AU777653B2 - Health supplements containing phyto-oestrogen, analogues or metabolites thereof - Google Patents

Description

Our Ref:7635600 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Novogen Research Pty Ltd 140 Wicks Road North Ryde New South Wales 2113 Australia DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Address for Service: Invention Title: Health supplements containing phyto-oestrogen, analogues or metabolites thereof The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 r 1 HEALTH SUPPLEMENTS CONTAINING PHYTO-OESTROGENS. ANALOGUES OR ME.

TABOLITES THEREOF TECHNICAL FIELD This invention relates to natural products containing phyto-oestrogens, or phyto-oestrogen metabolites, which have various beneficial physiological effects in man, and which have a variety of uses, such as to promote good health and as a dietary additive, for example.

BACKGROUND ART The particular product in accordance with the invention is an extract of certain plants with the particular purpose of enrichment for phyto-oestrogens, both in their natural state and their closely related derivatives and metabolites.

Plants which are used as foodstuffs or medicinal herbs contain a wide variety of chemicals which are assimilated into the body following ingestion. Some of these chemicals are important nutrients for man and animals fats, carbohydrates, proteins, vitamins, minerals) while others have none, or little or no known nutntional value The phyto-oestrogens hitherto have fallen into this latter category of no known nutritional value.

There are 3 principal classes of phyto-oestrogens, viz. isoflavones, lignans, and coumestans.

The isoflavones are thought to have a broad range of biological functions in plants, although Sthese are poorly understood. However, two particular functions are recognised as phytoalexin or stressor chemicals which are secreted by the plant in response to attack by parasites such as insects, fungi, viruses, etc and which display activity against these parasites, and (b) chemicals which encourage colonisation of nitrogen-fixing bacteria on the roots of legumes.

The biological functions in plants of the lignans and coumestans is not generally understood.

The different types of phyto-oestrogens are as follows.

Type 1 phyto-oestrogens (isoflavones) S" Isoflavones appear to be widely distributed in the plant kingdom and over 700 different isoflavones are described. However, the isoflavones which display oestrogenic activity belong to a small sub-group and are restricted almost exclusively to the Leguminosae family. The known oestrogenic isoflavones are daidzein, formononetin, genistein and biochanin A. In common human foodstuffs such as soya, chickpeas, lentils and beans, the total levels of the oestrogenic isoflavones range between about 40 and 300 mg per 100 g dry weight.

In the raw plant material, isoflavones occur principally as glycosides. Following ingestion by man and animals, the glycoside moiety is hydrolysed free by a combination of gastric acid hydrolysis and fermentation by intestinal bacteria Some of the isoflavones in the aglucone form are absorbed directly and circulate in the blood, while the remainder are metabolised by intestinal 'ermentation to a variety of compounds which are also absorbed. The absorbed isoflavones and their metabolites appear to undergo little or no further metabolism in the body, being readily transported in the bloodstream, and ultimately being excreted in the urine.

Type 2 phyto-oestrogens (lignans).

Lignans are widely distributed in the plant kingdom. Over one hundred lignans are described and they are reported in common human foodstuffs such as cereals, fruits and vegetables.

ilseeds such as flax (lseed) have te highlest known levels at 20-60 mgO1n g dry weight, while cereals and legumes have much lower levels at 0.3-0.6 mg/100 g, and vegetables even lower levels at 0.1-0.2 mg/100 g. The most common lignan described is metairesinol. Dietary lignans also appear to be metabolised fairly efficiently within the gut by bacterial fermentation, yielding metabolites such as enterodiol and enterolactone which are absorbed into the bloodstream and excreted in the unne.

Type 3 phyto-oestrogens (coumestans).

Compared to isoflavones and lignans, oestrogenic coumestans appear to have a relatively restricted distribution in plants and generally occur at much lower levels. Alfalfa, ladino clover and some other fodder crops such as barrel medic may have significant levels and have been reported to cause reproductive dysfunction in grazing animals. In the human diet, the important sources of coumestans are sprouts of soya and alfalfa where levels up to 7 mg/100g dry weight are reported. Whole soyabeans and other common foodstuff legumes contain levels of'approx. 0.12 mg/100 g dry weight and most of that is concentrated in the seed hull which commonly is removed in the preparation of human foodstuffs.

Type 4 phyto-oestrogens (oestrogens).

These are compounds closely related to animal oestrogens such as oestrone, oestradiol and oestriol. These have been described in plants such as liquorice, apple, French bean, pomegranate and date palm. Little is known of the metabolism and biological significance of these chemicals in humans and animals.

The full range of biological effects in anirmials of these dietary phyto-oestrogens has received only recent study. A primary effect appears to be associated with their close structural relationship to naturally-occurring oestrogens which allows the phyto-oestrogens to mimic the effects of the endogenous oestrogens. The known biological effects of phyto-oestrogens can be summarised thus: In vitro bind to both cytoplasmic and nuclear membrane (Type II) oestrogen receptors on human tissues; strongly compete with oestrogens for oestrogen receptors, but only weakly stimulate those receptors; l(c) tur, gly satimulatie ue Uproducution Of sex ihoiu1c-Ubiding globulin (SHBG) from human cells; In vivo weakly oestrogenic in animals; competitively-inhibit the response of tissue to oestrogens.

The three major types of phyto-oestrogens appear to act at the cellular level in a similar manner, that is through interaction with cell surface oestrogen receptors. In the body, naturally-occurring oestrogens circulating in the blood largely exert their activity by interaction with oestrogen receptors on cell surfaces; such interactions then triggering a particular biological function of that particular cell. Phyto-oestrogens are able to bind to those oestrogen receptors because the structure of these compounds so closely resembles the endogenous oestrogens, but unlike the animal oestrogens, phyto-oestrogens only weakly activate the oestrogen receptor.

As a result of phyto-oestrogens and endogenous oestrogens competing for the oestrogenbinding sites on cells, the more weakly oestrogenic phyto-oestrogens can be considered to have an anti-oestrogenic effect. This phenomenon is known as competitive-inhibition, by which is meant that the biological effect of an active substance is impaired by the competitive binding to a target receptor of a similar but less active compound.

Thus a primary biological effect of phyto-oestrogens is held to be competitive inhibition of endogenous oestrogcns. However, another more direct effect is the stimulation of synthesis of SHBG in the liver, as occurs with orally administered synthetic steroidal oestrogens. High levels of dietary phyto-oestrogens are thought to be responsible for the higher SHBG levels seen in vegetarians and in cultures maintaining traditional (high legume-containing) diets.

At high levels, dietary phyto-oestrogens can have profound physiological effects. An example of this is sheep and cattle grazing pastures containing a high proportion of subterranean clover or red clover which can contain levels of phyto-oestrogens as high as 5% of the dry weight of the plant. As a result of the competitively-inhibitory effect of the dietary phyto-oestrogens on endogenous oestrogen function in the hypothalamus, male and female sheep and cows can develop androgenic symptoms.

Such high dietary levels of phyto-oestrogens, however, are rare. It is far more common that most animal and human diets contain low to moderate levels of phyto-oestrogens, and there is growing epidemioiogicai evidence that such levels have a beneficial effect on human health.

In most traditional human diets in developing countries, the principal phyto-oestrogens consumed are isoflavones because of the generally high reliance on legumes (also known as pulses) as a source of protein. Tue general consumption rates (g/day/person) for legumes for different regions currently are approximately: Japan (50-90), India (40-80), South America North Africa (40-50), Central/Southern Africa (20-50) and Southern Mediterranean Legumes also are a source of lignans and, to a much lesser extent, coumestans, and the additional cereal and vegetables in the diet would also boost the lignan intake. However, the isoflavone intake in these traditional cultures with high legume consumption would typically be much in excess of either lignan or coumestan intake.

The major types of legumes used in traditional diets include soya, chickpeas, lentils, ground nuts, beans broad, haricot, kidney, lima, navy), and grams (bengal, horse and green).

In Western, developed countries, the daily intake of dietary phyto-oestrogens generally is

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negligible to low. In Western Europe, North America and Australasia, legumes were a major source of protein for the majority of the populations up to the end of the 19th century. From that time, legume consumption has declined significantly, being replaced in the diet with protein of animal origin. Average legume consumption in these regions currently is between 5-15 g/day/person with a significant proportion of the population ingesting little to no legumes or other phyto-oestrogen containing foods on a regular basis. Moreover, the types of legumes consumed in these regions garden peas, French beans) have a typically lower isoflavone content than legumes such as soya and chick peas.

Based on typical consumption rates and types of foodstuffs consumed, the typical phytooestrogen intake (mg/day) for different regions can be calculated approximately as Iseflavones Lignans Coumestans Japan 50-300 2-5 Australia 2-25 1-5 0.2 Thus it can be seen that regions which have maintained traditional diets have a higher average daily intake of phyto-oestrogens, particularly isoflavones, compared to western countries.

People in communities such as Japan or developing countries with high legume intake excrete substantially higher phyto-oestrogen metabolites in their urine compared to people in Western countries. Within the latter, vegetarians also excrete higher phyto-oestrogen metabolite levels than do those consuming a more typical, omnivorous Western diet.

The presence of relatively large amounts of phyto-oestrogen metabolites in urine serves to highlight their potential biological significance. It has beer shown that total urinary excretion of isoflavones and their active metabolites in people consuming moderate amounts of legumes is greatly in excess (up to 10,000 x) of steroidal oestrogen levels. So that while the oestrogenicity of isoflavones to oestrogen receptors is only about 1% that of endogenous oestrogens, this weaker effect is off-set by the much higher blood levels of the isoflavones.

It is known that legumes have formed an important part of the human diet over the past 20,000-30,000 years. It therefore follows that human metabolism has evolved over at least this period in the presence of relatively large levels of dietary phyto-oestrogens, particularly 6 isoflavones. Given the known biological effects of phyto-oestrogens, it also follows that endogenous oestrogen metabolism and function has evolved in the face of significant competitive inhibiting effects of phyto-oestrogens. It has been speculated that the presence of significant dietary levels of phyto-oestrogens in recent human evolution has led to a degree of adaption by tissues responsive to reproductive hormones to these dietary components. That is, both the rate of production and/or the function of endogenous oestrogens may be either dependent upon or influenced by the presence of phyto-oestrogens in the body. It follows therefore that a relative deficiency of dietary phyto-oestrogens could be expected to lead to an imbalance of endogenous oestrogen metabolism.

There is increasing interest in the likely contribution of a relative deficiency of dietary phytooestrogens to the development of the so-called "Western diseases", especially cancer of the breast, benign (cystic) breast disease, cancer of the uterus, cancer of the prostate, cancer of the bowel, pre-menstrunal sndrome, menonausal syndrome, and atherosc!erois. of these diseases are associated to a greater or lesser extent to oestrogen metabolism, and oestrogen function is either known or is suspected to play a role in their aetiology and/or pathogenesis.

Each of these diseases occurs at much higher incidence in Western, developed countries than it does in developing communities. Moreover, it is thought that in Western communities, the incidences of each have risen over the past century. It is also generally held, that of all the environmental factors likely to be contributing to this phenomenon, diet is the principal factor.

Of those dietary components with the potential to influence the aetiology of oestrogen-related disease, there is a growing awareness that phyto-oestrogens may have important potential.

The beneficial effects of phyto-oestrogens on human health are thought to derive from at least two principal function, those being competitive-inhibition of the function of endogenous oestrogens, and (ii) the stimulation of production of SHBG. SHBG plays an important role in primates in binding and transporting the reproductive hormones (oestrogens, androgens) in blood so that the availability of reproductive hormones is regulated to a large degree by SHBG levels. Higher SHBG levels are considered beneficial in leading to a reduction in both blood levels of 'nbound (and unregulated) reproductive hormones and metabolic clearance rates of the hormones. Although isoflavones are potent stimulators of SHBG synthesis, they only weakly bind to SHBG, so that the increased SHBG levels resulting from the dietary 7 isoflavones are largely available for binding to endogenous oestrogens.

In terms of directly identifying the beneficial effects of phyto-oestrogens in amelioration of any or all of the "Western diseases", there are only two examples. In one example, the diets of women, with menopausal syndrome were supplemented with foodstuffs (soya, linseed, red clover) high in phyto-oestrogens, and an alleviation of menopausal symptoms to an extent similar to that obtained with replacement therapy with synthetic oestrogens was achieved; that effect was ascribed to the phyto-oestrogen content of the supplement. In the other example, legumes such as soya and various pulses have been shown to have a hypocholesterolaemic effect in humans; this effect has not been ascribed to phyto-oestrogens, although purified isoflavones do have a hypocholesterolaemic effect in animals with artificially-induced hypercholesterolaemia.

in summary. it could reasonably be deduced that the inclusicn of greater levels of foodstuffs high in phyto-oestrogens in the standard diets of men and women in developed countries could be expected to redress a general imbalance of endogenous reproductive hormone metabolism, thereby reducing the predisposition of those communities to the above diseases. While there are various types of phyto-oestrogens which may be suitable to this end, the large discrepancy in isoflavone consumption between communities with Western and traditional diets suggest that foodstuffs with high isoflavone content are of prime interest.

However it is unrealistic to expect that public education programmes would readily convert communities in developed countries from a diet where the protein content is predominantly animal-derived, to one where the protein is predominantly legume-derived. Moreover, the legumes which are commonly consumed in developed countries are relatively poor sources of phyto-oestrogens and the general acceptance in the community of less well-known legumes with higher phyto-oestrogen content would be necessarily a slow process. Also, the highly variable levels of phyto-oestrogens in foodstuffs relating to plant strain type, degree of plant maturity, and climatic and other environmental conditions suggests that the supply of an assured amount of phyto-oestrogens through the use of whole foodstuffs may be difficult.

An alternative strategy is to make available either phyto-oestrogens in a purified form, or (ii) foodstuffs which are enriched for phyto-oestrogens. In this way, the phyto-oestrogen could Fax sent by 61 2 9262 1088 DAVIES COLLISON CAVE 10/09/04 11:27 Pff 4/10 8 be added to the diet in a conven~ient form as a supplement without requiring any subtanivechange to the diet.

DISCLOSURE OF INVENTION The present invention concerns in one aspect an isoflavone containing extract of clover or soya prepared by water/organic solvent extraction of said clover or soya followed by recovery of concentrated isoflavones therefromn, which extract comprises any two or more of the concentrated isoflavones: genistein, daidzein, hiochianin A. formononctin, and/or their glycosides.

In a preferred aspect the invention relates to use of a pliyto-oestrogcn isoflavone extract of soya or clover comprising: genistein and its methylated derivative biochanin A to daidzein and its methylated derivative formononctin; or genistcin to daidzcin and its methylated :derivative formnononetin; or genistein to forinononetin; or genistcin. and its methylated 1 do ivailLvc OliOLL 1jiti ilu LU 1 U -IIIIJ)VIS andIEUIL t.4UI~ALLU14..*,'4 it-. methylated derivative biochanirt A to duidzein for: i) lowering levels of blood lipoprotcin leading to reduced risk of development of atherosclerosis: ii) reduced risk of development of cancer of the prostate; iii) reduced risk ol'canceTs, to the breast; iv) reduced risk of development of cancer to the uterus, v) reduced risk of cancer of the large bowel; vi) reduced risk of developmient of preenrstrual syndrome; and vii) reduced risk of symnptom-, associatedI with menopause, In a further preferred aspect the invention provides a mecthod of treating: high levels of blood lipoprotein atherosclerosis, cancer of the prostate, cancer of the breast, cancer of the utecrus, cancer of the large bowel, premenstrual syndrome wid symiptoms associated with menopause which comprises administering an effective amount of said phytoocstrogeri isoflavone composition.

P Mll 34 X X 9,,7.%f2i 904-1 COMS ID No: SBMI-00907743 Received by IP Australia: Time 10:44 Date 2004-09-10 27- 8-04: 4:27PM:DAVIES COLLISON CAVE :612 92621080 9/ 16 P referably the extract contains an excipient. a diluent. a carrier or the like, or else tie supPlemrnt is mixed with food or can be consumed directly. It is also preferred that foodstuffs are readily available, have no known toxic components. and are rich sotrces of isotlavortes: such foodstuaffs preferably being red clover or soya. It is also preferred that the ratio of genistein and/or its methylated derivative biochanin A to daidzein andfor its inethylated derivative formononctin is between 1:2 to 2: 1. Other plant components with oestrogt aic activity including lignans. coumestans and flavones may also be present in the extract, but it is held that these are of secondary importance to the predominant isoflavories.

The c- in phyto-oestrogcns is used hereafter to indicate a predominance of isoflavones with lesser amounts of lignans. coumestans and tiavones.

The invention also concerns a method for the treatment or prevention of benign breast disease,-premenstrual syndrome symptoms associated with menopause, cancer of the prostate. cancer of thie bowel, or elevated blood cholesterol, which comprises administering to a subject an eilectivc atinuuaa U' l tiI du i.JEVIC t1Sill I CAUIMA Ul LI1UWC1 Ul bJYA PFCPJI1EU by water/organic solvent extraction of said clover or soya followed by recovery of concentrated isoflavones therefrom, which extract comprises any two or more of the co-accntrated isoflavones: gen-6stein4. d.-idzci.n, bioch'-anin A. fomnntn and/or their glycosides. optionally in association with a pharmaceutically acceptable carrier. Ideally. the extract is administered reguarly on a daily basis over a sufficient period such as at least a month. The health conditions which may be prevented or ameliorated include cancer of the COMS ID No: SBMI-00BB9446 Received by IP Australia: Time 16:46 Date 2004408-27 9 However, any source rich in phyto-oestrogens may be used instead, if desired.

Various different isoflavones have been identified from these sources they are principally genistein. biochanin A. daidzein, formononetin and glycitein In plants these compounds occur principally in a glycoside form bound to sugars such as glucose. with smaller amounts present as the aglucone forms. The formulae of the isoflavones are: daidzein R-H genistein R OH HO" v O The structure of biochanin A is the same as for genistein but with a 4'-methoxy group, and similarly formononetin has the same structure as daidzein. but with a 4'-methoxy group.

Following ingestion by humans, the glycosidic isoflavones are hydrolysed to the aglucone form and biochanin A and formononetin are demethylated by bacterial fermentation to genistein and daidzein respectively. A small proportion of these free isoflavones are absorbed directly from the bowel and circulate in the blood. The bulk of the isoflavones, however, remain in the bowel and undergo fermentation to form various metabolites which also are absorbed into the bloodstream. The principal metabolites which have been identified are equol i and O-desmethylangolensin.

In vitro and in vivo studies have indicated that genistein, biochanin A, equol, daidzein, formononetin all have oestrogenic activity in descending order. O-desniethylangolensin is only very weakly oestrogenic and glycitein is non-oestrogenic.

In animal and in vitro studies, genistein has been shown to have greater oestrogenic/antioestrogenic activity and SHBG-stimulating capacity than the other isoflavenes or their metabolites (approximately 10 times that of daidzein and formononetin). However, the full range of biological effects of the different isoflavones have yet to be fully determined, and in particular their relative efficacies in the different biological effects such as oestrogenicity, hypocholesterolaeni a. anti-angiogenesis. anti-oxidation. ant-.-carcinogenests for example are not yet fully known It is thought that be-,ause the methyl forms (biochanin A and formononenin) ultimately are larely demethylated to their principals, gen stein and daidzein. with improved biological efficacy. then it is unimportant whether the isolffkvoncs are present in the claimed product in the methylated or demethvlated forms.

Given that the relative biological importa.nce of the twvo isolavone groups (being genistein and daidzein) to human health remains unclear. and that each might indeed have different importance. plus the fact that both isoflavones are present in the dict in approximately equal proportions, then it is prudent that both isoflavones be present in the claimed product in approximately entigl nrorflrtiofl Any leguminous plants such as detailed here could be used as sources of phyto-oesoirogens (principally isoflavoncs With lesser amounts of lignans and coumestans)- Indian liquorice (A bnis pn.'catonus). various species of Acacia spi) including. .A wweum. A. ctbaa A.

Iongifol'a. and.A owaldii, ground nut (Apio tubervsa). ground pea (A nwhis h/npogca). milk v'etch (A sirug alus editbs). marama bean (Bauihinza esculenta). sword bean (Ciaitis caytri indicus).- jack bean (Caaiaia ensijioris)-; sword bean (Ca'noJala ghiiara), seaside sword bean (Cazvalia rusca). various Cassia spp. including~ C lonbundia. C. Iaevigata. and C occidentalts. car'obbean (Ceruionia siliqua). chick pea (Cicer anctinunt). vebnut (Cordeauxia eduh/s),- various Crvtalana spp. including C labirnifolia. and C. 1%dh~da. cluste!r bean (C ywncpsis psoraltoides). tallow tree (Deranmim senegakensc) swo,,d bean (Entada scandens).

balu (Eyphnna edulis). soyabean (Clycine may.;) inga (lngardults). Polynesian chestnut (Inocarpus.fagipfr). hyacinth bean (LdaW purpureus); grass pea or Indian vetch (Laz'hvnts saliriis); cyprus v'etch (LathYnis ochrus). lentil (Lens cu/mis), jumping bean (LeucarnaI eulcocephala).' various Lupinus spp. including L. albuy, L. hlits, L. angushfoliuni. L.

mutabilis, and L. cosenrinii.- ground bean (Alxcorvlia geocwpa).- horse gram (Mxcrotylorna uniflorum). alfalfa (Ml edicago saiiva). velvet bean (Aluvcunaprunens);, yam beans (Pachvrh1:11: emusu.s. P. tuberusus).- African locust bean (Pariua clappenioniwia).; Pw*ia speciosa-, oil bean tree (Pentaclethram nacropkyla). various Phaseodis spip. including P. acutifolrus, P. vulganis.

P. jun rus, P. coccine us. P. 1 xienathus, angulns. P. aurrus, P. calcarar us, P. mungo. and P.

11 polystachyus; garden pea (Pisum sativum); djenko bean (Pithecolobium lobatum); mesquite (various Prosopis spp.); goa bean (Psophocarpus scandens, P. tetmgonolobus); various Psoralea spp.; Sesbania bispinosa; yam bean (Sphenostylis stenocarpa); tamarind (Tamarinduis indica); fenugreek (Trigonellafoenum-gmecum); vetches (various Vivia spp. including V. sativa, V.

atropurpurea, V. cn'ilia, and V. monantha); broad bean (Vicia faba); black gram (Vigna mungo); various Vigna spp. including V. mdiata, V. c.'onitifolia, V. adanatha, V. angularus, V. tribolata, V. umbelata, and V. unguiculata, and, earth pea (Voandzeia subterrnmea).

The ideal sources of phyto-oestrogens for preparation of a supplement in accordance with the invention are preferably those which are readily available, (ii) are relatively inexpensive, (iii) are readily and economically processed so as to yield the extract, (iv) have a high isoflavone content so as to provide high yields, and have no known toxic components requiring selective removal or inactivation.

Certain clovers, such as red clover praense) and subterranean clover subterranean) are the preferred sources. On a dry weight basis, these clovers contain the highest amounts of oestrogenic isoflavones of all legumes tested to date with levels of 3-5 g% subterrnean) and 1-3 g% praensc). In comparison, soya flour has a level of 0.15-0.30 lentils (0.08- 0.12 chick peas (0.07-0.13 and garden peas (0.02-0.03 Thus it can be seen that clovers contain approximately at least 10-30 times by weight the isoflavone content of other commonly available, human leguminous foodstuffs meaning that for manufacturing purposes, the yield of isoflavones per unit weight of plant material is many times greater from clover than from other legumes.

Red clover and subterranean clover also are common fodder crops and are readily grown and are widely available. Clovers also are comparatively cheaper (S200/tonne) than crops such as soya and lentils (S500/tonne).

With clovers, the isoflavones are recovered from the leaf rather than from the seed in the case of soya, beans, nuts and grams. This provides a substantially higher yield of isoflavones per unit area of pasture for clovers compared to other legumes because of the greater leaf matter compared to seed matter recovered per plant.

Clovers also have an extended growing season, and faster growth rates compared to those legumes such as soya, lentils or chick peas where the seed is the end-product. Clover can be cropped for its leaf content repeatedly over a single growing season. An additional benefit of this is that as phyto-alexins, the isoflavone content increases in response to the stress of cropping.

Thus it c?.n be seen that in clovers versus other legumes provide a combination of higher isoflavone content per dry weight of plant, a higher yield of dry matter containing isoflavones per plant, and a higher yield of dry matter per hectare.

An additional feature of clovers is that there are wide varieties of cultivars with widely differing isoflavone levels and types. This allows blending of different cultivars to achieve the desired ratio of the different isoflavones, although it is equally possible to use a single cultivar which provides the desired ratio.

Other legumes such as soyabean flour may be used for enrichment of phyto-oestrogens but the substantially poorer (approx. 10%) yield of isoflavones compared to clovers means that the manufacturing costs are substantially greater and there is substantially greater amounts of waste products which requires disposal or further treatment for re-use as a foodstuff. An alternative, however, to the use of whole soya for this purpose, is to use the hull and hypocotyl (or germ) of the whole soyabean. The hull and hypocotyl represent only a small proportion by weight and 2% respectively) of the intact bean. However, the coumestrol content of soya is concentrated in the hull, and the daidzein content of soya is concentrated in the hypocotyl. The two cotyledons which comprise the bulk of the soyabean (90% by weight) contain the bulk of the genistein content of soya. During standard processing of soyabeans, the hulls being a fibrous component with little or no perceived nutritional value normally a-e separated and removed by physical means. The hypocotyls become separated following the splitting of the cotyledons, and while these currently generally are not deliberately isolated, they may be separated and isolated by passing the disturbed soyabeans over a sieve of sufficient pore size to selectively remove the small hypocotyl. The hypocotyl cont.ins approx. 1.0-1.5 g% isoflavones (95% daidzein, 5% genistein). The raw hypocotyl and hull material can be ground or milled to produce, for example, a dry powder or flour which then could be either blended or used separately as a dietary supplement in a variety of 13 ways including, for example, as a powder, in a liquid form, in a granulated form, in a tablet or encapsulated form, or added to other prepared foodstuffs. Alternatively, it could be further processed to yield an enriched extract of phyto-oestrogens. Either or both of these materials also could be added to other leguminous material such as clover to provide the invention.

In plants, the oestrogenic isoflavones are restricted principally to the leaf, fruit and root; the stem and petiole contain very little. With soya and other common human legume foodstuff crops, the leaves are rarely regarded as foodstuff; indeed with these crops, the plants normally are allowed to die and dry out before the seed crop is harvested. Nevertheless, the fresh leaves of these crops could be regarded as a source of phyto-oestrogens for the invention although the much lower isoflavone content of the leaves of these crops compared to clovers, plus their generally slow growth compared to clovers, suggests that they would not be a preferred source of large-scale isoflavone enrichment.

To provide a similar amount of isoflavone to that contained in most iraditional legume-rich diets (50-100 mg oestrogenic isoflavones/day) would require an average daily consumption of 3-6 g dry weight or 15-30 g wet weight of specially selected cultivars of clover with particularly high isoflavone levels. Clover grasses generally are not eaten by humans, except to a limited extent as sprouts of some of the pleasanter tasting varieties. Isoflavones are intensely astringent and are responsible in large part for the bitter taste of legumes. Thus the types of bean sprouts, clover sprouts and alfalfa sprouts generally available have been relected on the basis of cultivar and of age for pleasant taste, and in so doing inadvertently have been selected for low isoflavone content. Of the sprouts currently available in Western countries for human consumption, between approx. 100-250 g would need to be consumed daily to provide a dosage of 50-100 mg isoflavones. Certainly clovers and other legume sprouts are not generally eaten in such sufficient quantities by humans to obtain the advantages of the present invention.

The invention also concerns formulations containing the o strogens discussed above together with a dietary suitable excipient, diluent, carrier, or with a food. Ideally the S formulation is in the form of a pill, tablet, capsule, or similar dosage form.

The formulations may be a variety of kinds, such as nutritional supplements, pharmaceutical 14 preparations, vitamin supplements, food additives or foods supplemented with the specified active phyto-oestrogens of the invention, liquid or solid preparations, including drinks, sterile injectable solutions, tablets, coated tablets, capsules, powders, drops, suspensions, or syrups, ointments, lotions, creams, pastes, gels, or the like. The formulations may be in convenient dosage forms, and may also include other active ingredients, and/or may contain conventional excipients, carriers and diluents. The inclusion of the subject phyto-oestrogens in herbal remedies and treatments is also a preferred part of the invention The invention is also directed to the amelioration, prevention, or of various conditions responsive to treatment with the phyto-oestrogen substances of the invention. The preferred amounts to be administered to the human fall within 20 200 mg on a daily basis. More preferably the dosage is from 50 150 mg on a daily basis, and most preferably at a dosage of about 100 mg. If desired greater dosages can be administered for therapeutic reasons. In contrast to prior practices such high dosages were not possible. For example, dosages of un to or greater than 1 000 mo may be suitable to treat some conditions. n order to cbtain the benefits of the invention, the treatment with the isoflavones should continue for a considerable period, ideally for at least a month, and ideally continuously for the whole period for which the health improvement advantages should accrue.

The product according to the present invention yields a constant and accurately known amount of isoflavones. The product is also ideally a natural product, which has advantages for Sconsumer acceptance, and in accordance with the supposed theory behind the invention may S..very possibly be one of the main causes for its beneficial effects. Whole legumes have a widely variable isoflavone content due to two main causes: the type of legume and the environmental effect. The type of legume typically has a wide range of isoflavone content.

The miligram of isoflavone per hundred gram of whole foodstuff (dry weight) is given in the following table: Soya Products Whole Soya 150 300 Soya Milk 25 40 (mg per 200 ml) Tofu 55 Lentils 80 120 Chickpeas 70- 130 Broad beans 15 Garden peas 15 Thus common leguminous foodstuffs consumed in Western countries (broad beans, garden peas etc) have relatively low oestrogenic isoflavone content and exceptionally large amounts of these would need to be consumed daily to approximate those isoflavone levels consumed in traditional diets. Most Western cultures do not traditionally eat legumes with high isoflavone contents, and those soya products (milk, tofu etc) which are becoming increasingly popular in Western countries, also have reiatively low isonfavone !sve!s ccrm.pred tc '-hol soya, indicating that relatively large amounts of these would need to be consumed on a regular basis to deliver the required isoflavone levels.

The enviromental effect arises because the isoflavone levels in any species of plant depend greatly on the age of the plant, the climatic conditions where it is grown, the fertiliser and so forth. Therefore constant and consistant dosage is very difficult with ordinary whole foodstuffs. The accurately determined quality and quantity of the active isoflavones in the S S product, and its easy consumability when compared with the almost impossible task of eating @go huge amounts of often practically inedible foods, is therefore an import feature of the invention for preventing and helping in overcoming various health problems.

Among the various health problems, the treatment or prevention of high blood cholesterol levels, and the treatment of PMS and menopausal symptoms is especially important.

The product of the invention modulates the production and/or function of endogenous sex hormones in humans to modify or produce health improving effects, including the following:

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lowered levels of various blood lipoproteins including, for instance, low-density and very-low-density cholesterol leading to reduced risk of development of atherosclerosis; (ii) reduced risk of development of cancer of the prostate; (iii) reduced risk of cancer of the I breast; (iv) reduced risk of development of cancer of the uterus; reduced risk of development of cancer of the large bowel; (vi) reduced risk of development of the syndrome in women commonly referred to pre-menstrual syndrome (PMS), which includes pre-mensirual tension (PMT); (vii) reduced risk of development of many untoward symptoms (including dry vagina, peripheral flushing, depression etc) commonly associated in women with menopause; and for treating benign breast disease in women (benign or cystic breast disease associated with non-malignant swelling and tenderness of breast tissue). The invention therefore is directed to a method for the prophylaxis or treatment of a human, to combat conditions associated with phyto-oestrogen deficiency, which comprises administering to the human an effective amount of phyto-oestrogen principally isoflavone but which might also include relatively smaller amounts of lignans and coumestans, ideally in a concentrated form, wherein the isoflavones include genistein, and/or biochanin A, and/or daidzein, and/or formononetin.

Cancer of the breast generally is considered to be associated with oestrogenic dysfunction.

Breast CaniCcr ccss mydspy oYS rc ctgc rcccptors :3ar. rhl iona ucast Cec'Is aid stimulation of these receptors by endogenous oestrogens is thought to be a prire source of stimulation of their malignant growth. Currently synthetic anti-oestrogens are being used to prevent or treat the growth of malignant breast cells. Isoflavones are potent anti-oestrogens that could be expected to help prevent or to successfully treat breast cancer. It has been reported that the risk of breast cancer in western societies is indirectly proportional to the level of phyto-oestrogens in the diet and to the amounts of phyto-oestrogen metabolites excreted in the urine.

Cancer of the prostate generally is considered to be associated with sex hormone dysfunction and the growth of prostatic cancer cells is influenced by oestrogens and androgens. The incidence of prostatic cancer is low in communities with high legume intake and, conversely, is high in Western socieites. Phyto-oestrogens are though to protect from development of prostatic cancer. One mechanism may be the effect of phyto-oestrogens on lowering the.

proportion of unbound:bound reproductive hormones in the blood. However, there is other evidence to suggest that phyti-oestrogeni, particularly isoflavones, can have a direct influence on certain cellular enzymes within prostatic cells.

Pre-menstrual syndrome has uncertain aetiology and pathogencsis, although most certainly is Pre-menstrual syndrome has uncertain etiology and pathogenesis, although most certainly is 17 associated with reproductive honnone dysfunction. It also is a syndrome which has reportedly lower incidence in communities maintaining traditional high-legume diets. It is proposed that phyto-oestrogens will alleviate this condition by restoring balance to oestrogen metabolism.

Menopausal syndrome is associated with changes in the oestrogen profile in the body with advancing age. Adverse clinical symptoms may be treated with oestrogen replacement therapy. There is evidence that foodstuffs high in phyto-oestrogens are a suitable alternative to synthetic hormones in this respect, producing alleviation of adverse clinical symptoms.

Again, it is proposed that phyto-oestrogens will function by restoring balance to oestrogen metabolism.

Benign (or cystic) breast disease has unknown aetiology. However, its association in women with certain stages of the menstrual cycle is strongly suggestive of oestrogen dysfunction.

There currently is no successful treatment of this condition. Phyto-oestrogens are proposed to successfully treat this condition by restoring balance to oestrogen metabolism.

Atherosclerosis is associated with cholesterol metabolism which in turn is associated closely with oestrogen metabolism. The generally higher incidence of atherosclerosis in young men versus young women, the rising incidence in women following menopause, and the lower incidence in post-menopausal women receiving oestrogen replacement therapy, all point to the moderating influence of oestrogens on cholesterol metabolism. A prime effect of oestrogens on cholesterol metabolism is stimulation of the liver to process cholesterol, particularly the highly atherogenic low-density lipoproteins and very low-density lipoproteins, into bile salts.

It is proposed that phyto-oestrogens have an important hypocholesterolaemic effect in humans.

There may be a variety of mechanisms involved, but one may be the stimulation by phytooestrogens of cholesterol catabolism by the liver.

MODES FOR CARRYING OUT THE INVENTION S The invention is now described with reference to various examples.

EXAMPLE 1 Preparation of Red Clover Product Tablets were prepared using red clover in accordance with the following procedure. The raw plant material is harvested and dried; such drying being either sun-drying or from applied heat.

4 i I 18 The dried product is then preferably chaffed, before the following extraction step, although this can be omitted if desired.

The dried material is extracted in an aqueous: organic solvent mix. The aqueous phase is required to extract the water-soluble glycoside form of isoflavones, while the organic solvent is required to solubilise the water-insoluble aglycone form. The organic solvent can be either alcohol, chloroform, acetone or ethyl acetate. The ratio of solvent in the water can be between 0.1% and 99.9%. The preferred method is to use 60% alcohol in water.

The isoflavones are extracted by exposing the plant material to the water:solvent mix. The exposure time in general terms is indirectly proportional to the temperature of the mixture.

The temperature of the mix can range between ambient temperature and boiling temperature.

The exposure time can be between 1 hour and 4 weeks or even longer. It has been determined that the adequate times for maximal recovery of i nlavonres are 2 weeks at and 24 hours at 90°C. The supernatant is separated from the undissolved plant material and the organic solvent removed by distillation. The aqueous supernatant then is concentrated, typically by distillation.

Additional processing steps can be used, if desired, to convert the extracted natural product to capsule, tablet, or other convenient form for ingestion, using normal techniques for doing this. Otherwise the product can be packaged as a convenient food additive.

EXAMPLE 2 preparation of soya hypocotyl product.

Soyabeans were heated in dry air so that the hull became brittle. The beans then were processed through a tumble mill which removed the hull and split the bean the two cotyledons and the small-sized hypocotyl which separated from each other. The light-weight hulls then S. were removed by an air stream. The small-sized hypocotyls were separated from the larger cotyledons by sieving through a steel wire mesh with apertures of 1 mm x 1 mm. This yielded approximately 87% purity of hypocotyls with 13% contamination by small cotyledon chips.

Normal soybean processing steps isolate the hulls and then these are discarded or processed 19 separately for use in human and animal foodstuffs. The hypocotyls normally are not separated and are processed along with the cotyledons. However, a small number of soybean processors are separating hypocotyls by the above methods in order to reduce the astringent taste of soyflour for human consumption, and currently these hypocotyls are either discarded or processed to flour for use in animal feed.

EXAMPLE 3 effect of administering red clover extract to humans Seven normal individuals were studied for the comparative effects of red clover extract and whole legumes on blood cholesterol levels. All the individuals were consuming a standard Western diet with minimal levels of legumes.

Three men consumed between 100-150 g haricot or navy beans daily for 3 weeks as a supplement to their normal diet. This yielded an approximate daily isoflavone dosage or between 60-100 me.

Four other individuals (3 men, 1 woman) consumed 5 g of red dover extract containing 100 mg isoflavones daily for 3 weeks.

Total serum cholesterol 'evels were determined immediately before and immediately following the challenge.

Pre-treatment Post-treatment change Beans only Patient 1 5.77 5.46 5.4 Patient 2 6.24 6.12 1.9 Patient 3 7.45 8.51 +14.3 Red clover extract Patient 5 6.53 5.90 9.6 Patient 6 7.43 6.63 -10.8 Patient 7 6.33 5.50 -13.1 Patient 8 6.98 7.28 4.3 The red clover extract had a significantly (P 0.05) greater hypocholesterolaemic effect than did the whole beans.

Neither of the treatments produced any untoward side effects, although the whole bean eaters reported greater difficulty with compliance of treatment than did those taking the red clover extract.

EXAMPLE 4 effect of administering soy hypocotyls to humans Fifteen volunteers (8 women, 7 men) were given 5 g of soy hypocotyl containing (45 mg daidzein and 5 mg genistein) daily for 2 months. The hypocotyl was consumed as a powder added to the diet.

The effects on cholesterol levels are shown in the following table. The individuals are gruuped according to their pre-rreatment choiesteroi levels (high, medium, low).

Range (mean) unmol/L n Pre-treatment Post-treatment Group 1 6 6.3 8.4 5.4 6.5 (6.1) Group 2 6 5.0 6.2 4.7- 5.9 (5.1) Group 3 3 3.3 4.7 3.4 4.6 (4.1) The results show a significant fall in total cholesterol levels in those individuals with cholesterol levels considered to be at the upper end of the normal range.

In addition, 1 woman reported substantial amelioration of her benign breast disease problem associated with mid-cycle swelling and tenderness, and another woman reported regularisation S of her menstrual cycle and reduced menstrual bleeding. Both of these effects were regarded as beneficial.

No other side-effects were reported as a result of the treatment.

y I -21 EXAMPLE 5 further therapeutic treatments Compositions Compositions comprising an extract of soya or clover were prepared in accordance with Examples 1 and 2. These compositions, for convenience referred to as "the inventive composition", were prepared comprising 40 mg, 80 mg, 120 mg, 160 mg and 240 mg of phyto-oestrogen.

Treatments Prostate Cancer Two patients diagnosed with prostate cancer were treated initially with the inventive composition comprising 240 mg per day, and subsequently 120 mg per day phyto-oestrogen.

TIte PSA levels, a m-arke fr for prostate cancer, ere aiie in these patiepnts and there has been no rise in the PSA levels subsequently. This demonstrates the treatmentof prostatic cancer in these individuals.

A further patient diagnosed with malignant prostate cancer (PSA 13.1 pg/L) was treated with the inventive composition. The patient was treated with the composition comprising 160 mg per day phyto-oestrogen. seven days prior to prostatectomy. Histological comparison was 20 made of the pre-operative needle biopsy and the prostatectomy specimen. The needle biopsy revealed low grate infiltrating adenocarcinoma. The prostatectomy sp n showed mild patchy microvacuolation and prominent apoptosis (programmed cell death). Lymph nodes were negative for malignancy. The degenerative changes in the prostatectomy specimen, especially the apoptosis. show treatment of the prostatic cancer.

Benign or Cystic Breast Disease A patient with benign or cystic breast disease was treated with 160 mg of the inventive composition administered orally on a daily basis. The patient exhibited no breast tenderness, which was maintained when the dosage level was reduced to 80 mg. Her symptoms did not return and she continues to have relief from mastalgia.

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-22- Pre-Menstrual Syndrome (PMS) Nine women were treated with 80 mg per day of the inventive composition and were screened for the well-described symptoms of PMS including psychological, psychiatric, gynaecological and personal status. Relief from PMS in these various symptoms was observed across the treatment group.

Menopause Eight menopausal women were divided into two groups of four and treated with either 40 mg or 160 mg of the inventive composition administered orally on a daily basis. Four patients were also treated with a placebo composition. Indicators measured were incidence or severity of hot flushes, night sweats. Green score, vaginal pH, vaginal cytology and mean cholesterol levels across the treatment groups. A significant change in menstrual symptoms was observed aind a Uos response change as observed betwe enI ithe 40 mg and 1i60 mg dousage range. This indicating that 160 mg per day was the most effective dosage for treatment of menopausal symptoms.

Throughout this specification, unless the context requires otherwise, the word "comprise", 20 or variations such as "comprises" or "comprising" or the term "includes" or variations thereof, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. In this regard, in construing the claim scope, an embodiment where one or more features is added to any of claim is to be regarded as within the scope of the invention given that the essential features of the invention as claimed are included in such an embodiment.

Claims (8)

1. Use of a pbyto-oestrogefl isoflavone extract of soya or clover comprising: genistein arid its mnethylated derivative hiochanin A to daidzein anid its niethylated derivative formorionctill; or Zcnistein to daidzcin and its muethylated derivative forrnononetin; or gcnistein to formononetin; or gcnistein and its methylated derivative hiochanin A to Cormononetin; or biochanin A to formonionetin; or genistcin and its methylated derivative biochanin A to daidzein in a ratio b etween 1: 2 to 2: 1, for: i) lowering levels of blood lipoprotein leading to reduced risk of development of atherosclerosis; ii) reduced risk of development of cancer of the prostate; iii) reduced risk of cancers to the breast, 15 iv) reduced risk of development of cancer to the uterus; v) reduced risk of cancer of the large bowel; v i) reduced risk ofidevelopment of premcnnstnMaisndo and vii) reduced risk of symptoms associated with menopause. 20

2. Use according to claim 1, wvherein the isoflavotne is extracted from red clover.

3. Use according to claim 1, wherein the isoflavone is extracted from soya hypocotyls.

4. Use according to any one of claims I to 3, wherein the isoflavone extract is [ormulated with a conventional excipient, carrier and/or diluent.

Use according to any one of claimns 1 to 4, for reduced risk of development of premenstrual syndrome or rcduced risk of symptoms associated with menopause.

6. A method of treating: high levels of blood lipoprotein, atherosclerosis, cancer of the prostate, cancer of the breast, cancer of the uterus, cancer of the large bowel, premenstrual syndrome and symptoms associated with menopause, which comprises COMSID No: SBMI-00907743 Received by IP Australia: Time 10:44 Date 2004-09-10 Fax sent by 61 2 9262 1088D0E OLSNCU 1/98 12 :61 DAVIES COLLISOM CAVE 10/09/04 11:27 Pff: 6/10 24 administering an effective amount of a phyto-ocstrogefl isoflavone composition comprising: gcnistein and its methylated derivative biochanin A to daidzcin and its methylated derivative formononetin; or genistein to daidzein and its methylated derivative formononetin; or gcnistein to formononetin; or gcnistein and its mecthylated derivative biochianin A to formononetin; or hiochanin A to formononctin; or genistein and its miethylated derivative biochanin A to daidzcin in aratio of betweenl 1:2 to 2:1.

7. A method accordiny, to claim 6 wherein an effective amount is from about 50 mg to 150 mg per day. 1 5

8. A method or use according to claim 1 or 6, substantiaUy as desc-ribcd her-ein by reference to the Examples. DATED this I10th day of September 2004 20 Novogen Research Pty Ltd By its Patent Attorneys DAVIES COLLISION CAVE COMS ID No: SBMI-00907743 Received by IP Australia: Time 10:44 Date 2004-09-10