Link:  Pharm/Biotech Resources

Title:  Method for modulating steroidogenic activity

United States Patent:  6,905,714

Issued:  June 14, 2005

Inventors:  Ong; Yek Cheng (Singapore, SG); Yong; Eu Leong (Singapore, SG)

Assignee:  National University of Singapore (Singapore, SG)

Appl. No.:  793772

Filed:  February 27, 2001

The present invention relates generally to a method for modulating steroidogenic activity and a composition useful for same. The present invention further relates to a composition comprising a steroidogenic modulator useful for modulating physiological processes mediated by the androgen receptor or an active form thereof or complex comprising same and/or for modulating physiological processes mediated by estrogen receptors. The composition of the present invention preferably comprises an extract of herbs or botanical or horticultural equivalents of the herbs or chemical or functional equivalents of one or more components of the herbal extract thereof.

FIELD OF THE INVENTION

The present invention relates generally to a method for modulating steroidogenic activity and a composition useful for same. The present invention further relates to a composition comprising a steroidogenic modulator useful for modulating physiological processes mediated by the estrogenic or androgenic receptor or an active form thereof or complex comprising same and/or for modulating physiological processes mediated by the estrogen and androgen receptors. The composition of the present invention preferably comprises an extract of herbs or botanical or horticultural equivalents of the herbs or chemical or functional equivalents of one or more components of the herbal extract.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

Herbal formulations comprising extracts of more than one herbal plant have been used for centuries in Traditional Chinese Medicine (TCM). There is now an increasing acceptance of their value and therapeutic efficacy in Western medicine. TCM has its own unique and philosophical theory in etiology, pathology, diagnosis, pharmacology and therapeutics. Many concepts surrounding TCM have particular relevance to Western medicine such as viewing parts of the body as an organic whole, considering inter-relations and influences between organs and being aware of relevant adaptation of the human body to the natural environment.

Despite the effectiveness of many herbal formulations in the treatment of a range of conditions, little is known about how the formulations work. Information on the mechanisms of action for herbal formulations would permit the rational design of particular compositions or chemical synthetic production of one or more components of the compositions as well as ensuring that the appropriate composition is selected for a particular ailment or condition.

Androgens are one of a group of steroid hormones which include testosterone and dihydrotestosterone (DHT). The androgens stimulate the development of male sex organs and male secondary sexual characteristics such as beard growth, deepening of the voice and muscle development. The principal source of these hormones is the testis but they are also secreted in small amounts from the adrenal cortex.

Androgens act through an X-linked androgen receptor (AR) to regulate androgen-responsive genes. This in turn leads to a cascade of metabolic events which manifest as androgenic effects including male sexual development in the fetus, secondary sexual development and sperm production at puberty, anabolic processes including muscle growth and bone density, male sex drive (i.e. libido), hair growth, skin condition, and physical stamina in adults (Wilson, 1992).

Naturally occurring and synthetic androgens are used in replacement therapy such as to treat delayed puberty in boys, hypogonadal men, impotence and as anabolic agents and in the treatment of cancer. However, only limited number of natural and synthetic androgens are known. As stated above, testosterone and DHT are examples of natural androgens. Miborelone and mesterolone are examples of synthetic androgens. The chemical structure common to steroidal hormones, including androgens, is the 1,3 -cyclopentanophrenanthrene ring system.

Androgens in the cell bind to the ligand-binding domain (LBD) of the androgen receptor. Upon ligand binding, the androgen receptor which comprises the transactivation domain (TAD), DNA-binding domain (DBD) and the ligand bound LBD adopts a transactivational conformation and translocates to the nucleus where it binds specifically to the androgen-responsive element (ARE) of the androgen-regulated gene. Following the recruitment of DNA polymerase and co-activators to form the quaternary transcription complex, the gene downstream of the ARE is expressed. Hence, the prerequisite of androgen receptor activity is the specific binding of a ligand into the hydrophobic core of the androgen receptor LBD.

As stated above, there are many conditions associated with low androgen levels, hypofunction of the androgen receptor (Yong, 1994; Tut, 1997; Lim, 1997; Yong, 1998; Wang, 1998, Ghadessy, 1999; Ong, 1999; Dowsing, 1999), declining androgen action associated with aging and other conditions.

There is a need to identify naturally occurring compounds and materials generally from non-mammalian sources, which interact with or activate the androgen receptor and/or the androgen-androgen receptor complex leading to transcription of an androgen-responsive gene. There is also a need to identify estrogenic modulating agents, as well as agents modulating the effects of other members of the steroid/nuclear receptor superfamily of proteins. The identification of such compounds and molecules is needed for the development of therapeutic compositions and/or nutraceutic applications.

In work leading up to the present invention, the inventors sought an edible plant extract with steroidogenic properties.

Eucommia Ulmoides OLIVER (Du-Zhong) is a large deciduous tree which originated in China. The bark of the tree (commonly referred to as Cortex eucommiae) has been used for natural medicine since ancient times (Wei, 1955; Li, 1987).

Decoctions of E. Ulmoides (EU) bark have been used for, amongst other things, the relief of back pain, to increase strength, to make bones and muscle strong, to increase recovery from fatigue, to increase ability to remember and to induce an anti-aging effect. Mechanical training and the use of EU leaf extracts co-operatively can increase the ability of rats to avoid lactate accumulation in skeletal muscle and the administration of the EU leaf extract along with light intensity training enhances the ability of a muscle to resist fatigue (Li, 1996b). EU leaves contain compounds similar to the bark and are reported to have similar pharmacological effects. Since irridoid monoglycosides, such as geniposidic acid and aucubin in EU can stimulate collagen synthesis in aged model rats (Li, 1991a), it is thought that the active compound is actually geniposidic acid or aucubin.

Crude extracts of Tochu tea, an aqueous extract of EU leaves, have a suppressing effect on the induction of chromosome aberrations in CHO cells and mice. Out of 17 Tochu tea components, five irridoids (geniposidic acid, geniposide, asperulosidic acid, deacetyl asperulosidic acid and asperuloside) and three phenols (pyrogallo, protocatechuic acid and p-trans-coumaric acid) were found to have anti-clastogenic activity (protective effect against chromosomal aberrations). Since the anti-clastogenic irridoids had an alpha-unsaturated carbonyl group, this structure was considered to play an important role in the anti-clastogenicity (Nakamura, 1997).

Ingestion of EU bark and leaves, and/or their extracts, cause no known side effects.

In accordance with the present invention, the inventors have determined that certain extracts of EU exhibit steroidogenic activity. The identification of the activity in EU extracts permits the rational design of therapeutic protocols and compositions useful in ameliorating the symptoms of disease conditions. It also permits the production of the active agents in the extracts in purified or chemical synthetic form.

SUMMARY OF THE INVENTION

In the context of the present invention, a composition "consisting essentially of" recited ingredients will elicit physiological conditions and responses mediated by estrogen or estrogen receptor or androgens or androgen receptors, preferably in a synergistic manner compared to the response obtained using estrogen or androgen alone as the eliciting compound. As a preferred instance of synergistic effect, a basal degree of activity of steroid alone or of the composition alone might be two-fold activation of the steroid receptor activity. Synergistic action is observed when greater than four-fold activation is observed, preferably when greater than six-fold activation is observed, more preferably when greater than eight-fold activation is observed, even more preferably when greater than 10-fold activation is observed when the steroid and the composition of the invention are applied together in the assay.

One aspect of the present invention is a method of extracting active steroidogenic compounds from EU plants. The steps comprise macerating the tissues of the EU plant, extracting the active compounds with steroidogenic activities with a solvent system, separating the liquid from the solid phase and adding water to precipitate the undesired compounds that may cause side-effects and/or reduce efficacy of the main active compounds with steroidogenic activities. As tissues of the EU plant, all tissues can be used; bark or leaves are preferred, and bark is most preferred.

According to this invention, the term ethanolic EU extract refers to EU extract using a solvent system consisting only of ethanol. The term hydroethanolic EU extract refers to the EU extract using a solvent system comprising ethanol and 20% water, in which the water component could be added before or after the extraction process. For the purpose of the examples, the extract was dried, weighed and resuspended in ethanol at a known concentration.

Another aspect of the present invention contemplates a method of modulating a steroidogenic-mediated physiological condition in a subject, said method comprising administering to said subject an effective amount of a formulation comprising an extract of EU or botanical or horticultural equivalents of EU or chemical or functional equivalents of the extract or a purified, or chemically synthesized form of one or more components of the extract. Another aspect of the present invention is directed to a composition comprising a part of EU or a botanical or horticultural equivalent of EU or an extract thereof or chemical or functional equivalents of the extract or a purified, or chemical synthetic form of one or more components of the extract wherein said composition is effective in modulating a steroidogenic-mediated condition in a subject.

Another aspect of the invention is an article of manufacture that comprises an extract of the invention, or a purified or chemically synthesized molecule that is a component of the extract, that has steroidogenic activity, preferably synergistic activity together with a steroid compound (especially with an androgen or estrogen compound). In such an article of maufacture, the extract or purified or synthesized component is packaged together with written materials that provide instructions or describe or urge use of the extract or purified or synthesized component to modulate, and especially to enhance, a physiological condition or response mediated by a steroid, especially a condition or response mediated by an androgen receptor or by an estrogen receptor.

Yet another aspect of the present invention is directed to a purified or chemical synthetic molecule form of EU, or a botanical or horticultural equivalent thereof, or an extract thereof which molecule is capable of modulating a steroidogenic-mediated condition.

Still another aspect of the present invention provides for the use of EU or a botanical or horticultural equivalent thereof or an extract thereof or a chemical or functional equivalent of the extract or a purified or chemical synthetic form of one or more components of the extract in the manufacture of a medicament for the treatment of steroidogenic-mediated conditions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the identification of steroidogenic-modulating properties in a formulation comprising parts of EU or an extract thereof.

Accordingly, one aspect of the present invention is a method of extracting active steroidogenic compounds from EU plants. EU plant parts are macerated prior to the actual extraction process. Fresh EU plant parts or preferably dried plant parts are macerated using any of the known process such as chopping into small pieces, grinding into powder or breaking up into fine particles using a high speed blender. Although active compounds with steroidogenic activities can be extracted from different parts of the EU plant, the preferred part of the EU plant for extraction is the bark.

A soaking method is one of the several methods of extracting the active compounds from the EU plant. In this method, the macerated plant is soaked with a solvent system and left for a period of time to allow the active compounds to dissolve into the solvent system. To enhance the diffusion of the active compounds into the solvent system, the mixture can be mechanically agitated and/or heated to a pre-determined temperature. The mechanical agitation methods include but are not limited to the following: shaking, vortexing, swirling, stirring and ultrasonicating.

After a sufficient period of time for the diffusion of the active compounds into the solvent system, the liquid is separated from the solid by any one of the well-known techniques such as filtration and centrifugation.

The solvent system can comprise any of the well-known systems such as an organic solvent or a combination of organic solvents. Preferably, the organic solvent is alcohol and more preferably, ethanol. Other organic solvents such as hexane, dichloromethane, ethyl acetate will also be effective. Water can be part of the solvent system. The percentage of water can range from 0% to 100%. If water is not part of the initial solvent system, water can be added to the liquid phase during any part of the soaking period. It is also possible to add water to the liquid phase after separation from the solid phase. One of the effects of the addition of water is to cause precipitation of non-steroidogenic compounds that may have adverse side-effects. Water can be added up to 20% by volume.

Another method of extracting active compounds from the macerated EU plant is to percolate the macerated EU plant with a continuously refluxed solvent system such as the soxlet-type method for extraction. After completion of the extraction process, the liquid containing the active compounds may be mixed with water (up to 20% by volume) to precipitate the undesired compounds. The precipitate can be separated from the liquid containing the active compounds with steroidogenic activity using any one of the well known separation methods such as filtration and/or centrifugation.

The solvents in the liquid phase containing the active compounds with steroidogenic effects can be evaporated off using any of the well known drying methods including but not limited to, rotary evaporation, speed-vacuum centrifugation or open-top drying. The dried extract is then suitable for use or storage. If desirable, the dried extract can be resuspended in suitable solvents prior to use.

For further purification, the extract, either dried and then redissolved in an appropriate solvent, preferably water or an alcohol, can be applied to a reverse phase chromatography column and the steroidogenic compounds can be eluted with a mobile phase comprising water and acetonitrile. Alternatively, or in combination with the reverse phase purification, the extract can be applied to a chromatography matrix comprising dextran crosslinked by an alkyl ether and the steroidogenic compounds can be eluted with an alcohol, preferably a C₁-C₆ alkyl alcohol, more preferably an n-alkyl alcohol.

Accordingly, another aspect of the present invention contemplates a method of modulating a steroid-mediated physiological condition in a subject, said method comprising administering to said subject an effective amount of a formulation comprising an extract of EU or botanical or horticultural equivalents of EU or chemical or functional equivalents of the extract or a purified or chemical synthetic form of one or more components of the extract.

A "steroid-mediated physiological condition" includes an androgen-mediated physiological condition, an estrogenic-mediated physiological condition and/or other physiological condition mediated through the steroid/nuclear receptor family of proteins. A "steroidogenic agent" includes an androgen, an estrogen and/or any other ligand interacting with the steroid/nuclear receptor family of proteins. Estrogenic agents are useful in hormonal therapy in hypoestrogenic states such as but not limited to menopause, osteoporosis and cardiovascular disease. An "estrogen-mediated physiological condition" also includes and encompasses conditions mediated via an estrogenic receptor or active or complex forms thereof.

Reference herein to an "androgen-mediated physiological condition" includes reference to the induction of physiological processes mediated via the androgenic receptor. These physiological processes include biological, endocrinological and other bodily processes which are induced, stimulated, enhanced or otherwise facilitated by the androgen receptor or androgen receptor complexes and/or its activated forms which are responsive to natural or synthetic androgens or other compounds which have androgenic properties such as being able to activate the androgen receptor.

The ability to activate or otherwise modulate the androgen and/or estrogen receptor may be tested in vitro or in vivo. Reference to in vivo includes the practice of the present invention in humans, primates, livestock animals (e.g. sheep, cows, pigs, goats, horses, donkeys), laboratory test animals (e.g. rabbits, mice, rats, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals.

For convenience, a "steroid receptor" encompasses the androgen receptor and/or estrogen receptor and/or other receptor belonging to the steroid/nuclear receptor family of proteins. A "steroidogen" encompasses an androgen, an estrogen and/or any other ligand interacting with the steroid/nuclear receptor family of proteins.

The term "modulate" and its variations including "modulating" and "modulates" includes the up-regulation and down-regulation of steroidogenic receptor activity or the activity of steroidogen-steroidogen receptor complex activity. This may be conveniently determined at the level of up-regulating or down-regulating target gene expression following modulation of steroidogen receptor activity.

The present invention is particularly directed to a formulation comprising EU or parts thereof or an extract thereof or botanical or horticultural equivalents of EU or chemical or functional equivalents of the extract from EU extract or purified or chemical synthetic forms of one or more components in EU or an extract thereof.

Reference herein to "Eucommia Ulmoides" or "E. Ulmoides", or "Du Zhong", or "EU" includes reference to botanical and horticultural equivalents thereof. Botanical and horticultural equivalents includes herbs and other plants related at the genetic, biochemical, or medicinal level to EU. For example, a medicinally functional equivalent plant may be indigenous to another country. Such a plant is encompassed by the present invention. Botanical equivalents of EU are described by Gu et al. (Z. M. Gu et al., Chung Kuo Chung Yao Tsa Chih 14:714-7171 (1989)) and encompass plants that are used among different ethnic groups as substitutes for cortex eucommiae. Their study and identification of samples of all original plants and materia medica from all locations producing Du Zhong identified 48 species of ethnic substitutes for Du Zhong from 17 genera of 10′ families:

Reference to the Du Zhong herb also encompasses natural and artificially created variants of EU. An artificially created variant includes a variant made by selective breeding or by genetic manipulation. A part of EU includes the bark, leaf, stem, root, flower, seed or other reproductive or vegetative portion of the plant or a combination of two or more of these portions.

The term "formulation" includes an extract of EU or parts thereof in liquid, solid or aerosol or vapour form. In a preferred embodiment, the formulation comprises an ethanolic or aqueous extract of EU.

Herbal extraction techniques were designed to maintain maximal levels of active components. The steps comprise macerating the preferred bark of the EU plant, extracting the active compounds with steroidogenic activities with a solvent system, separating the liquid from the solid phase and optionally adding water of up to 20% v/v to precipitate the undesired compounds that may cause side-effects and/or reduce efficacy of the main active compounds with steroidogenic activities. The solvent system is preferably an C₁-C₆ alcohol, which can also include water in an amount up to 20% v/v. The separation of the solvent extract from insoluble solids can be performed by any method typical in the art. Filtration is preferred, but centrifugation can also be efficiently employed.

The term "ethanolic EU extract" as used herein refers to EU extract using a solvent system consisting only of ethanol. A "hydroethanolic EU extract" refers to the EU extract using a solvent system comprising ethanol and 20% water, in which the water component could be added before or after the extraction process.

As defined herein, the EU extract is considered to exhibit steroidogen activity. More particularly, the extract itself or one or more components therein are considered herein to be "androgen modulators" or "estrogen modulators" (i.e. steroidogen modulators) in that the extract or its components are capable of modulating the activity of the androgen receptor or a complex comprising same and/or the activity of the estrogen receptor or a complex, etc. The steroidogen modulators of the present invention are isolatable or obtainable from EU are defined as being "phyto-androgens" or "phyto-estrogens" (i.e. phyto-steroidogens) due to their botanical origin. Reference to an "androgen" in the term "phyto-androgen" is not to imply any limitation as to the structure of the phyto-androgen and the term extends to any component of EU or any extract of EU or any component or extract from a botanical or horticultural relative of EU which is capable of modulating androgen-receptor activity. Androgen receptor activity is conveniently measured in vitro or in cell culture by assays of transactivation and/or downstream target gene expression. "Downstream target genes" whose expression is regulated by androgen receptor activation are known in the art. Transactivation assays, in which androgen or other steroid receptor binding sites are coupled to reporter genes and then these constructs are placed into cells either in vitro or in vivo, are well-known in the art. Androgen receptor activity can also be assessed in vivo by measurement of physiological or anatomical parameters, e.g. development of male sexual organs, increase in muscle mass or spermatogenesis, known in the art to be regulated by androgen activity. Accordingly, the term "androgen modulator" includes a formulation or composition or extract of EU or a part of EU or a purified or chemical synthetic form of a component of the extract or part of EU or its botanical or horticultural equivalent. Similar comments, in relation to development of feminine characteristics, including regulation of serum levels of hormones related to the estrus cycle, apply in relation to "phyto-estrogen".

Reference herein to the "androgen receptor" or "estrogen receptor" includes reference to the naturally occurring receptor or its recombinant forms as well as splice variants or other genetic variants including polymorphic variants. Furthermore, the term encompasses complexes comprising the receptor and other molecules (e.g. androgen or estrogen) as well as the receptor's monomeric, dimeric, trimeric or multimeric forms including homodimeric, homotrimeric, homomultimeric, heterodimeric, heterotrimer and heteromultimeric forms. The receptor may be membrane associated or it may have translocated to the nucleus or be associated with chromosomal DNA.

The androgen receptor when activated by androgens, including phyto-androgens, has the intrinsic ability to bind to specific DNA sequences. Following binding, the transcriptional activity of a target gene (i.e. a gene associated with the specific DNA sequence) is modulated as a function of the ligand bound to the receptor. Functional activity of androgen receptors can be measured with reporter gene(s), such as firefly luciferase, coupled to steroid response elements, that are co-expressed with the androgen receptor (Quigley, 1995).

Processes capable of being modulated by an androgen or via an androgen receptor, in accordance with the present invention, include but are not limited to, the in vivo modulation of male sexual development in the fetus, secondary sexual development at puberty and anabolic processes (muscle growth, bone density), male sex drive (libido), skin condition, hair growth and physical stamina in adults, lipid metabolism, modulation of androgen-related processes (e.g. aging, stamina, muscle tone, spermatogenesis and the like). As readily recognized by those of skill in the art, the availability of selective phyto-androgen(s) in EU extracts for the androgen receptor makes it possible, for the first time, to develop nutraceutics in the form of, for example, food supplements and natural medicines for human and animal consumption without the need for special prescriptions. Such in vivo applications of the invention process may allow the daily modulation of various biological processes related to androgen action with reduced occurrence of undesirable side effects and the like. Processes capable of being modulated by estrogen or via an estrogen receptor include menopause, osteoporosis and cardiovascular disease.

The ability of compounds of the invention to modulate such processes may be evidenced in any number of ways. For example, EU extracts, in the presence of a ligand (e.g. DHT) exert a potentiating effect on the expression of genes under the control of androgen-response elements.

Accordingly, another aspect of the present invention is directed to a composition comprising a part of EU or a botanical or horticultural equivalent of EU or an extract thereof or chemical or functional equivalents of the extract or a purified or chemical synthetic form of one or more components of the extract wherein said composition is effective in modulating a steroidogenic-mediated condition in a subject.

The composition of the present invention may also be referred to as a herbal composition, natural medicine, a formulation and/or a formulation or composition with medicinal or ameliorating properties. The terms "formulation" and "composition" are used herein interchangeably.

The subject formulation in the form of a part of EU or an extract thereof may be administered in any suitable form including ingestion, topical application or via vapour or aerosol means. The term "ingestion" includes administering the herb or extract via edible or liquid means.

For in vivo applications, the extract or plant parts can be incorporated into a pharmaceutically acceptable formulation including a carrier or diluent for administration. Those skilled in the art will readily determine suitable dosage levels.

Reference herein to "suitable dosage levels" includes reference to levels of phyto-steroidogens sufficient to provide circulating concentrations high enough to effect activation of steroidogen receptor(s) or to agonize activity of a steroidogen-steroidogen receptor complex. Such a concentration typically falls in the range of about 1 nM up to 2 nM; with concentrations in the range of about 100 nM to 200 nM being preferred. Generally, however, the concentration is measured in terms of w/w of dried extract or v/v of liquid extract.

Exemplary mass amounts are from 1 to about 80% w/w or more particularly from 5 to about 50% w/w or even more preferably from 5 to about 20% w/w.

Exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, subcutaneous, intramuscular, subcutaneous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.

For the preparation of oral liquids, suitable carriers include emulsions, solutions, suspensions, syrups and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, sweetening, flavouring and perfuming agents, and the like.

For the preparation of fluids for parenteral administration, suitable carriers include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain additional ingredients such as preserving, wetting, emulsifying and dispersing agents. Formulations may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured as solutions in sterile water, or some other sterile injectable medium, immediately before use.

The present invention further contemplates purified or chemical forms of one or more components of EU or its extracts. A "purified" form means a component which has undergone at least one purification step such as HPLC, electrophoresis, immunoprecipitation, ammonium sulphate precipitation or high speed centrifugation.

Accordingly, another aspect of the present invention provides a purified or chemical synthetic molecule form of EU or a botanical or horticultural equivalent thereof or an extract thereof which molecule is capable of modulating an steroidogenic-mediated condition.

Purification of components in the EU extract may be readily accomplished by any convenient means. For example, the extract may be fractionated and fractions then tested in a cotransfection bioassay (e.g. see Ong, 1999). In this assay, an EU extract or a fraction thereof is contacted with a cell cotransfected with a steroidogen receptor expression plasmid and a luciferase-receptor plasmid containing two steroidogen responsive elements. Luciferase activity is then a measure of steroidogenic activity. Any other reporter molecule may be used.

Accordingly, another aspect of the present invention contemplates a method for identifying a component of EU having steroidogenic properties, said method comprising contacting an extract of EU or a fraction of said extract with cells cotransfected with a steroidogen expression plasmid and a genetic sequence encoding a reporter molecule containing an steroidogen responsive element and determining the level of activity of the receptor molecule.

Yet another aspect of the present invention is directed to the use of EU or a botanical or horticultural equivalent thereof or an extract thereof or a chemical or functional equivalent of the extract or a purified or chemical synthetic form of one or more components of the extract in the manufacture of a medicament for the treatment of steroidogenic-mediated conditions.

Still yet another aspect of the present invention provides a method for hormonal therapy in a subject, said method comprising administering to said subject an effective amount of an extract of EU or botanical or horticultural equivalents of EU or chemical or functional equivalents of the extract or a purified or chemical synthetic form of one or more components of the extract for a time and under conditions to modulate steroidogenic activity.

In one embodiment, the steroidogenic activity is androgenic activity. This is important for modulating male sexual development, sexual function and infertility amongst other conditions.

In another embodiment, the steroidogenic activity is estrogen activity. The latter is important for the treatment of menopause, osteoporosis and cardiovascular disease amongst other conditions.

Claim 1 of 18 Claims

1. A composition comprising:

a preparation of Eucommia ulmoides prepared by ethanol extraction thereof and chromatography a) over a hydroxypropylated, cross-linked dextran matrix having an exclusion limit for peptides of 4 kilodaltons and an exclusion limit for small organic molecules of 5 kilodaltons with elution using n-butanol or b) over a reverse phase matrix with elution by an aqueous buffer and acetonitrile gradient or c) over a diol matrix with elution using at least one organic solvent;

that lacks fluorescent components exhibiting R_f values of 0 and 0.4 and also lacks vanillin-sulphuric acid reactive components exhibiting R_f values 0.47, 0.62, 0.71 and 1 when the composition is separated by two dimensional thin layer chromatography on silica gel and eluted in the first dimension with dichioromethane and in the second dimension with hexane.

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