AU777632B2 - Health supplements containing isoflavones - Google Patents

Description

22- 8-01; 6:01 612 93645173 5/ 6 1 636 0~ Our Ref:7635620 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Address for Service: Invention Title: 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 Health supplements containing isoflavones The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 22- 8-01; 6:01 612 93645173 6/ 36 HEALTH SUPPLEMENTS CONTAINING ISOFLAVONES NOTE: This application is a divisional application of Australian Patent Application No. AU-B- 40525/93 (Acceptance No. 683838). Application 40525/93 is based on Australian Provisional Patent Application No. PL 2511 filed 19 May 1992.

TECHNICAL FIELD This invention relates to natural products containing isoflavones which have various beneficial physiological effects in man, and which have a variety of uses, such as to promote good health (and thus may be regarded as health supplements) and as a dietary additive, for example.

The invention also relates to methods of treatments or prevention of various disorders.

The particular product in accordance with the invention is an extract of plants, particularly a health supplement extract with the particular purpose of enrichment for isoflavones.

BACKGROUND ART 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 nutritional value. The isoflavones hitherto have fallen into this *latter category of no known nutritional value.

There are 3 principal classes ofphyto-oestrogens, viz. isoflavones, lignans, and coumestans. The isoflavones are thought to have a broad range of biological functions in plants, although these are poorly understood. However, two particular functions are recognised as phyto-alexin 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 chemicals which encourage colonisation of nitrogen-fixing bacteria on the roots of legumes. The biological function in plant of the lignans and coumestans is not generally understood.

The different types ofphyto-oestrogens are as follows: Type 1 phyto-oestrogens (isoflavones) P~\WPDOCS\GRS\SPECNovogen.doc 22/8/01 22- 8-01; 6:01 612 93645173 7/ 36 2 Isoflavones appear to be widely distributed in the plant kingdom and over 700 different isoflavones are described. The isoflavones contemplated in this invention are those which display oestrogenic activity being principally represented by daidzein, genistein, and also formonentin and biochanin A. These compounds are restricted almost exclusively to the Leguminosae family.

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 fermentation 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).

eLignans 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. Oilseeds such as flax (linseed) have the highest known levels at 20-60 mg/100 g dry weight, while cereals and legumes have much lower levels at 03-0.6 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 urine.

*o 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 P.\WPDOCSGRSSPECnNovogen.doc 22/801 22- 8-01; 6:01 612 93645173 8/ 36 3 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 animals 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; S* strongly compete with oestrogens for oestrogen receptors, but only weakly stimulate those receptors; S. strongly stimulate the production of sex hormone-binding globulin S (SHBG) from human cells; In vivo weakly oestrogenic in animals, competitively-inhibit the response of tissue to oestrogens.

The known biological effects of oestrogen include proliferation of breast and uterine tissue; stroke and increased cancer disposition particularly of the breast and uterus associated with long term use such as HRT; mood disturbances, flushing, and thermoregulatory disturbances associated with aberrant oestrogen levels; and femminisation.

The three major types of phyto-oestrogens appear to act at the cellular level in a similar manner, P*\WPDOCS\GRSSPECNovogen.0OC 22/8/01 22- 8-01 6:01 612 93645173 9/ 36 4 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. As stated previously herein, phyto-oestrogens appear to be 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 oestrogen binding 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 oestrogens. However, it has been postulated that another more direct effect of the phytoestrogens may be 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.

SA confounding variable in the theories on the action of phyto-oesterogens is the well-known observation in sheep and cattle exposed to oestrogenic isoflavones. At high levels, dietary phyto-oestrogens can have profound detrimental 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-oestrogen on endogenous oestrogen function in the hypothalamus, male and female sheep and cows can develop androgenic symptoms; particularly feminisation with proliferative response of the uterine and breast tissue and severe reproduction dysfunction. Such high dietary levels of phyto-oestrogens, however, are rare and are contraindicated. This points clearly away from an anti-oestrogenic effect. This PAWPDOCSGRS\SPECIMovgen.doc 22/8/01 22- 8-01; 6:01 612 93645173 10/ 36 behaviour in animals suggests that oestrogenic isoflavones would have a similar pro-oestrogenic effect in humans, an assumption confirmed by Wilcox et al who showed that feeding plants containing phytoestrogens to women induced increased vaginal maturation, a sensitive marker of oestrogenicity (Wilcox et al (1990) British Medical Journal vol. 301, 905-906).

The presence of phytoestrogens, including isoflavones, in the human diet may have implications for human health, particularly as a range of relatively common disorders in Western communities are thought to be associated with estrogen metabolism. For example, cancer of the breast and uterus and bowel, pre-menstrual syndrome and benign (cystic) breast disease are generally considered to be associated with excess estrogen activity. It is well known that treating women with breast cancer or cystic breast disease with estrogen exacerbates those conditions. Adding further oestrogen in the form of phytoestrogen, particularly isoflavones, to a woman's diet therefore might be considered also to be able to contraindicated in those conditions.

Further to the above, oestrogen administration for the treatment of menopause may play a role in increasing the risk of breast cancer and uterine cancer, and has been implicated in proliferative responses in these conditions. The oestrogenic activity ofisoflavones may thus play a role in the aetiology and pathogenesis in these conditions.

On the other hand, menopausal syndrome, prostate cancer and atherosclerosis are generally considered to be associated with low estrogen activity. Oestrogen is known to be beneficial for these conditions, while it is known that anti-oestrogens can exacerbate these conditions. Adding further anti-oestrogen in the form of phytoestrogen to ones therefore might be considered also to exacerbate those conditions.

The likelihood that phytoestrogens in general and isoflavones in particular may be involved detrimentally in the aetiology and/or pathogenesis of these estrogen-associated disorders has been raised by workers involved in isoflavone studies in humans. Setchell et al. (The American Journal of Clinical Nutrition 40, Sept 1984, pages 569-578) and Kaldas Hughes (Journal of Reproductive Toxicology Review, Vol. 3, Number 2, 1989, pages 81-89) raise concern over the use of phytoestrogens with oestrogenic activity in pre-menopausal women.

P:\WPDOCStGRS\SPECriNovogen.doc 22/8/01 22- 8-01; 6:01 612 93645173 11/ 36 6 Further, Wilcox et al (British Medical Journal 301, 905-906, 1990) raise concerns over the use ofphytoestrogens with anti-oestrogenic activity in menopausal women.

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. The general consumption rates (g/day/person) for legumes for different regions currently are approximately: Japan (50-90), India (40-80), South America (30-70), North Africa (40-50), Central/Southern Africa (20-50) and Southern Mediterranean (30-60). 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 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 Isoflavones Lignans Cournestans Japan 50-300 2-5 P:\WPOOCS\GRSSPECftNovogen.doc 22/8/01 22- 8-01; 6:01 612 93645173 12/ 36 7 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 ofphyto-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 been 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. The inventor further reasoned that human metabolism has evolved over at least this period in the presence of relatively large levels of dietary phyto-oestrogens, particularly isoflavones. Given the known biological effects ofphyto-oestrogens it also followed to him that endogenous oestrogen metabolism and function has evolved in the face of significant competitive inhibiting effects of phyto-oestrogens. The inventor 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. Therefore 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 phyto-oestrogens to the development of the so-called "Westem diseases", especially cancer of the P:\WPDOCSGRSSPECMNovogen.doc 22//01 22- 8-01; 6:01 612 93645173 13/ 36 8 breast, benign (cystic) breast disease, cancer of the uterus, cancer of the prostate, cancer of the bowel, pre-menstrual syndrome, menopausal syndrome, and atherosclerosis. All 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 functions, those being competitive-inhibition of the function of endogenous eoesti-ogens, 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 unbound (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 isoflavones are largely available for binding to endogenous oestrogens.

S" 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 P:\WPDOCS\GRS\SPECINovogen.doc 22/8/01 22- 8-01: 6:01 612 93645173 14/ 36 9 hypocholesterolaemic effect in animals with artificially-induced hypercholesterolaemia.

In summary, it could reasonably be deduced that the inclusion 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.

However, there is the concern of the corresponding effect on western diseases.

If it were desired to change the diets of men and women in western countries 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.

In circumstances where the beneficial effect of increased diet of food stuffs high in phytoestrogens was not shown to be directly attributed to the phytoestrogens themselves and where it had been suggested that increased phytoestrogens increased the risk of, inter alia, cancer of the breast, benign (cystic) breast disease, cancer of the uterus, cancer of the bowel, feminisation of men and reproductive dysfunction in pre-menopausal women, it was a commonly held view that increasing the level of phytoestogens was not only contraindicated, it was also potentially S* dangerous. Further, it was a commonly held view that any advantage was in the increase in the relevant foodstuffs in the diet.

Contrary to the commonly held view it was appreciated by the inventor as an alternative strategy to make available either phyto-oestrogens in a purified that is, an enriched form, or (ii) foodstuffs which are enriched for phyto-oestrogens. In this way, the phyto-oestrogen could be P-AWPDOCSGrS-SPECrlovgen.doc 22/W01 22- 8-01: 6:01 612 93645173 15/ 36 added to the diet in a convenient form as a supplement without requiring any substantive change to the diet.

The inventor has surprisingly found that plant extracts containing concentrated isoflavones with oestrogenic activity provided a beneficial health effect in conditions associated with excess oestrogenic function (viz. cancer of the bowel, benign breast disease and pre-menstrual syndrome (PMS)) and in conditions associated with low oestrogenic function symptoms associated with menopause, atherosclerosis, and cancer of the prostate). Such extracts may be administered orally, for example to women with disorders where estrogen is contraindicated and to other women where an anti-estrogen is contraindicated, or in individual women suffering dual disorders in which estrogens and anti-estrogens are both contraindicated, or males suffering from prostate cancer. Such extracts may be regarded as health supplement or nutritional supplement extracts, which may be orally administered, and are safe and effective in providing health benefits and in treating various health disorders.

The inclusion of greater levels of isoflavone in the diet of men and women in developed countries, free of problems associated with reproductive dysfunction and other problems previously referred to; and to provide the aforementioned beneficial health effects in a manner •which does not require dietary modification or substantive change of legume-denied diet, is met according to the present invention.

DISCLOSURE OF INVENTION The present invention concerns in one aspect an isoflavone containing extract of clover or soya prepared by extraction of said clover or soya with a mixture of water and organic solvent followed by recovery of concentrated isoflavones therefrom, which extract comprises any two or more of the concentrated isoflavones: genistein, daidzein, biochanin A, formononetin, and/or their glycosides. The isoflavone containing extract, which may be considered a natural product, is a health supplement or nutritional supplement derived from clover or soya. The extract is in a form suitable for oral administration.

PA.WPOOCSGRSSPECf ovogen.dc 22/=1 Fax sent byj 61 2 9262 1080 DAVIES COLLISOM CAVE la- 18/89/04 11:25 Pq: 4/7 in a preferred aspect th .e ivnonprovides a pharrriaccutical. composition of a phytooestrogenl isoflavoile extracted fromi clover or soya, which extract comprises: gcnistein and its mcttbylated derivative biochax'if A to daidzcein arnd its methylated derivative formononetin; or genistein to daidzcin and its mcthylatcd derivative forniononetin; or gcnistein to formononCtirl; or genistein and its mcthylated derivative biochanin A to forniononetin; Or biochanin A to forruononetin; or gcnistein and its rnethyiated derivative hiochanin A to daid7eifl in aratio between1: 2 to 2 :l1.

P WV1XX-!ATXS\.%j*ON7d33A-.- I- C-OMS ID No: SBMI-00907742 Received by IP Australia: Time 10:41 Date 2004-09-10 22- 8-01; 6:01 612 93645173 16/ 36 11 As used herein, the terms "mixture of water and organic solvent", or "water organic solvent" has the same meaning as the terms "mixture of aqueous and organic solvent", or "aqueous organic solvent".

By recovery of concentrated isoflavones is meant solvent removal and concentration of the aqueous phase containing extracted isoflavones. Solvent removal and concentration of the aqueous phase containing extracted isoflavones enriches the isoflavone content. An example of recovery of concentrated isoflavones is provided in Example 1. The extract after recovery of concentrated isoflavones is in a form which enables it use as a health supplement.

Extraction of clover or soya by a mixture of water and an organic solvent is a chemical extraction which enriches or concentrates isoflavone levels, for example on a weight/weight basis over the original plant material. Organic solvents which may be used in the extraction process of the invention are exemplified in Example 1 herein. These include ethyl acetate and alcohol.

The extract according to an aspect of the invention may be considered a health support because organic solvent is removed in its production, it contains concentrated isoflavones, it can be safely administered to human subjects, preferably orally, and is a natural product in the sense of being prepared from plant material.

Preferably the extract contains an excipient, a diluent, a carrier or the like, or else the supplement is mixed with food or can be consumed directly. It is also preferred that foodstuffs from which an extract is prepared are readily available, have no known toxic components, and are rich sources of isoflavones, such foodstuffs preferably being red clover or soya. It is preferred that the ratio of genistein and/or its methylated derivative biochanin A to daidzein and/or its methylated derivative formononetin in the extract is between 1:2 to2:1. Other plant components with oestrogenic 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 isoflavones. The term phytoestrogens is used hereafter to indicate a predominance of isoflavones with lesser amounts of lignans, coumestans and flavones.

P-%WPDOCSkGRSSPEC"No0en.doc 22/8101 22- 8-01; 6:01 612 93645173 17/ 36 12 The extract in accordance with the invention may be obtained from a number of 10 different sources. Preferably the extract is prepared from clover such as red clover or subterranean clover or from soya which contain high levels of phyto-oestrogens. However, any source rich in isoflavone phyto-oestrogens may be used instead if desired.

Various different isoflavones have been identified from the above sources they are principally genistein and diadzein and also biochanin A, formononetin and glycitein. In plants these compounds occur principally in a glycoside from bound to sugars such as glucose, with smaller amounts present as the aglucone forms. The formulae of the isoflavones are:

SOH

R o -r daidzein R- H istcin R OH

S..HO

*H

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 blochanin 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 and 0-desmethylangolensin.

In vitro and in vivo studies have indicated that genistein, biochanin A, equol, daidzein, formononetin all have oestrogenic activity in descending order. 0-desmethylangolensin is only very weakly oestrogenic and glycitein is non-oestrogenic.

P-AWPDOCS\GRSSPECt&Noogen.Ooc 22/8/01 22- 8-01; 6:01 612 93645173 18/ 36 13 In animal and in vitro studies, genistein has been shown to have greater oestrogenic/antioestrogenic activity and SHBG-stimulating capacity than the other Asoflavenes 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, hypocholesterolaemia, anti -angiogenesis, anti-oxidation, anti-carcinogenesis for example are not yet fully known.

The biological effects of the different oestrogenic isoflavones, and their protential antioestrogenic effect are therefore uncertain. However, it has been found by the inventor that an isoflavone extract according to the invention is useful in the treatment of benign breast disease, premenstrual syndrome (PMS), symptoms associated with menopause, cancer of the prostate, cancer of the bowel, free of side effects which may have been expected to be associated with oestrogenic and antioestrogenic compounds It is thought that because the methyl forms (biochanin A and formononetin) ultimately are largely o demethylated to their principals, genistein and daidzein, with improved biological efficacy, it is unimportant whether the isoflavones are present in the claimed product in the methylated or demethylated forms.

Given that the relative biological importance of the two isoflavone 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 diet in approximately equal proportions, then it is prudent that both isoflavones be present in the claimed product in approximately equal proportions.

The ideal sources ofphytoestrogens for preparation of an extract, or as may otherwise be referred to as a health supplement extract in accordance with the invention are preferably those which (i) 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 a to provide high yields, and (v) have no known toxic components requiring selective removal or inactivation.

P*WPOOCSGRS\SPECINovogen.doc 22/8/01 22- 8-01: 6:01 612 93645173 19/ 36 14 The extract in accordance with the invention may be obtained from a number of different sources.

Soya (soya max, otherwise known as soy) and clovers (Trifollium Spp) containing significant levels of isoflavones as mentioned above. Preferably the phytoestrogens are extracted from a clover, such as red clover or subterranean clover or from soya that contain high levels of phytoestrogens.

Certain clovers, such as red clover pratense) and subterranean clover suberranean) are 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 up to 3-5 (T subterranean) and 1-3 g% pratense). 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 ($200/tonne) than crops such as soya and lentils ($500/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. In contrast clover flowers or 'tops', traditionally used by herbalists as a source of'red clover', contain essentially no isoflavones.

Thus it can be seen that 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.

P"WPDOCSGR=SPECNovogn.doc 2218/01 22- 8-01; 6:01 612 93645173 20/ 36 An additional feature of clovers is that there are wide varieties of cultivars with widely differing isoflavone levels and types. Accordingly, isoflavone analysis is required to be carried out on clovers as clover cultivars may contain negligible isoflavones 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 phytoestrogens but the substantially poorer yield of isoflavones compared to clovers means that the manufacturing costs are substantially greater and there is substantially greater amounts of waste products which required disposal of 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 represents only a small proportion by weight of the intact bean. However, 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 perveived nutritional value, normally are separated and removed by physical mean. 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 removed the small hypocotyl. The hypocotyl contains 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 0000 be either blended or used separately as a dietary supplement in a variety of 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 ofphyto-oestrogens, for example by chemical extraction with a mixture of water and organic solvent. This extract could be added to other leguminous extracts such as from 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 PF:WPDOCSGRS\SPECNovogen.doc 22/01 22- 8-01; 6:01 6 12 93845173 21/ 36 16 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 isoflavones; 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.

Any leguminous plants such as detailed here could be used as sources of isoflavones; Indian liquorice (Abrus precatorius); various species of Acacia spp. including A. aneura, A, cibaria, A.

longifoblia and A. os-walii; ground nut pio tuberusa); ground pea rachis hypogea); milkc vetch A. srstragalus edults); marama bean Bauhinia esculenta); sword bean Cajanus cajan indicus); jack bean (Canavalia ensi/ormis); sword bean (Canavalia gladiata); seaside sword bean Canavalia rosea); various Cassia spp. including C floribunda, C laevigata, and C occidentalis, carobbean Ceratonia siliqua); chick pea (Cicer arietinum); yebnut (Cordeauxia edulis); various Crotalaraia spp. including C laburnifoblia and C. pallida; cluster bean (Cyamposis psoraioides); tallow tree Edtariaum senegalense); sword bean (Entada scandens); balu Erythrina edulis); soyabean (Gycline max); inga (Ingaedulis); Polynesian chestnut (Indarpus fagjfier); hyacinth bean Lablab purpureus); grass pea or Indian vetch Lathyrus sativus); cyprus vetch (Lathyrus ochrus); lentil (Lens culinaris); jumping bean (Leucaenal eucocephala); various Lupinus spp. including L. albus, L. luteus, L. angustifiblium, L. mutabilis, and L. cosentin ii, ground bean (IVauuuysIma geocarpa); horse gram (Macrotylorna unifoblium); alfalfa (Medicago sativa); velvet bean (Mucuna pruriens); yamn beans (Pachyrhyzuz erosus, P.

tuberosus); African locust bean (Parkia clappertoniana); Parkia speciosa; oil bean tree .:(Pentaclethra macrophylla); various Phasecius spp. including P. aculifiblius, P. vulgaris, P.

luntus, P. coccineus, P. adenathus, P. anguiris. P. aureus, P. calcaratus, P. mungo, and P.

polystachyus; garden pea (Pisum sativum); djenko bean (Pithecolobium lobatum); mesquite (various Pros opis spp.); goa bean (Psophocarpus scandens, P. tetragonolobus); variousPsoralea spp.; Sesbania bispinosa; yarn bean (Sphenostylis stenocarpa); tamarind (Tamarindus incida); fenugreek (Trigonella foenum-graecum); vetches (various Vivia spp. including V. sativa, V atropurpurea, V ervilia, and V. monantha); broad bean (Viciafaba); black gram Vigna mun go); P'.AWPDOCSGRSPECJ~b..gentdoc 22/Mi1 22- 8-01; 6:01 612 93645173 22/ 36 17 various Vigna spp. including V. radiata, V. aconitifolia, V adanatha, V angularus, V. tribolata, V. umbelata, and V unguiculata; and, earth pea Voandzeia subterranea).

To provide a similar amount ofisoflavone to that contained in most traditional 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 selected 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 benefit of clovers as a source of isoflavones has traditionally be limited due to the fact that is not normally considered a food for human consumption as above mentioned. The present invention, being an extract, now allows clovers to be utilised as a major source of high levels of isoflavones. It is somewhat surprising that such could be utilised in the method of the invention based on the fact that high levels of consumption of clovers containing isoflavones have been linked with reproductive problems in sheep, as herein before described.

The invention also concerns health supplements in the form of formulations or compositions containing the extract discussed above together with standard dietary suitable excipients, diluents, carriers, or with a food. Ideally the formulation is in the form of a pill, tablet, capsule or similar dosage form. Standard formulation methodology employed in the art may be utilized to achieve such formulations according to the invention.

The formulations may be a variety of kinds, such as nutritional supplements or health supplements, pharmaceutical preparations, vitamin supplements, food additives or foods P:\WPDOCSAGRS\SPECHNovogen.doc 22/8/01 22- 8-01; 6:01 612 93645173 23/ 36 18 supplemented with the isoflavone containing extracts of the invention, liquid (for example after alcohol removal) 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 as above mentioned. The inclusion of the subject isoflavone extract in herbal remedies and treatments is also a preferred part of the invention. Herbal treatments are traditionally fraught with uncertainty as activity is rarely standardised or analysed, and the content or material source of products is uncertain. The extracts of this invention are of defined content, origin and activity.

It will be appreciated that standard methodology used in the art may be utilized to ensure the content of the isoflavones both within the starting material, for example the clover or soya, and the end product (the extract health supplement).

S.Compositions or formulations (as above mentioned) in accordance with the invention preferably contain isoflavones in an amount from 20 mg to 200 mg.

The invention is also directed to the amelioration, prevention or treatment of various conditions responsive to treatment with the isoflavone substances of this 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. For example, dosages of up to or greater than 1000 mg may be suitable to treat some conditions. In order to obtain 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.

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: PA.IWPDOCSRS1SPECI2ovcgen.doc -22/8/01 22- 8-01; 6:01 612 93645173 24/ 36 19 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 The environmental 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.

As previously discussed herein, common leguminous foodstuffs consumed in Western countries i* (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 relatively low isoflavone levels compared to whole soya, indicating that relatively large amounts of these would need to be consumed on a regular basis to deliver the required isoflavone levels.

The product according to the present invention yields a constant and accurately known amount of isoflavones. The accurately determined quality and quantity of the active isoflavones in the product, and its easy consumability when compared with the almost impossible task of eating 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 PMS and menopausal symptoms is especially important. The product and methods of the invention modulates the production and/or function of endogenous sex hormones in humans to modify or produce health P:\WPDOCSGRS\SPECINovogen.doc 22/8/01 22- 8-01; 6:01 612 93645173 25/ 36 improving effects, including the following: reduced risk of development of atherosclerosis; (ii) reduced risk of development of cancer of the prostate; (iii) reduced risk of cancer of the 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-menstrual tension (PMT); (vii) reduced risk of development of many untoward symptoms commonly associated in women with menopause; and (viii) treatment of benign breast disease in women (benign or cystic breast disease associated with non-malignant swelling and tenderness of breast tissue).

Symptoms associated with menopausal are well known and may otherwise be referred to herein as "menopausal symptoms" or "menopausal syndrome". These include hot flushes, night sweats S. and depression.

The invention is also directed to a method for the treatment or prevention of benign breast disease, premenstrual syndrome (PMS), symptoms associated with menopause, cancer of the prostate, or cancer of the bowel as previously referred to.

Cancer of the breast generally is considered to be associated with excess oestrogen activity. Most breast cancer cells display more oestrogen receptors than normal breast cells and stimulation of these receptors by endogenous estrogens is thought to be a prime source of stimulation of their malignant growth. Currently synthetic anti-estrogens are used to prevent or treat the growth of malignant breast cells. The use of estrogens such as HRT exacerbates breast cancer.

Surprisingly, the oestrogenic extract of the invention is effective in the treatment of breast cancer.

Cancer of the prostate generally is considered to be associated with sex hormone dysfunction and the growth of prostatic cancer cells is influenced by estrogens and androgens. The standard therapy for prostate cancer is the administration of oestrogen. The use of compounds having antioestrogenic activity such as isoflavones would be contraindicated for prostate cancer. The inventor has surprisingly found that an isoflavone extract is effective in the treatment of prostate cancer.

P:\WPDOCSGRSSPEClNovogendoc 22/8/01 22- 8-01; 6:01 612 93645173 26/ 36 21 Pre-menstrual syndrome has uncertain aetiology and pathogenesis, although most certainly is associated with excess oestrogen function. It has surprisingly been found that an isoflavone extract is effective in the treatment ofpre-menstrual syndrome.

Menopausal syndrome is associated with declining oestrogenic activity with advancing age.

Adverse clinical symptoms may be treated with oestrogen replacement therapy, which may cause side effects. Anti-oestrogen therapy is contraindicated for treating symptoms of menopause. It has been surprisingly found that a plant extract containing isoflavones with oestrogenic and antioestrogenic properties is beneficial in the treatment of menopausal symptoms, producing alleviation of adverse clinical symptoms, free of side effects with long term health benefit..

Benign (or cystic) breast disease has unknown aetiology. However, its association in women with the mid- stages of the menstrual cycle is strongly suggestive of increased oestrogen activity. The S* •use of estrogens would be contraindicated in this condition. It has been surprisingly found that an isoflavone extract is effective in the treatment of benign (or cystic) breast disease.

Atherosclerosis is associated with cholesterol metabolism which in turn is associated closely with S. *oestrogen metabolism. The generally higher incidence of atherosclerosis in young men versus young women, the rising incidence in women receiving oestrogen replacement therapy, all point to the moderating influence of estrogens on cholesterol metabolism.

Oestrogenic isoflavones (such as genistein, daidzein, biochanin A and formononetin) belong to the category of plant chemicals referred to as phytoestrogens. The term "phytoestrogen" embraces a variety of plant chemicals including isoflavones, resorcyclic lactones, lignans and coumestans ('non-steroidal' phytoestrogens) and 'steroidal phytoestrogens' such as oestradiol.

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

PAWPDOCSAGRSLSPECftNovoendCC 22/&/01 22- 8-01; 6:01 612 93645173 27/ 36 22 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.

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

The dried material is subject to chemical extraction, by extracting in an aqueous:organic solvent mix. The aqueous phase is required to extract the water-soluble glycoside from ofisoflavones, 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 watersolvent 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 week or even longer. It has been determined that the adequate times for maximal recovery ofisoflavones are 2 weeks at 50 0 C and 24 hours at 90 0

C.

The supernatant is separated from the undissolved plant material and the organic solvent 'removed by distillation. The aqueous supernatant is then concentrated, by 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.

Soya may be subject to the same chemical extraction and processing.

Example 2 Preparation of a 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 were PAWPDOCSGRS'SPECINovogen.doc 22/8/01 22- 8-01; 8:01 612 93645173 28/ 36 23 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.

This hypocotyl product can be further processed by the extraction procedure of example 1 to yield a further enriched extract containing phytoestrogens.

Normal soybean processing steps isolate the hulls and then these are discarded or processed 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 (prepared according to Example 1) 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 mg.

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

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

0 Pre-treatment Post-treatment change Beans only Patient 1 5.77 5.46 5.4 P.\WPDOCS\GRSSPECKNovogen.doc 22/8/01 22- 8-01; 6:01 612 93645173 29/ 36 24 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 6.998 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 Adminsitering Soy Hypocotvl to Humans Fifteen volunteers (8 women, 7 men) were given 5 g of soy hypocotyl extract produced according to Example 2 containing (45 mg daidzein and 5 mg genistein) daily for 2 months. The extract was consumed as a powder added to the diet.

The effects on cholesterol levels are shown in the following table. The individuals are grouped according to their pre-treatment cholesterol 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) P'WPDOCSGRSPECN~ovogen4oc W2MI1 22- 8-01; 6:01 612 93645173 30/ 36 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 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.

Example 5 Further Therapeutic Treatments Compositions Compositions comprising an extract of clover were prepared in accordance with Example 1.

These compositions, for convenience referred to as "the inventive composition", were prepared comprising 40 mg, 80 mg, 120 mg, 160 mg and 240 mg ofisoflavones.

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-estrogen. The PSA levels, a marker for prostate cancer, were stabilised in these patients and there has been no rise in the S. :PSA levels subsequently. This demonstrates the treatment of prostatic cancer in these individuals.

A further patient diagnosed with malignant prostate cancer (PSA 13.1 ug/L) was treated with the inventive compositions. The patient was treated with the composition comprising 160 mg per day phytoestrogen, seven days prior to prostatectomy. Histological comparison was made of the pre-operative needle biopsy and the prostatectomy specimen. The needle biopsy revealed low grate infiltrating adenocarcinoma. The prostatectomy specimen showed mild patchy microvacuolation and prominent apoptosis (programmed cell death). Lymph nodes were negative P\WPDOCSGRS\SPECNovogen.do 22/8101 22- 8-01; 6:01 612 93645173 31/ 36 26 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.

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 were 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 compositions 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 and a dose response change was observed between the 40 mg and 160 mg dosage range. This indicating that 160 mg per day was the most effective dosage for treatment of menopausal symptoms.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that that prior art forms part of the common P:\WPOOCS\GRS\SPECIlNovogen.doc 22/8/01 22- 8-01; 6:01 ;612 93645173 #3/3 32/ 36 general knowledge in Australia.

P:%WPDDCSGRSSPECZhNovcgen.doc 2211VOI

Claims (8)

1. A pharmaceutical composition of a phyto-oestrogcn isuflavone extracted rom clover or soya, which extract comprises: genistein and its methylated derivative biochanin A to daidzein and its methyiated derivative formononetin; Or genisteiri to daidzein and its methylated derivative formononetin;, or genistein to formOonnein; or genistein and its methylated derivative biochanin A to formorionetin; or hiochanii A to formononein; or genistein and its methylated derivative biochanin A to daidzein in aratio between 1:2 to 2:1. A composition according to claim I which is an extract from red clover.

3. A composition according to claimn 1 which is an extract from soya hypocotyls.

4. A composition according to any one of claims 1. to 3 in thec form of tablets, coated tablets or capsules.

5. A composition according to claim 4, wherein the composition contains R0mg, 120mng, 160mg or 240mg of said isoflavone extract.

6. A composition according to claim 4, whercin the composition contains I1000mg, Or greater of said isoflavone extract.

7. A composition according to any one of claims 1 to 3 in the form of a solution, drink, suspension Or syrup.

8. A composition according to any one of claims 1 to 7 which further comprises at least one cxcipient, diluent and/or carrier. ),IPO5T%%Sp~kd%2 IO'/(J4 COMS ID No: SBMI-00907997 Received by IP Australia: Time 13:34 Date (Y-KM-d) 2004-09-10 Fax sent by 61 2 9262 1080 DAVIES COLLISON CAUE 10/09/04 14:19 Pf: 5/8 -29-

9. A composition according to claim 1, substantially as described herein by reference to the Examples. DATED this 10th day of September 2004 Novogen Research Pty Ltd By its Patent Attorneys DAVIES COLLISON CAVE oooo oooo ooo o P'.\WPDOCSTX5'Sy\Cpn7WC b2Orvv3 MOMM COMS ID No: SBMI-00907997 Received by IP Australia: Time 13:34 Date 2004-09-10