trials onWestern populations

 This may be due to timing and duration of exposure, the type of flavones ingested, variable absorption, equol-producer status or otherfactors Further research is required

iso-Key Facts for Estrogen Receptors

 ERs are hormone receptors, which are activated by the hormone estradiol and potentially by compounds of similar stearic structure Thereare at least two estrogen receptors: ERa and ERb

17b- ERs are predominantly cytoplasmic receptors in the un-liganded state,although a portion resides in the nucleus

 Although widely distributed, ERa is preferentially expressed in trium, breast, ovarian stroma and hypothalamus, and ERb in kidney,brain, bone, heart, lungs and mucosa

endome- 17b-Estradiol binds equally well to both receptors Estrone and raloxifenebind preferentially to the a receptor, whereas estriol and isoflavones, such

as genistein, bind preferentially to the b receptor

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