WO2007031140A2

WO2007031140A2 - Extracts from epimedium species method for production and use thereof

Info

Publication number: WO2007031140A2
Application number: PCT/EP2006/006929
Authority: WO
Inventors: Egon Koch; Clemens Erdelmeier; Hermann Hauer
Original assignee: Dr. Willmar Schwabe Gmbh & Co. Kg
Priority date: 2005-07-15
Filing date: 2006-07-14
Publication date: 2007-03-22
Also published as: EP1904075A2; WO2007031140A3

Abstract

The invention relates to special purpose extracts (liquid and dry extracts) from Epimedium species, methods for production thereof, medicaments and foodstuffs comprising the same, in particular, dietary products and adjuncts including Medical Food and Dietary Supplements and use of the special purpose extracts for the prophylaxis and treatment of disease states caused by a lack of oestrogen or by a dysregulation of sex hormone metabolism, in particular, oestrogen metabolism.

Description

EXTRACTS FROM Epimedium TYPES, METHOD FOR THEIR PREPARATION AND USE

The present invention relates to special extracts (liquid and dry extracts) from Epimedium species, to processes for their preparation, to medicaments and foods containing them, in particular dietetic foods, nutritional supplements and "medical food" and "dietary supplements", and to the use of the special extracts for Prophylaxis and treatment of disease states that are caused by a lack of estrogens or by a dysregulation of the sex hormone metabolism, in particular of the estrogen metabolism.

Irregular menstrual cycles typically occur in women around the age of 45 and mark the onset of menopause. This period characterized by changes in the endocrine system, which may extend over a period of up to 10 years, is known as menopause or climacteric. It is triggered by the decreasing responsiveness of the ovaries to gonadotropins and the cessation of regular follicular maturation. As a result, there is a decrease and finally the final discontinuation of ovaπellen Ostrogensynthese. Due to the lack of estrogen production develop various deficiency symptoms, such as hot flashes, palpitations, depression, anxiety, headaches, sleep disorders, irritability or concentration disorders. In addition to acute climacteric symptoms, long-term postmenopausal health problems such as osteoporosis and atrophy of the urogenital mucosa and increased susceptibility to cardiovascular disease, stroke, Develop Alzheimer's disease or certain tumor diseases.

It is natural to replace the existing deficiency state with the replacement of the missing hormone, and indeed, menopausal symptoms can be effectively treated by estrogen replacement (estrogen replacement therapy, ÖET). However, this desired effect is paid for by an increased rate of endometrial carcinomas, but apparently this can be reduced by the combination (cyclic or continuous) with progestin (hormone replacement therapy, HET). This treatment is highly effective in relieving vasomotor and mental discomfort during the menopause. Also, a favorable influence of postmenopausal osteoporosis by a HET is considered proven. Controversial, however, is whether HET positively affects other health problems that occur during the peri- and postmenopausal period. In recent years, however, there have been increasing numbers of reports that not only do these expectations have not been met, but that hormone replacement therapy is even associated with grave, detrimental health consequences. The HERS study (Hulley et al., 1998; Graday et al., 2000) first demonstrated disillusionment by demonstrating that HET in women with pre-existing coronary artery disease does not reduce the incidence of cardiac events, and on the contrary, the risk of thromboembolic disease and increased gallbladder surgeries. An increased rate of myocardial infarction was also found in the HERS II study (Graday et al., 2002, Hulley et al., 2002), and in the ERA study, a favorable influence on the progression of existing atherosclerosis by an OET or HET undetectable (Herrington et al., 2000; SCRIPT, 24 March 2000, p. 21). In 2002, the US National Heart, Lung, and Blood Institute announced the premature and immediate discontinuation of the Women's Health Initiative (WHI) study announced. This study examined the benefits and risks of HET in a total of 16,608 women between the ages of 59 and 79 (Writing Group for Women's Health Initiatives Investigators, JAMA 288, 321, 2002). After a similar trend had already emerged in earlier interim evaluations, it became apparent that the incidence of breast cancer increased by 26% under this treatment. In addition, the rate of strokes (+ 41%), heart attacks (+ 29%), thromboembolism (+ 100%) and total cardiovascular disease (+ 22%) increased. Although a reduction in colon carcinoma (-37%), femoral neck fracture (-33%) and total fracture (- 24%) was observed, the benefit / risk balance was clearly negative (Russouw et al., 2002; Fletcher and Colditz, 2002). The One Million Women Study in England also showed that women who were currently taking hormones were at an increased risk of developing breast cancer and dying (Million Women Study Collaborators, Lancet 362, 419, 2003).

Due to the severe side effects that have become clear, the use of HET for the treatment of vasomotor and mental climacteric complaints is no longer responsible and is now rejected by many women. Similarly, the use of HET for the prophylaxis of osteoporosis is to assess, especially since it is usually a long-term treatment. For these reasons, there is a great need for alternative therapy options with a better benefit-risk profile.

Substances which interact with the endocrine system in humans or animals and thereby produce an estrogen-like effect by influencing hormone-regulated signaling pathways have been found in many plants. At least 20 different substance groups with estrogenic / anti-estrogenic egg properties from more than 300 plants from more than 16 plant families have been found so far. Most known phytoestrogens belong to the groups of isoflavones, lignans or cumestanes. For numerous phytoestrogens, selective estrogen receptor modulating (SERM) properties are described. In contrast to natural estrogen, these compounds have partial, organ-specific estrogenic activity. For the use for the prophylaxis and therapy of menopausal symptoms, osteoporosis or cardiovascular diseases phytoestrogens are particularly advantageous, the agomstisch on certain target organs act (eg bones, vessels) or target symptoms (eg hot flashes) act, but at the same time no or estrogen have antagonistic effects on the mammary gland or the uterus.

Traditional Chinese medicine (TCM) knows some epi-medium species (ferns Berberidaceae) whose herb is used medicinally. These are Epimedium brevicornum, E. sagittatum, E. pubescens, E. wushanense and E. koreanum, E. davidu, E. hunanense. The first five Epimedium species are part of the Herba Epimedii monograph of the Chinese Pharmacopoeia (Pharmacopoea of the Peoples Republic of China, Vol. I, 2000) and are formulated as a preparation with renal fat for the treatment of impotence, limb weakness, rheumatic pains and climacteric Used complaints.

In the TCM, herbs of the Epimedium Gattunq are used by the physicians in the form of various preparations, but not in the form of extracts from extraction with organic solvents for the treatment of impotence, spermorrhea, micturition, fatigue, lumbar and knee pain, infertility, amenorrhoea , Senile depression, rheumatoid arthritis, cardiovascular failure, hypertension, chronic bronchitis, angina pectoris and other diseases. (H. Wu, EJLien and LL Lien, Progress in Drug Research, Vol.60, 2-57, Ed E. Jucker, 2003).

Epimedium species contain primarily Prenylflavonglyko- side as main ingredients. Icariin 1 is a major glycoside and is found in many Epimedium species. Other constituents are its desmethyl analogue epimedoside A 2. Furthermore, free prenylflavones such as icaritine and free nonprenylated flavones such as luteolin occur. Finally, the chemically non-uniform group of icarisides, e.g. Icarisid I3, as a major component group (H.Wu, E.J.Lien and L.L. Lien, in Progress in Drug Research, Vol.60, 2-57, Ed E. Jucker, 2003).

1 R = Me icariin

2 R = H epimedoside A

3 R = Me Icariside I

For preparations of Epimedium species containing icariin and the total flavonoid fraction, a number of Pharmacological Effects: Influencing Hormone Regulation and Immunological Functions, Antibody Properties, Anti-Aging and Anti-Atherosclerosis Activity (H. Wu, EJLien and LL Lien, in Progress in Drug Research, Vol.60, 2-57 , Ed. E. Jucker, 2003).

US6238707 describes a composition of at least 10 different drugs which also contains epimedium for the treatment of hormonal disorders in women. It is obvious that only a small concentration of the epimedium ingredients can be present in this composition.

No. 6,750,248 (corresponding to WO03040134) describes micro-preparative methods for the production and isolation of estrogen-active substances from plants such as, for example, Epimedium species. Also claimed are three estrogen-active compounds that have been microproperatively isolated and characterized from Epimedium herb. It is not known which Epimedium species were included in the herb drug used. The described methods for isolating the claimed estrogenic compounds are very expensive, uneconomical and technologically unusable. These include liquid-liquid distribution, solid-phase fractionation, thin-layer chromatography, flash chromatography, and high performance or high pressure liquid chromatography. For liquid-liquid distribution, however, no concrete suitable solvents are mentioned. As solid phase fractionation concrete (see Example 8) polyamide, diol, and C18 (= RP18) phases are mentioned and applied. These are pure adsorption or distribution chromatographic phases and have no ion exchange character. Ion exchangers are not mentioned in US Pat. No. 6,750,248. Surprisingly, amino acid-like or alkaloid-like ingredients in simple Epimedium extracts could now be clearly detected by means of thin layer chromatography (Dragendorff and iodoplatinate reagent) and HPLC. The main compound of this amine or Alkaloidfrakti- on the Aporphinalkaloid Magnoflorin was identified. Magnoflorm has weak curare-like and hypotensive properties. Such compounds may also have excelled cytotoxic properties.

It is an object of the present invention to provide plant extracts with improved pharmacological actions which are suitable for the prophylaxis and treatment of disease states caused by a lack of estrogens or by a dysregulation of sex hormone metabolism, in particular of estrogen metabolism. These plant extracts are characterized by the fact that they have lower undesirable side effects compared to conventional estrogen-effective extracts and are free of toxicologically questionable amines or alkaloids.

Another object of the invention is to provide such extracts which also have improved pharmaceutical properties and thus are better galenically processable and optically acceptable.

Another object of the present invention is to provide methods of making these special extracts, as well as drugs and foods containing these special extracts.

According to the invention, these objects are achieved by the special extracts (liquid and dry extracts) of Epimedium species measured according to claims 1-9, the process for their preparation. ment according to claims 10-24, the medicament according to claim 25, the food according to claim 26, and the use of the special extracts according to claims 27-29.

Corresponding special extracts may e.g. from the following epi-medium species (Fern. Berberidaceae): Epimedium brevicornum, E. sagittatum, E. pubescens, E. wushanense and E. koreanum, E. davidii, E. hunanense, E. grandiflorum, E. acuminatum, E. lotorrhizum, E. wanshanense, E. fragesii, E. sutchuenense, E. diphyllum, E. sempervirens, E. baicaliquizhounense, E. caotum, E. glandulospilosum, E. zushanense, E. reticulatum, E. baojingenensis, E. simplicifolium, E. elongatum, E. ecalcaratum, E. truncatum, E. haiyangense, E. platypetalum and E. cantabrigiense.

In the context of the present invention, particularly preferred Epimedlum species are Epimedium brevicornum, Epimedium sagittatum, Epimedium pubescens, Epimedium wushanense and Epimedium koreanum, which are approved as plants for the drug Herba Epidemii of the Chinese Pharmacopoeia. For special extracts according to the invention from the leaves of Herba Epimedii, surprisingly, in the context of the present invention, particularly strong estrogenic effects were found.

The special extracts according to the invention could be obtained from primary extracts by further separation and purification. It was found that the observed pharmacological effects are caused by the interaction of the prenylated flavone compounds present in the extracts. Due to the strong depletion, in particular of the aporphinal cocoa magnoflorin, the special extracts according to the invention also have lower undesired side effects. The Magnofloringehalt lies in the special invention ene ckenextrakten at less than 0.1 wt .-%, preferably less than 0.05 wt .-% and in particular less than 0.01 wt .-%, based on the dry extract.

The preparation of the special extracts according to the invention is carried out by a method comprising:

A) an optional cleaning step to degrease the drug

B) a primary extraction step C) at least one further purification step and optionally

D) the concentration of the liquid extract obtained in C) to the dry extract.

Usual plant extracts are often sticky due to lipophilic accompanying substances (fats, waxes, chlorophylls, etc.) or have a semi-solid consistency and are dark-colored. According to a preferred embodiment, extracts according to the invention having improved pharmaceutical properties can be prepared by pre-extracting (degreasing) the epimedium drug prior to the actual extraction of the special extract by means of an apolar solvent and thus removing lipophilic accompanying substances.

The cleaning step A) for the purpose of degreasing can be carried out by pre-extraction of the drug with apolar organic solvents, for example with alkanes, more preferably heptane, or with ethers, more preferably tert. Butyl methyl ether, or with mixtures of alkanes with esters, in particular mixtures of heptane with ethyl acetate of up to 50 wt .-%, or with supercritical CO ₂ . The degreased drug thus obtained is then available for further processing.

The primary extraction step (B) can be carried out by methods known per se with inorganic or organic solvents. like P. List and PC Schmidt, Technology of Herbal Remedies, Wissensch. Verlagsgesellschaft mbH, Stuttgart 1984, Chapter 3, pages 85-95 (the disclosure of which is incorporated herein by express reference), for example with water, alcohols, esters and ketones, in particular methanol, ethanol, ethyl acetate, acetone, CO ₂ etc ., And their mixtures, in particular with water, at temperatures from room temperature to 100 ⁰ C with gentle to vigorous mixing within 10 Hin. up to 24 hours.

The further cleaning step (C) according to the invention extracts extract constituents with undesired effects. For this, either a Flussig-Flussig distribution with e.g. Ethyl acetate / water, butanone / water, n-butanol / water or methyl ethyl ketone / water, etc., or adsorption desorption on ion exchanger, e.g. Sephadex LH-20, Diaion HP20, Dowex or Lewatit performed. As solvents for the chromatographic separation, depending on the particular solid phase, different polarity solvents, e.g. Water, methanol, ethanol, other alcohols, acetone, other ketones, ethyl acetate and mixtures thereof. Those fractions which contain the phytosterol components without the constituents with undesired effects are combined, more or less concentrated in vacuo to give liquid or spissum extracts and optionally (D)) freeze-dried or, at elevated temperature and reduced pressure, e.g. freed of solvent in a drying oven to obtain a dry extract.

The dry extracts according to the present invention have a dry residue level of at least 95% (m / m) based on the total weight of the dry extract. According to F. Gaedcke, B. Steinhoff, H. Blas ius, Phytopharma ka, Wis sen ttl. Verlagsgesellschaft mbH Stuttgart, 2000 are dry extracts (Extracta sicca) solid preparations which are prepared by evaporation of the solvent used for their preparation.

The ratio of drug to solvent in each extraction ranges from about 1: 7 to about 1:10.

In a preferred embodiment, the extraction in step B) is carried out twice.

The alcohols and ketones used in step B) are preferably used as 10-96% (v / v) or (w / w), especially 50-92% (v / v) or (w / w) aqueous mixtures.

The solvent used in step B) is preferably selected from the group consisting of aqueous ethanol, aqueous methanol, aqueous acetone and aqueous or water-saturated ethyl acetate. In particular, the solvent used in step B) is 70% (v / v) (62% (w / w)) ethanol, 60% (w / w) ethanol or water saturated ethyl acetate.

The special extracts obtainable according to the invention, together with customary pharmaceutically acceptable excipients, can be converted into medicaments in the form of e.g. Capsules, film-coated tablets, tinctures and dragées can be processed, wherein filling, binding, blasting, lubricating and coating compositions for film-coated tablets and dragees as well as oils and fats can be used as filling compositions for capsules as customary pharmaceutically acceptable auxiliaries.

Furthermore, the special extracts obtainable according to the invention, together with customary food additives, can be used to produce foods, in particular dietetic foods, food supplements and "medical food" and "dietary supplements" in the form of, for example, capsules, film tablets, tablets. the usual food additives, colorants, preservatives, antioxidants, emulsifiers, stabilizers, acidulants, gelling agents, excipients, and other substances according to EU Framework Directive 89/107 and EU Directive 95/2 / EEC. and amendments 96/85 / -, 98/72 / - and 2001/5 / EC.

The special extracts obtainable according to the invention can be used (for the preparation of medicaments or foods) for the prophylaxis and treatment of disease states which are caused by a lack of estrogens or by a dysregulation of the sex hormone metabolism, in particular estrogen metabolism.

The disease states may be selected from the group consisting of climacteric complaints, sex hormone dependent cancers, benign prostatic hyperplasia, osteoporosis, Alzheimer's disease and cardiovascular diseases, as well as erectile dysfunction and urinary incontinence, the cancers comprising breast cancer, prostate cancer and uterine cancer.

With the prenylated flavones comprising special extracts according to the invention, there is thus provided a medicament which is very well suited for the prevention and / or control of disease states which are caused by a lack of estrogen or by a dysregulation of the sex hormone metabolism, in particular estrogen metabolism ,

Suitable dose ranges for the special extract according to the invention are 10 mg to 5 g, more preferably 50 mg to 2 g of extract per day. The examples given below illustrate the invention and are not intended to be limiting. All percentages are by weight unless otherwise specified.

HPLC system for the determination of the nitrogen-containing compound

Magnoflorin:

HPLC method (detection limit 0.05% by weight)

DC system for the detection of the nitrogenous compound Magnoflorin in Epimedium extract

Stationary phase: silica gel F 254 Merck Mobile phase: ethyl acetate-formic acid-acetic acid-water

(100: 11: 11: 27)

Detection: 1. Dragendorff reagent (compare H. Wagner, S. Bladt, Plant Drug Analysis, 2 ^nd ed., Springer-Verlag Berlin, Heidelberg, New York 1996, p 360) 2. Iodoplatinate reagent (compare H. Wagner, S. Bladt, Plant Drug Analysis, 2 ^nd ed., Springer-Verlag Berlin, Heidelberg, New York 1996, p 361)

To 1.: When spraying the DC plate with Dragendorff reagent, the magnoflorin (Rf range 0.2 - 0.3) turns orange.

Ad 2: When spraying with Jodplatmat the Magnoflorin (Rf range 0.2-0.3) turns purple. Detection limit: 0.01% by weight

Example 1 Production of a Special Dry Extract from Herba Epimedii by Flussig-Flussig Distribution with Ethyl Acetate

a) extraction (process step B))

300g ground leaves (4 mm) of Herba Epimedii were two times each with 2.1 kg of 70% (v / v) (62% (w / w)) of ethanol, and 1 h at 50 ⁰ C with vigorous stirring , After extraction, each was filtered with suction through a Supra Seitz 1500 filter. The two extract solutions were combined and from each of them an aliquot was dried on a rotary evaporator to determine the dry residue. The result was a dry residue level of 47 g corresponding to a yield of 15.7% (= extract 1, total extract).

The magnoflorine content, determined by HPLC in the total extract (extract 1) was 1.1% by weight, based on the dry extract.

b) purification and concentration to dryness (process steps C) and D)) The combined extraction solution obtained in a) was removed from the ethanol on a rotary evaporator at 50 ^° C. The aqueous residue was shaken out 3 × with 225 ml of ethyl acetate each time, and the ethyl acetate phases were combined and concentrated to dryness on a rotary evaporator.

Ethyl acetate extract (= special dry extract 2) = 7.86 g (2.62% based on drug, 16.7% based on total extract)

According to TLC and HPLC analysis, the ethyl acetate extract contains a large number of flavonoid-type compounds, while amino- or alkaloid-type compounds are no longer detectable (detection limit 0.01% by weight). The latter are clearly detectable in the separated water phase.

Example 2: Preparation of a special dry extract from Herba Epimedii by Flussig-Flussig distribution with methyl ethyl ketone

a) extraction (process step B))

was carried out as described in Example 1.

b) purification and concentration to dryness (process steps C) and D))

The combined extraction solution obtained in a) was removed from the ethanol on a rotary evaporator at 50 ^° C. The aqueous residue was shaken out 3 × with 270 ml of methyl ethyl ketone each, the methyl ethyl ketone phases were combined and concentrated to dryness on a rotary evaporator.

Methyl ethyl ketone extract (special dry extract 3) = 12.2 g (4.1% based on drug, 26.0% based on total extract) The methyl ethyl ketone extract contains according to TLC and HPLC analysis a variety of flavonoid-like compounds, while amine or alkaloid-like compounds are no longer detectable (detection limit 0.01 wt .-%). The latter are clearly detectable in the separated water phase.

Example 3: Production of a special dry extract from Herba Epimedii by liquid-liquid distribution with n-butanol

a) extraction (process step B))

was carried out as described in Example 1.

b) purification and concentration to dryness (process steps C) and D))

The combined extraction solution obtained in a) was removed on a rotary evaporator at 50 ⁰ C, the ethanol. The aqueous residue was shaken out 3 × with 270 ml of n-butanol each time, and the n-butanol phases were combined and concentrated to dryness on a rotary evaporator.

n-butanol extract (special dry extract 4) = 21.1 g (7.0% on the drug, 45% on the total extract)

The n-butanol extract contains according to TLC and HPLC analysis

Variety of flavonoid-like compounds, while amine or alkaloid-like compounds are no longer detectable

(Detection limit 0.01% by weight). The latter are clearly detectable in the separated water phase.

Example 4: Extraction of Epimedium brevicornum with water-saturated ethyl acetate and preliminary degreasing of the drug 300 g of ground Epimedium brevicornum drug was initially tert with 2.1 kg. Butyl methyl ether at 50 ⁰ C extracted (step A)). The obtained liquid extract was discarded. The remaining defatted drug was extracted after intermediate drying twice with in each case 2.1 kg of water-saturated ethyl acetate with intensive stirring (process step B)). The combined extract solutions were then filtered off and the resulting ethyl acetate extract solution was concentrated to about 200 ml and extracted by shaking three times with 80 ml of ethyl acetate-saturated water in a ratio (process step C)). The separated and combined ethyl acetate phases were concentrated to dryness (process step D)):

Yield: 5.6 g (1.9% based on the drug)

The extract obtained is comparatively brightly colored and has a very good powder flowability, characterized by a very low angle of repose.

In addition, the extract according to DC and HPLC analysis contains a variety of flavonoid-like compounds, while a- or alkaloid-like compounds are no longer detectable (detection limit 0.01 wt .-%). The latter are clearly detectable in the separated water phase.

Example 5: Preparation of a special dry extract from Herba Epimedii by adsorption of alkaloid-like ingredients to an ion exchanger

200 g of an extract solution (containing 5% dry matter content) obtained with 60% (m / m) ethanol from E. koreanum according to Example 1 (process step B)) were passed through a hydrochloric acid-regenerated cation exchanger Dowex HCR-W2 (20 ml). It was then washed with two column volumes of 60% ethanol (column dimension: 2 cm ID, 6 cm long). The collected acidic run was adjusted to pH 5.0-5.5 with 1M sodium hydroxide (step C)). Finally, it was concentrated and dried (process step D)).

Yield: 9.46 g (94.6% of the extract used)

The extract obtained from amines or alkaloids thus obtained contains the same spectrum of flavonoid-like compounds as before treatment with the cation exchanger. Alkaloids or amines were no longer detectable (detection limit 0.01 wt .-%).

Example 6 Testing of the Prepared Specialty Dry Extracts For Estrogenic Activity Was Performed With A Cellular Estrogen Receptor Assay Assay Using Human Endometrial Carcinoma Cell Lines ECC-I (Bergeron et al., Mol., Cell Endocellol 150, 179-187, 1999). The cells were incubated (37 ° C., 5% CO ₂ in air) in DME medium (ICN, 1033126) with addition of fetal calf serum (5%), glutamine (2 mM), antibiotic / antimycotic solution (1%). , Sigma, A-9909) and insulin (0.25 U / ml). Before carrying out the experiment, the adharently growing cells were removed by trypsinization from the bottom of the tissue culture bottles and adjusted to a density of ⁵ × 10 ⁵ cells / ml in complete culture medium. Per well of a 24 well tissue culture plate (TPP, # 9224), 500 μl of cell suspension was filled. After 24 h, the plates were centrifuged (350 g, 5 min, RT), the medium was discarded and the cells were washed twice with test medium (DMEM-F12 [Sigma, D-2906] with addition of Charcol-stripped bovine calf serum [5%], Glutamine [2 mM] and antibiotic / antimycotic solutions were then cultured for a further 24 hours in 500 μl of fresh test medium, and after extracting 100 μl of medium, the extracts were added to 10 μl final concentration in a volume of 50 μl allocated to the cells give. A hour later 2, 4, 6, 7- ³ H-estradiol (50 .mu.l of 0.01% ethanol xn, Amersham, TRK 322) is also 10 times the final concentration (3 nM) is added by pipette to all substance Ansatzen After incubation for 35 min at 37 ⁰ C, the Uber- supernatants discarded and 500 ul of phosphate buffer (5 mM, pH 7.4 with addition of 0.5% bovine serum albumin, 0.25 mM sucrose, and 10% glycerol) was added. After 30 minutes at room temperature, the cells were washed twice with ice-cold phosphate buffer without BSA and then added to 500 μl of ice-cold 96% ethanol. The cells suspended in ethanol were transferred into 4 ml each of Quicksafe A and the bound radioactivity was measured in a β-counter (Packard, Tri-Carb 2300 TR). All approaches were done in duplicates. To determine nonspecific binding, cells were incubated in the presence of 100 nM unlabelled estradiol. The blank used was samples without labeled estradiol. Percent inhibition of receptor binding was calculated in each case in comparison to a parallel tested solvent control (DMSO, 0.1%). The determination of the half-maximal inhibitory concentration (IC ₅₀ ) was carried out by non-linear regression calculation.

Results :

Table 1:

The special dry extracts according to the invention are approximately 3 to 6 times more effective with respect to the reference extract. With respect to the value of pure 17β-estradiol they are less effective. However, the concentrations of the phytostenes in the organism are many times greater than those of the body's own steroids (17β-estradiol) in the above-mentioned conventional dosages, so that the effect of the special dry extracts in the organism corresponds approximately to that of 17β-estradiol.

Claims

claims

1. Special extract of Epimedium species containing less than 0.1% by weight of magnoflorin, based on the dry extract, obtainable by

B) a primary extraction step of the epimedium species with an inorganic or organic solvent or a mixture thereof to obtain a primary extract solution, C) at least one purification step of the primary extract solution obtained in B), whereby to deplete the substances with undesirable side effects the cleaning step C) a Flussig-liquid distribution, characterized by one or more times distribution of nachsetzlich added with water or water-containing Primarextraktlosung between water and an organic solvent selected from the group consisting of ethyl acetate, n-butanol, methyl ethyl ketone and Butanone and combining the organic solvent phases obtained, or an adsorption-desorption is carried out on ion exchanger, and optionally

D) a step of concentration to the dry extract of the organic solvent phase (s) obtained in C).

2. Special extract of Epimedium species according to claim 1, wherein A) is preceded by a purification step for degreasing the drug the primary extraction step B).

3. Special extract of Epimedium species according to claim 2, wherein the purification step for degreasing the drug A) is characterized by extracting one or more Epimedium species one or more times with a solvent selected from the group consisting of i) alkanes, comprising heptane, ii) ethers, comprising tert. Butyl methyl ether, iii) mixtures of alkanes with esters, comprising mixtures of heptane with ethyl acetate of up to 50%; iv) supercritical solvents comprising supercritical CO2.

4. Special extract of Epimedium species according to any one of claims 1 to 3, wherein the primary extraction step B) is characterized by one or more extractions of one or more Epimedium species with a solvent selected from the group consisting of i) alcohols and ketones including aqueous alcohols and aqueous ketones; and ii) esters including aqueous or water-saturated esters.

5. Special extract of Epimedium species according to claim 4, wherein additionally filtered after step B) and the organic solvent is stripped from the obtained in step i) alcoholic or ketonic extraction solution and a residue or aqueous residue is obtained.

6. Special extract of Epimedium species according to claim 4, wherein additionally filtered after process step B) and process step C) is characterized by one or more direct distribution of the ester extract obtained in step ii) with water or ester-saturated water and combining the obtained organic solvent phases.

7. Special extract of Epimedium species according to one of claims 1 to 6, wherein the obtained in step C) organic solvent phase (s) in process step D) to dryness to a dry residue level of at least 95% (m / m ) based on the total mass of the dry concentrated to obtain a special dry extract.

8. A special extract according to any one of claims 1 to 7, wherein the Epimedium species is selected from Epimedium brevicornum, E. sagittatum, E. pubescens, E. wushanense, E. koreanum, E. davidii, E. hunanense, E. grandiflorum, E. acuminatum, E. Iotorrhizum, E. wanshanense, E. fragesii, E. sutchuenense, E. diphyllum, E. sempervirens, E. baicaliquizhounense, E. caotum, E. glandulospilosum, E. zushanense, E. reticulatum, E. baojingenensis, E. simplicifolium, E. elongatum, E. ecarcaratum, E. truncatum, E. haiyangense, E. platypetalum and E. cantabrigiense.

9. Special extract according to one of claims 1 to 7, wherein Epimedium brevicornum and / or E. sagittatum and / or pubescens and / or E. wushanense and / or E. koreanum is used.

10. A process for the preparation of an extract according to claims 1-9, comprising

A) an optional cleaning step to degrease the drug,

B) a primary extraction step of the epimedium species with an inorganic or organic solvent or a mixture thereof, whereby a primary extract solution is obtained,

C) at least one purification step of the primary extract solution obtained in B),

wherein for the depletion of the substances with undesired side effects in the purification step C) a Flussig-Flussig distribution, characterized by one or more times distribution of the post-added with water or containing water Primarextraktlosung between water and an organic nisches solvent selected from the group consisting of ethyl acetate, n-butanol, methyl ethyl ketone and butanone and combining the resulting organic solvent phases, or an adsorption desorption is performed on ion exchanger, wherein the desired ingredients in organic solvent phase (s) are obtained as a liquid extract, and possibly

11. The method of claim 10, wherein process step A) is characterized by: one or more times extracting one or more Epimedium species with a solvent selected from the group consisting of i) alkanes comprising heptane, ii) ethers comprising tert. Butyl methyl ether, iii) mixtures of alkanes with esters, comprising mixtures of heptane with ethyl acetate of up to 50% by weight, iv) supercritical solvents comprising supercritical CO2.

12. The method of claim 10 or 11, wherein process step B) is characterized by: one or more times extracting one or more Epimedium species with a solvent selected from the group consisting of i) alcohols and ketones including aqueous alcohols and aqueous ketones; and ii) esters including aqueous or water-saturated esters.

13. The method of claim 12, wherein after step B) is additionally filtered and the organic solvent ses obtained in step i) alcoholic or ketonischen extraction solution is withdrawn and a residue or aqueous residue is obtained.

14. The method of claim 12, wherein after the process step B) additionally filtered and process step B) is characterized by one or more direct distribution of the ester extract obtained in step n) with water or ester-saturated water and combining the resulting organic solvent phases.

15. The method according to any one of claims 10 to 14, wherein the obtained in step C) organic (s) solvent phase (s) in process step D) to dry to a dry residue level of at least 95% (m / m) based on the Total mass of the dry extract are concentrated to obtain a special dry extract.

16. The method according to any one of claims 10 to 15, wherein the extraction in step B) is carried out twice.

17. The method according to any one of claims 10 to 16, wherein the solvent used in step B) is aqueous methanol or aqueous ethanol.

18. The method according to any one of claims 10 to 16, wherein the solvent used in process step B) is aqueous acetone.

19. The method according to any one of claims 10 to 18, wherein the alcohol used in step B) or the ketone used in step B) is present as 10-96% (v / v) or (w / w) aqueous mixture.

20. The method according to any one of claims 10 to 18, wherein the alcohol used in process step B) or the in used in step B) ketone is present as 50-92% (v / v) or (w / w) aqueous mixture.

21. Process according to claim 17, wherein the solvent used in process step B) contains 70% (v / v) or 60%

(w / w) is watery ethanol.

22. The method according to any one of claims 10 to 16, wherein the solvent used in process step B) is ethyl acetate or aqueous or water-saturated ethyl acetate.

23. The method according to any one of claims 10 to 22, wherein the Epimedium species is selected from the group Epimedium brevicornum, E. sagittatum, E. pubescens, E. wushanense and E. koreanum, E. davidu, E. hunanense E grandiflorum, E. acumatum, E. leotorrhizum, E. wanshanense, E. fragesu, E. sutchuenense, E. diphyllum, E. sempervirens, E. baicaliquizhounense, E. caotum, E. glandulospilosum, E. zushanense E. reticulatum, E. baoj ingenensis, E. simplicifolium, E. elongatum, E. ecarcaratum, E. truncatum, E. haiyangense, E. platypetalum and E. cantabngiense.

24. The method according to any one of claims 10 to 22, wherein E- pimedium brevicornum and / or E. sagittatum and / or pubescens and / or E. wushanense and / or E. koreanum is used.

25. A pharmaceutical composition comprising a special extract according to any one of claims 1 to 9 and conventional pharmaceutically acceptable excipients.

A food comprising a special extract according to any one of claims 1 to 9 and common food additives.

27. Use of a special extract according to any one of claims 1 to 9, a medicament according to claim 25 for the prophylaxis and treatment or a food according to claim 26 for the prophylaxis of disease states caused by a deficiency in estrogens or by a dysregulation of sex hormone metabolism, in particular estrogen metabolism - sels are caused.

28. Use according to claim 27, wherein the disease states are selected from the group consisting of climacteric complaints, sex hormone-dependent cancers, benign prostate hyperplasia, osteoporosis, Alzheimer's disease and cardiovascular diseases, as well as erectile dysfunction and urinary incontinence.

29. Use according to claim 28, wherein the sex hormone dependent cancers are selected from the group consisting of breast cancer, prostate cancer and uterine cancer.