WO2017065515A1 - Composition for preventing or treating gynecological cancers and menopausal symptoms containing flemingia strobilifera extract or compound isolated therefrom as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating gynecological cancers and menopausal symptoms containing a Flemingia strobilifera extract, an active fraction thereof, or a single component isolated from the active fraction and acceptable salt thereof as active ingredients. The present invention confirms for the first time that the Flemingia strobilifera extract, active fraction thereof, and single components derived therefrom exhibit characteristics of the female hormone, and thus female hormonal substances provided by the present invention can be utilized in the medicinal and functional food fields to treat and prevent gynecological cancers and menopausal symptoms.

Description

Composition for the prevention and treatment of female cancer and menopausal symptoms containing Flemingia strobiviera extract or a compound isolated therefrom as an active ingredient

The present invention Flemingia ( Flemingia) strobilifera ) extract, an active fraction thereof, a compound isolated therefrom and an acceptable salt thereof as an active ingredient, and relates to a composition for the prevention and treatment of female cancer and menopausal symptoms.

Women's cancers such as endometrial cancer and breast cancer and diseases such as menopausal syndrome are associated with female hormones such as excessive or reduced estrogen and imbalance of estrogen and progesterone.

Endometrial cancer

In Korea, endometrial and ovarian cancers have increased by about five times since 1991, and the increase in the incidence of female hormone cancer is expected to continue in the future. Risk factors for abnormal proliferation of the endometrium are prolonged exposure to estrogens without sufficient antagonism of progesterone, overweight, and westernized life. Primary treatment for endometrial cancer is surgery, and female hormone therapy is effective in patients who have both positive estrogen or progesterone receptor after surgery and chemotherapy. Excessive estrogen exposure, which is not balanced with progesterone as a major cause of endometrial cancer, is known to increase endometrial cancer by more than eightfold. ™) is widely used. However, side effects of megestrol include high blood pressure, thrombophlebitis, and weight gain. Therefore, if a substance that can balance the effects of progesterone and estrogen from natural products is discovered, it can be an effective endometrial cancer drug with reduced side effects.

Breast cancer

As with the development of endometrial cancer, breast cancer caused by excessive estrogen has increased significantly in Korean women over the past decade and is currently the highest among women. The main causes of the increase in breast cancer are high fat and high calorie westernized diet and obesity, late marriage and lower fertility rate, avoiding lactation, and overexposure to estrogen. The western region is more than three times more likely to develop breast cancer than Korean women, and the demand for breast cancer drugs is very high worldwide. Breast cancer treatment is primarily performed after surgery, radiation therapy, chemotherapy, hormone therapy, etc. to prevent recurrence. Hormonal therapy is effective in lowering recurrence rates, but there is a problem that resistance is expressed after long-term administration over many years. In the case of tamoxifen, the first-choice drug, endometrial cancer induction and expression of cellular resistance after long-term administration have been pointed out in many patients with metastatic cancer. Anastazole, Letrazole, and Exemestane are currently used as aromatase inhibitors, which are secondary anti-hormonal drugs that have improved side effects and recurrence, but their use is limited to postmenopausal women. An important aspect in the development of therapeutics for breast cancer is to first target molecules related to carcinogenesis in order to reduce toxicity, and to minimize the expression of drug resistance for long-term use and prevention of recurrence. As ER / PR positive patients make up two-thirds of all breast cancer patients, ER / PR is the best molecular target. Therefore, developing selective estrogen receptor modulators (SERMs) and selective progesterone receptor modulators (SPRMs) with molecular targets of ER / PR while minimizing side effects and drug resistance expression have been developed to treat breast cancer. It is a key goal of the company.

Menopause syndrome

The rapid decrease in post-menopausal estrogen can lead to symptoms of cerebral depression, mental instability and sudden emotional changes, vaginal dryness, including hot flashes, worsening osteoporosis, hyperlipidemia, poor memory, and reduced cognition. . For these postmenopausal symptoms, the treatment of estrogen alone or a combination of estrogen / progesterone is almost the only treatment. This is called hormone replacement therapy (HRT). However, long-term use of HRT is known to be directly involved in increasing the incidence of cancer in tissues containing ER, such as breast, uterus and ovary. Women's Health Initiative (WHI) study found that taking Premarin and Prempro, one of the most prescribed HRT products in North America, is associated with increased breast cancer and the development of cardiovascular disease. In addition, various small-scale clinical trials suggest that the use of HRT is closely related to hypercellular growth and carcinogenesis, such as increased uterine proliferation in postmenopausal women who are taking HRT.

As a carcinogenic mechanism related to HRT, excessive cell proliferation due to E2 / ER interaction and accumulation of DNA mutations have been suggested, and genotoxicity by reactive metabolites occurring during in vivo metabolism of estrogens is also carcinogenic. It is presented as a mechanism. The pharmacological effects of these tissue-specific estrogens have been known to be due to the variety of molecular mechanisms involved in estrogen and ER and transcription factors involved in specific tissues / cells (Jordan, 2007). For example, tamoxifen, which is used as a breast cancer drug, acts as an ER antagonist in the breast, but acts as an ER agonist in the uterus and bone, so long-term use of tamoxifen may cause side effects of endometrial cancer. Therefore, the development of a therapeutic agent or a postmenopausal symptom treatment agent for cancers in which ER is involved in carcinogenic mechanisms, such as breast cancer or endometrial cancer, has been described as a substance capable of having a tissue-specific differential effect on ER, that is, selective female hormone receptor modulators (SERMs) Developing is the ultimate goal of drug development.

On the other hand, pomegranate extract, soybean extract and isoflavone formulations, evening primrose oil, etc. are used as health foods for improving women's menopausal symptoms and preventing / treating osteoporosis. These products, made from vegetable raw materials, are rapidly expanding in the market, giving consumers a sense of safety. However, there is no scientific basis for the recognition that it is safe because it is vegetable, and these products are composed of complex extracts composed of various kinds of chemical ingredients, and many of the dietary supplements have not been subjected to strict toxicity tests. none. The side effects of these products are actually impossible to monitor in government. In addition, there is a lack of scientific basis for pharmacological efficacy in its efficacy, and it is difficult to judge whether it complies with strict and precise quality control regulations. Therefore, there is a need for a safer and more potent natural material derived from the existing plant extracts.

Flemingia Flemingia strobilifera ) is a legume (Leguminosae , Fabaceae) plant known to be found in the lower Indus River in southeastern Pakistan, Bengal region in northeastern India, and the Rajputana region in northwestern India. Flemingia strobillipera contains flavonoids, flavonoid glycosides, chalcones, epoxychromenes, pterocarpans, and the like (Madan S et al.). , 2008). The plant's bracts, leaves, flowers and roots are used as folk medicines. The leaves and flowers of the plant are used to treat tuberculosis and the roots are used to treat epilepsy, hysteria and edema. Root juice has been reported to be used for the treatment of diarrhea and dysentery (Duthic JS et al ., 1994). In Burma, the roots of Flemingia strobilifera are used to treat epilepsy, and it is reported that plants of the same genus are used as folk medicines for fever, diarrhea, indigestion and vomiting (Chopra RN et al ., 1965; Kirtikar KR et al ., 1935. Also Flemingia Strobillipera Studies have shown that plants have antimicrobial, antifungal, antiparasitic, anticancer, antirheumatic, anti-inflammatory, antioxidant and antihistamine effects (Duthic JS et al ., 1994; Mitscher LA et al ., 1985; Chowdhury A. et al ., 1974; Das B. et al ., 2006; Chen M. et al ., 1991; Kan WS et al ., 1981; Pan BS et al ., 2005).

[Preceding technical literature]

[Non-Patent Documents]

Madan S, Singh GN, Kumar Y. A New Flavanone from Flemingia strobilifera (Linn) R. Br. and its Antimicrobial Activity. Trop J Pharm Res. (2008)

Duthic JS. New Connaught place. Dheradoon: Bishen singh Mohendra pal singh publications; (1994).

Chopra RN, Nayer SL, Chopra IC. India: C.S.I.R. Publication; (1965). Glossary of Indian medicinal plants; p. 220

Kirtikar KR, Basu BD. Lacit mohan basu M.B 49, Leader road. Allahabad: Prabasi press Calcutta; (1935). Indian medicinal plants.

Duthic JS. New Connaught place. Dheradoon: Bishen singh Mohendra pal singh publications; (1994). Flora of upper gangetic plain

Mitscher L.A., Gollapudi S.R., Khanna I.K., Drake S.D., Hanumaiah T., Ramaswamy T., Rao K.V. J .: Phytochemistry 24, 12, 2885 (1985).

Chowdhury A., Mukherjee N., Adityachaudhury N .: Experientia 30, 1022 (1974).

Das B., Tandon V., Saha N .: Parasit. Int. 55, 17 (2006).

Chen M., Lou S. Q., Chen J. H .: Phytochemistry 30, 3842 (1991).

Kan W.S .: Pharmaceutical Botany. p. 316, National Research Institute of Chinese Medicine, Taipei 1981.

Pan B.S., Kuo Y.Y., Chen T.Y., Liu Y.C .: Life Sci. 77, 22, 2830 (2005).

The present invention is to provide a new natural medicine and health functional food for the prevention and treatment of female cancer and menopausal symptoms. In particular, the present invention isolates and identifies Flemingia strobiviera extract, an active fraction thereof, and a single component separated therefrom that exhibit estrogen activity, and thus new natural medicines and health functionalities for the prevention and treatment of female cancer and menopausal symptoms. It aims to provide food.

The development of drugs for treating cancer or postmenopausal symptoms in which estrogen receptors (ER), such as breast cancer or endometrial cancer, are involved in the carcinogenesis mechanism, are substances capable of having tissue-specific differentiation effects on ER, that is, selective female hormones. Developing receptor modulators (SERMs) is the ultimate goal. One of the important objects of the present invention is to provide a plant-derived material that can balance the effects of estrogen in the body while improving the side effects of conventional endometrial cancer therapeutics.

In the present invention, by identifying the therapeutic efficacy, pharmacological metabolism, and safety of female cancer of Flemingia strobiviera extract, its active fraction and the active ingredients isolated therefrom, endometrial cancer, breast cancer, etc. Provided are new pharmaceutical compositions or functional food compositions effective for prophylaxis and treatment.

Specifically, in the present invention,

Flemingia Flemingia strobilifera ) extract, its active fraction, the compounds represented by the formulas (I) to (IV) below, and any one or more selected from the group consisting of pharmaceutically acceptable salts thereof, female cancer and menopause Provided are pharmaceutical compositions for the prevention and treatment of symptoms. The pharmaceutical composition of the present invention may further include a compound represented by the following formula (V) or a pharmaceutically acceptable salt thereof.

In the present invention,

Flemingia Flemingia strobilifera ) extracts, active fractions thereof, compounds represented by the formulas (I) to (IV) below, and any one or more selected from the group consisting of salts acceptable for foods thereof, female cancer and menopausal symptoms Functional food compositions for the prevention and improvement of the present invention are provided. The functional food composition of the present invention may further include a compound represented by the following formula (V) or a salt acceptable with the food thereof.

(Ⅰ)

(Ⅱ)

(Ⅲ)

(Ⅳ)

(Ⅴ)

Flemingia Strobillipera It was first discovered in the present invention that the extract and the single components of the above formulas (I) to (IV) exhibit feminine hormones. (V) has been known as phytosetrogen as genistein.

The female cancer is preferably any one of endometrial cancer, breast cancer and ovarian cancer.

The menopausal symptoms, preferably include the symptoms of any one of hot flashes, hyperlipidemia, lower brain mental function, osteoporosis, venous thrombosis and atrophic vaginitis.

The food is preferably a health functional food having any one of tablets, capsules, powders, granules, liquids, and pills.

The present invention, Flemingia Strobillipera By obtaining plant extracts and confirming their effects on female hormones, we provide new natural medicines and dietary supplements useful for the prevention and treatment of female cancer and menopausal symptoms. In addition, the present invention, Flemingia Strobiviera By separating and identifying single fractions showing active fractions and female hormones from plants, we provide new plant metabolism-derived single-component drugs and health supplements useful for the prevention and treatment of female cancer and menopausal symptoms. In particular, the present invention is the first confirmation that Flemingia strobillifera and the single components derived therefrom exhibit female hormones, and the pharmaceutical composition of the present invention containing these components occurs most frequently among female cancers. It can be useful for breast cancer, which has an increased incidence, and endometrial cancer, which has a similar pathogenesis.

1 shows in vitro system Competitive binding of Flemingia strobillifera (◆) extract and tritium-labeled estrogen ([ ³ H] estrogen) (●) to pure recombinant human estrogen receptor alpha (hERα) to be.

Figure 2 shows the results of analyzing the hERα binding force at 20μg / ml concentration of the three fractions (20) of the Flemingia strobiviera extract.

3 is ER subtype selective gene transcriptional activity of 20 fractions (FS-1 to FS-20) of Flemingia strobillifera was measured. Flemingia Among 20 extracts and 20 fractions of strobilifera (FS-1-20), two fractions (FS-7, FS-9) showed an ERβ / ERα ratio of 2 or more.

Figure 4 MCF7 cell proliferation after treatment with vehicle, estrogen (Etrogen), Flemingia strobilifera extract at a concentration of 10 ^-6 g / ml ~ 5x10 ^-5 g / ml to evaluate the estrogen receptor-reactive MCF-7 cell cell proliferation effect Will confirm. Flemingia strobilifera extract induced cell proliferation at concentrations of 10 ^-6 g / ml and 5x10 ^-6 g / ml.

Figure 5 shows the results of experiments whether the Flemingia strobiviera extract changes the activity of the ERE gene in MCF-7 cells transfected with the estrogen responsive element (ERE). Significantly different values from vehicle group are indicated with *** (*** <0.001, ** <0.05).

FIG. 6 shows the results of analyzing the activity of ERE genes in subfractions (20) of Flemingia strobiviera extracts in MCF-7 cells transfected with Estrogen Responsive Element (ERE).

FIG. 7 shows the results of analyzing the activity of five ERE genes isolated from Flemingia strobiviera in MCF-7 cells transfected with an Estrogen Responsive Element (ERE).

FIG. 8 shows the results of analyzing the activity of PRE-genes of subfractions (20) of Flemingia strobolifera extracts in MCF-7 cells transfected with progesterone responsive element (PRE).

FIG. 9 shows the results of analyzing the activity of five PRE-genes isolated from Flemingia strobillifera in MCF-7 cells transfected with a progesterone responsive element (PRE).

FIG. 10 shows the results of experiments showing increased expression of ERE-induced pS2 gene of Flemingia strobilifera extract in MCF-7 cells transfected with Estrogen Responsive Element (ERE).

FIG. 11 shows the results of analyzing pS2 gene activity in immature rat uterine tissue by Flemingia strobillifera extract.

12 shows the results of analyzing protein expression of estrogen receptor alpha (ERα), progesterone receptor A (PRA) and progesterone receptor B (PRB) in immature rat uterine tissues by Flemingia strobillifera extract.

Figure 13 evaluates the effect of Flemingia strobiviera extract on neuroglobin (Ngb) promoter activity of human neurons SKNSH. In the case of vehicle, positive control, estrogen, genistein, and Flemingia strobiviera extract, Ngb promoter activity was increased by positive control and Flemingia strobiviera extract.

Figure 14 evaluates the effect of Flemingia strobiviera extract on Ngb mRNA expression of human neurons SKNSH. When estrogen, genistein, and flemingia strobiliifera extracts were treated as vehicle and positive controls, Ngb gene expression was increased by the positive control and flemingia strobiliifera extract.

Figure 15 evaluates the effect of Flemingia strobiviera extract on the Ngb promoter activity of mouse neurons N2a. In the case of vehicle, positive control, estrogen, genistein, and Flemingia strobiviera extract, the activity of Ngb promoter was shown to be positive control and Flemingia strobiviera extract.

In the present invention, "female cancer" is meant to include endometrial cancer, breast cancer, ovarian cancer, and other female genital cancers in which female hormones such as estrogen and progesterone are directly or indirectly involved in carcinogenesis.

In the present invention, "menopausal symptoms" is meant to include both hot flashes, hyperlipidemia, brain mental functioning, osteoporosis, venous thrombosis and atrophic vaginitis appearing in menopausal women.

In the present invention, "low brain mental function" is meant to include both memory loss and cognitive decline in menopausal women.

In the present invention, five single components showing feminine hormones isolated from Flemingia strobiviera are represented by the following formulas (I) to (V).

(Ⅰ)

Cajanin

(Ⅱ)

2-Hydroxygenistein

(Ⅲ)

Pisatin

(Ⅳ)

2 ', 3', 4 ', 6'-tetramethoxychalcone

(Ⅴ)

Genistein

Flemingia strobivipera is a legume (Leguminosae, Fabaceae) plant that is known to grow in East Asia and is used as a folk medicine for symptoms such as epilepsy, gastritis, stomach pain and rheumatism. Anti-convulsive, cytotoxic, analgesic, antiparasitic, antimicrobial, hepatoprotective, and antioxidant effects of Flemingia strobiviera extracts have been reported, whereas Flemingia strobiviera extracts have been studied for female hormonal activity. No results have been reported. Flemingia strobillifera used in the experiments in the present invention was sold from the overseas biological material hub center.

In the present invention, the process of confirming the effectiveness of Flemingia strobiviera extract and subfraction fraction and separating and identifying the components of (I) to (V) effective in Flemingia strobilifera effective for the prevention and treatment of female cancer and menopausal symptoms. Is as follows.

One. Flemingia Strobillipera  Female Hormonal Pharmacological Effectiveness Test of Extracts

Flemingia Strobillipera Competitive inhibition of human estrogen receptor alpha (hERα) binding to extracts, analysis of MCF-7 intracellular transcription through MCF-7 estrogen and progesterone reactive genes, uterine proliferative effect and pS2 gene expression in uterine tissue and Protein expression confirmation experiments of estrogen receptor alpha (ERα), progesterone receptor A (PRA) and progesterone receptor B (PRB) were performed.

2. Flemingia Strobillipera  Of extract Subdivision  And Separation of Active Ingredients and Confirmation of Pharmacological Activity

Isolation and identification of 20 subfractions were carried out by experimenting with the separation fraction of flemingia strobiviera extract and the active ingredient, and the pharmacological activity of 5 compounds among them was confirmed to be excellent. Of these, five are the compounds represented by the above formulas (I) to (V), and the compounds (I) to (IV) confirm the female hormonal pharmacological activity for the first time in the present invention. The compound of (V) was genistein, which has been confirmed to bind to estrogen receptor and its transcriptional activity in previous studies.

The pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms of the present invention is from the group consisting of Flemingia strobiviera extract, the compound represented by the formula (I) to (IV) and pharmaceutically acceptable salts thereof It includes any one or more selected as an active ingredient. The pharmaceutical composition of the present invention may further include a compound represented by Formula (V) or a pharmaceutically acceptable salt thereof.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical composition of the present invention may further include other pharmaceutically active ingredients or active mixtures.

The pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose ( Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, Whitepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for preferred effects, it is preferable to administer at 0.0001 to 1000 mg / kg per day based on the compound represented by the formula (I) to (IV). Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or Intracerebroventricular injection.

The term definitions of the excipients, binders, disintegrants, lubricants, copulation agents, flavoring agents, etc. of the present invention are those described in the literature known in the art and include those having the same or similar functions.

Functional food composition for the prevention and improvement of female cancer and menopausal symptoms of the present invention is an active ingredient selected from the group consisting of compounds represented by the above formulas (I) to (IV) and salts acceptable to these foods Include as. The functional food composition of the present invention may further include a compound represented by the formula (V) or a food acceptable salt thereof.

The functional food composition comprises 0.0001 to 20% by weight of the compound represented by the formula (I) to (IV) in the total weight. The functional food may include, in particular, a health functional food. The term "functional food" as defined in the present invention means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on the structure and function of the human body, and "health functional food" means the human body. Means food manufactured and processed using raw materials or ingredients with useful functional properties. The health functional food may have any one form of tablets, capsules, powders, granules, liquids, and pills.

In addition, the functional food composition of the present invention may be a functional food composition in the form of a functional ingredient added to various foods or beverages. The food, for example, may be in the form of any one of beverages, powdered drinks, solids, chewing gum, tea, vitamin complexes, food additives.

Functional food composition of the present invention is an essential ingredient, except for containing any one of the components (I) to (IV) there is no particular limitation to the other components, such as various flavors or natural carbohydrates, such as ordinary food or drink It may further contain various components of. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the functional food composition of the present invention may include a flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is preferably selected in the range of 0 to about 20% by weight of the total weight of the functional food composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, these examples are only for illustrating the present invention in more detail, the scope of the present invention is not limited by these examples.

[ Experimental Example ]

Experiment method

1. Female Hormone Receptor Binding Experiment: Estrogen Receptor (ER)

Recombinant human estrogen receptor alpha (hERα) was purchased from Invitrogen. 750 mol of each receptor protein was diluted with binding buffer and used as 3 nM. The composition of the binding buffer is 10 mM Tris / pH 7.5, 10% glycerol, 1 mM DTT and 1 mg / ml BSA. ERα 750 mol, 3 nM tritium estrogen ([ ³ H] estrogen, [ ³ H] E2) and a certain concentration of test substance (dissolved in DMSO) were placed in a microcentrifuge tube and the final reaction volume was 100 μl. After incubation at 28 ° C. for 3 hours, ER-test complex was obtained by filtration on glass filter using Harvester, and unreacted free tritium estrogen ([ ³ H] E2) was removed by washing. 3 ml of Ultima Gold scintillation cocktail was added to the glass filter, and the radioactivity remaining in the glass filter was measured using a liquid scintillation counter. 5 nM estrogen (E2) was used to measure non-specific binding. 10 nM estrogen (E2) was used as a comparative control. The obtained curve and IC ₅₀ also values for the radiation count-tested with a number of density values for the same test substance in every experiment radioactive (radioactive) estrogen (estrogen, E2) of the degree of concentration by interfering with receptor binding affinity Calculated. IC ₅₀ values were calculated using Prism 3.0 software.

2. Gene reporter evaluation test: estrogen (ERE), Progestrone  (PRE) reactive gene transcription experiment

For the ERE / PRE-luciferase assay, the breast cancer cell line MCF-7, which is known to have high ER content, was used. MCF-7 breast cancer cell lines were purchased from the American Tissue Culture Collection (USA). Twenty four hours before seeding, the cells were incubated in charcoal dextran-treated medium (CD-DMEM), and the cells that reached about 90% confluency were seeded in 12-well plates at a concentration of about 5 × 10 ⁵ / well. All tests were then performed in CD-DMEM medium. After 24 hours of incubation, the estrogen response element (ERE) -luciferase and the progesterone response element (PRE) -luciferase plasmid were transfected with Lipofectamine 2000 reagent (Invitrogen, USA). In the case of ERE-luciferase assay, estrogen (estrogen, E2, 1 nM) is treated as a positive control, ICI-182,780 (1 mM) as an antagonist control, and progesterone (P4, 10 nM) as a positive control for PRE-luciferase assay. As a antagonist control, mifepristone (mifepristone, MIF, 1mM) was treated. Other test substances dissolved in various concentrations of DMSO were diluted in the medium and treated with cultured cells. After incubation for 24 hours, cell culture was terminated, and water-soluble cell extracts were obtained using Passive Lysis Buffer (Promega, USA). Luciferase activity present herein was quantitatively measured using a Luciferase Assay System (Promega) and a Luminometer including a substrate of Luciferase and a reaction buffer. The gene activity represented by the test substance was set to 100% of the activity indicated by the positive control, and the relative activity was shown.

3. In vivo  ( in vivo Evaluation of female hormones: uterine growth test Uterotrophic  assay)

The uterine growth test is a method of indirectly evaluating female hormone sex by measuring the increase in uterine tissue mass induced by estrogen. The effect of uterine proliferation was measured by examining the effect of the test substance on the immature uterus when subcutaneous injection was administered to female rats at 21 days of age for 3 days. Immature female rats were used for the experiment. During the acclimatization period, the subjects were selected to be close to the average body weight, and grouped by random method. Individual identification of animals was done by tail marking and tag by breeding box. In the positive control group, estrogen (E2) was uniformly suspended in corn oil to 3 μg / kg, and then diluted in corn oil stepwise, and the test substance was also suspended in corn oil. The experimental group was divided into three groups, and each group was subcutaneously injected with vehicle (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), and Flemingia strobiviera extract (300 mg / kg) for 24 hours. Administered. The dosage was 0.05 ml per 10 g body weight of immature rats, and solution preparation of the substance was performed on the day of administration. The test items to be tested in this test are weight and uterine weight. Body weight is measured for all animals immediately before administration and just before autopsy, and uterine weight is sacrificed by cervical dislocation 25 days after birth about 24 hours after the last administration, and the uterus is carefully removed to remove fat and fibrous tissue. After thorough drying of the water on the room, the weight of the uterus obtained from each rat was accurately measured using a micro balance (Mettler microbalance).

4. Intracellular  Evaluation of Effect on Transcriptional Activity of Endogenous Hormone Reactive Target Genes

The 21-day-old immature female rats were divided into vehicle, estrogen, and flemingia strobiliifera groups and each group of vehicle (corn oil 5ml / kg), estrogen (0.003 mg / kg), flemingia strobiliifera MRNA was extracted from the rat uterine tissues obtained by subcutaneous injection of (300 mg / kg) extract at 24 hours intervals for 3 days using Trizol. cDNA was obtained by reverse transcription of a predetermined amount of mRNA (1 μg) using an iScript cDNA synthesis kit (Bio-Rad). The expression levels of the genes were quantitatively measured by real-time PCR using cDNA, a pair of primers capable of recognizing the cDNA of the target gene, and PCR SYBR green kit (Qiagen) reagent. In order to make up for the technical mistakes in the various stages of the reaction, the expression level of the housekeeping gene, GAPDH, was simultaneously measured and the ratio between the expression level of the gene and the expression level of the target gene was used for the final quantitative analysis. Primers that recognize GAPDH include forward 5′-CTCTCTGCTCCTCCTGTTCGAC; And reverse 5'-TGAGCGATGTGGCTCGGCT.

The expression level of Trefoil factor 2 (pS2) was measured as a target gene of estrogen. Primers that recognize pS2 for real-time RT PCR reactions are forward 5'-CGTGAAAGAC AGAATTGTGGTTTT; And reverse 5'-CGTCGAAACAGCAGCCCTTA. Real-time PCR was performed at about 40 cycles (95 ° C; 30 seconds, 60 ° C; 30 seconds, 72 ° C; 30 seconds). Each gene expression curve is expressed in logarithm, and then the threshold cycle (C _T ) is mathematically obtained. The value obtained by substituting this value with 2-DDC _T represents the relative expression level of a specific gene. The expression level of the specific gene divided by the expression level of the house keeping gene was selected as the final value for the gene expression level, and the ER antagonist or agent treatment value was compared with that of the test substance.

5. Intracellular  Evaluation of Effects on Changes in Endogenous Hormone Reactive Protein Expression

The 21-day-old immature female rats were divided into vehicle, estrogen, and flemingia strobiliifera groups and each group was vehicle (corn oil 5ml / kg), estrogen (estrogen, 0.003 mg / kg), and flemingia strobiliifera ( Changes in protein expression of estrogen receptor alpha (ERα), progesterone receptor A (PRA) and progesterone receptor B (PRB) in rat uterine tissues obtained after subcutaneous injection of 300 mg / kg Confirmed. Proteins were isolated by adding lysis buffer containing protease inhibitor and phosphatase inhibitor to rat uterine tissues, and the extracted proteins were quantified by BCA method at 20 μg, 7.5% SDS-PAGE gel at 80V for 2 hours. It moved. After electrophoresis, transfer to PVDF membrane at 25V for 3 hours, blocking with RT for 1 hour with BSA, and reacted with primary and secondary antibodies were detected as ECL solution. At this time, the primary antibody was used by diluting ERα (sc-7207, SANTA CRUZ BIOTECHNOLOGY, INC.) And PR (sc-538, SANTA CRUZ BIOTECHNOLOGY, INC.) In a 1: 500 ratio. Β-actin was used to correct the difference in protein amount due to experimental error.

6. Neuron based Ngb  promoter luciferase  assay and in cells and brain tissue Ngb  mRNA, protein expression change evaluation

(1) Ngb promoter assay: Estrogen and SERMS are suppressed in the neuron cell death and was known to induce the survival SERMs also improves the memory and indicates the stroke action after improvement. In the present invention, it was confirmed whether SERMs can exhibit brain protection through Ngb activation. Ngb is a subtype of globin found in neurons that has the function of binding and transporting O ₂ and is involved in the scavenge of reactive oxygen species (ROS). In addition, it is involved in signal transduction of G-protein coupled receptors and is known to regulate apoptosis and cell survival by inducing cytochrome C electron transfer in mitochondria. Recently, the neuroprotective activity of the estrogen-based plants of the present invention was tested based on previous studies showing that estrogen increases Ngb expression in neuron and astrocyte and is related to anti-inflammatory action. Luciferase experiments were performed using Neuro2a neuroblastoma cell line (CCL-131; ATCC, Manassas, VA, USA) and SKNSH (human neuroblastoma cell line) to confirm whether plant extracts have brain protective effects. The neuroglobin (Ngb) -luciferase sequence can be inserted into cellular genes to quantitatively evaluate the Ngb promoter transcriptional activity of plant extracts. Cells were cultured in 10% FBS DMEM and seeded in 96well plates with 3x10 ⁴ cells. After 24 hours, the media was replaced with 2% FBS DMEM when 40-50% confluency was achieved. After culturing the cells, the media was replaced by dissolving the drug in serum-free phenol red free DMEM when it became 70-80% confluency. Treat cells with extract (10 ^-4 to 10 ^-7 g / ml) using Vehicle (DMSO, 0.1%), 17β-ES (1nM), and genistein (1μM) as controls, for 6 hours (SKNSH) or 24 hours ( After N2a), 40 μl of lysis buffer was added thereto, followed by incubation at RT for 15 minutes, cells were collected to obtain lysate, and 30 μl of substrate was added to measure luminescence.

(2) Ngb mRNA level (quantitative PCR ) : SKNSH cells were incubated in 10% FBS DMEM, and the media were dissolved by dissolving the drug in phenol red free DMEM. Treated with cells (DMSO, 0.1%), 17β-ES (1nM), genistein (1μM) as a control, the extract (10 ^-4 ~ 10 ^-7 g / ml) to the cells and incubated with trizol after 24 hours Isolate from the vessel surface and destroy the cells. Isolate mRNA in cells using Qiagen RNeasy mini kit. cDNA was obtained by reverse transcription of a predetermined amount of mRNA (1 μg) using an iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using a pair of primers capable of recognizing cDNA and cDNA of a target gene and a PCR SYBR green kit (Qiagen) reagent to quantitatively measure the expression level of the gene. In order to make up for the technical mistakes in the various stages of response, the expression level of GAPDH, which is a housekeeping gene, was simultaneously measured, and the ratio between the expression level of the gene and the expression level of the target gene was used for the final quantitative analysis. Primers that recognize human GAPDH include forward 5'-GGCTGAGAACGGGAAGCTTGTCAT; And reverse 5'-CAGCCTTCTCCATGGTGGTGAAGA. Primers that recognize Mouse GAPDH include forward 5'-TGCCAAGTATGATGACATCAAGAA; And reverse 5'-GCCCAAGATGCCCTTCAGT. Primers that recognize human Ngb are forward 5'-TGGAAGACCTGTCCTTCACTG; And reverse 5'-GAGCAGAGACTCACCCACTG. Primers that recognize Mouse Ngb are forward 5'-TACAATGGCCGCCAGTTCT; And reverse 5'-TGGTCACTGCAGCATCA. Quantitative PCR was performed in about 40 cycles (95˚C; 15 seconds, 60˚C; 60 seconds). Each gene expression curve was expressed in logarithm, and then the threshold cycle (C _T ) was mathematically obtained. The value obtained by substituting this value into the 2 ^-ΔΔCT expression represents the relative expression level of the specific gene. The expression level of the specific gene divided by the expression level of the house keeping gene was selected as the final value for the gene expression level, and compared with that of the vehicel group and the test substance administration group.

7. Human for evaluation of anti-inflammatory activity hematopoietic  Evaluation of prostaglandin D synthase (HPDGs) inhibitory ability

Flemingia The anti-inflammatory activity of 16 low-molecular single compounds isolated from plant extracts such as strobilifera and plant extracts and fractions was evaluated by the inhibitory effect of human hematopoietic prostaglandin D synthase (HPDGs). Enzyme reactions included assay buffer (0.1M potassium phosphate with 1mM EDTA, pH 6.5), GSH 2.5mM, CDNB 1mM, human hematopoietic prostaglandin D synthase (10mg / ml). After mixing assay buffer, CDNB, and HPDGs in 96-well plate, put GSH and plant extract (250mg / ml) or single compound (10mM) separated from plant extract and fraction. DMSO was used as a control.

After reacting for 2 minutes, the amount of CDNB-glutathione-coupled material was measured at 340 nm using a VICTOR3 luminometer (Perkin Elmer) instrument, and compared with the control (DMSO) value to compare the activity of HPDGs.

8. Acute Toxicity Test

 Single dose acute toxicity experiments were performed to increase the clinical applicability of the candidate plant extracts. Animals were used as female mice and fasted 24 h before administration. Body weight (1% CMC in saline), 250mg / kg extract, 500mg / kg, 1000mg / kg, 1500mg / kg doses were administered in a single oral dose. The experiment was carried out for a total of 2 weeks and the administration volume was within 5μl / g.

9. Analysis of Active Ingredients on Plant Extract Fractions

By separating the active fractions from crude extracts, the active plant metabolites were identified and identified as the main single component with pharmacological effects. The specific experimental method is as follows.

(1) Extraction and separation: MeOH extract of each plant was subjected to C18-RP- medium pressure liquid chromatography to obtain several fractions.

(2) Bioassay-guided fractionation: For each fraction, fractions showing activity were selected by performing hormonal receptor efficacy and antagonism in cultured cells as standard tests.

(3) Detailed separation and purification of single component separation of active fractions: By fractionation and purification of active metabolites, fractions containing active plant metabolites are obtained by MPLC, HPLC, and LC-MS analysis. Was carried out.

(4) Chemical structure analysis and characterization of single component: UPLC, LC-MS / MS, etc. are used to separate single components on chromatogram and to obtain single components, followed by IR analysis, NMR analysis ( ¹ H, ¹³ C, DEPT, Various structural crystallographic methods such as COSY, HSQC, HMBC, NOESY, ROESY) were used. Existing medicinal plant DB was used to confirm whether the final substance was new or known.

result

One. Flemingia Strobillipera  Female Hormonal Pharmacological Effectiveness of Extracts

end. For ER binding Flemingia Strobillipera  extract, Subdivision Competitive inhibition of a single component

In vitro system Competitive binding of Flemingia strobillifera extract and tritium estrogen ([ ³ H] E2) to pure recombinant human estrogen receptor alpha (hERα) was measured. The results are shown in FIGS. 1, 2 and Tables 1, 2. In a competitive binding assay, the IC ₅₀ of estrogen (E2) (•) was 3.83 × 10 ⁻¹⁰ g / ml (hERα), respectively (FIG. 1, Table 1). The IC ₅₀ of the phytoestrogen, a type of phytoestrogen, was 1.60 × 10 ⁻⁵ g / mL (hERα). These results confirm that Flemingia strobillifera extract acts concentration-dependently on tritium estrogen ([ ³ H] E2) separation bonds and has a relative bioavailability (RBA) compared to endogenous estrogens. Was 0.0024% (FIG. 1, Table 1). Concentration-dependent ER binding capacity analysis of subfractions (Nos. 1-20 ) showed excellent ER binding strengths of fractions FS5, FS6, FS7, FS8 and FS20 ( Figure 2 , Table 2) .

Flemingia Evaluation of ER subtype selectivity for fractions of strobilifera

Flemingia Twenty fractions of strobilifera (FS-1-20) were dissolved in DMSO or water at an appropriate concentration, and then the ER subtype selective gene transcriptional activity was measured for 10 mg / ml samples. The relative comparison of luciferase activity of each sample is shown in FIG. 3. F lemingia extracts and fractions 20 species of strobilifera - in fractions two kinds (FS-7, FS-9 ) (FS-1 20) showed that the ERβ / ERα ratio is 2 or more, but ERα activity is not significant the lower the result (FIG. 3) .

Table 1 Flemingia ERα binding force of Blow Billy Straw Extract

(Table 2) Flemingia Strobillipera Comparison of 20 Types of ERα Avidity Subdivided from Extracts

RBA = (Ki _(E2) / Ki _{( Flemingia strobilifera )} ] × 100.

I. Flemingia Strobillipera  extract, Subdivision , Single component MCF -7 through estrogen response genes MCF -7 cell proliferation and Intracellular  Transcription analysis

In order to evaluate MCF-7 cell proliferation, MCF7 cell proliferation was confirmed after Flemingia strobilifera extract was treated with 10 ^-6 g / ml ~ 5x10 ^-5 g / ml concentration. The 17β-ES used as a positive control induced about 2.1-fold proliferation of MCF7 cells compared to the vehicle group, and the genistein-administered group also increased the proliferation of MCF7 cells by 1.8 times. Flemingia strobilifera extract was found to induce cell proliferation about 1.6-fold and 2-fold more at concentrations of 10 ^-6 g / ml and 5x10 ^-6 g / ml, respectively. Relatively high concentrations of 10 ⁻⁵ g / ml and 5 × 10 ⁻⁵ g / ml did not induce cell proliferation (FIG. 4) .

Experiment was not for the Fleming starvation Our Billy Blow extract change the activity of the gene ERE- in MCF-7 cells in which transfection of estrogen-responsive genes (Estrogen responsive element, ERE) results are shown in Fig. When treated with Flemingia strobillifera alone, the activity was different depending on the treatment concentration when compared to the vehicle at the concentration of 1-20 μg / mL. The maximum activity was found at the concentration of 5 μg / mL of the extract, and the ERE gene activity was increased by about 11.8 times more than the vehicle. In addition, ERE activity tended to increase with increasing concentration at concentrations of 1μg / ml, 2.5μg / ml, and 5μg / ml, and the concentration of ERE decreased with increasing concentrations above 10μg / ml. It was confirmed that the activity was high up to about 80% of ERE gene activity by estrogen (estrogen, E2) at the concentration of 5μg / ㎖ showing the maximum activity. These results show that Flemingia strobiviera extracts exhibit different ERE gene activity depending on the concentration. In this experiment using MCF-7 cells, it was confirmed that ERE gene activity by estrogen (estrogen, E2) was blocked when co-administered with ICI, an ER antagonist. Based on the results of this experiment, Flemingia strobivipera during gene transcription The extract was found to exhibit ERE gene activity as about 80% of estrogen (E2). Also of Flemingia Strobiviera Subfractions were performed for the ERE and PRE gene reporter experiments. Flemingia Strobillipera The subfractions were tested at 20 μg / ml concentrations of Flemingia strobiviera Ten subfractions increase transcriptional activity through ERE by more than 90% compared to the activity of estrogen (fraction number: FS-1, 2, 4, 5, 6, 7, 8, 9, 10, 11) (Fig. 6) . Furthermore, FS 5, 6, 7, fractions increased ERE gene activity more than three times compared to vehicle. Concentration-dependent ERE transcriptional activity of five single components isolated from Flemingia strobillifera was identified (FIG. 7) . SK-F4 was found not to exhibit ERE transcriptional activity, and SK-F1 showed ERE transcriptional activity similar to or higher than that of estrogen above 2.5 μM. SK-F2 and SK-F3 showed ERE transcriptional activity in a concentration-dependent manner, and concentration-dependent ERE transcriptional activity of SK-F5 (genistein) was also confirmed. On the other hand, no single fraction was found to increase transcriptional activity through the progesterone responsive element (PRE), but fractions that decreased PRE activity when co-treated with progesterone (P4) were found to be FS-9, 10, Turns 11, 12, 13, 15, 20 (FIG. 8) . FS-9, 10, 11, 12, 13, 15 and 20 were found to inhibit PRE gene transcriptional activity by P4 by 28%, 21%, 25%, 23%, 17% and 25%, respectively. These results suggest that these fractions contain substances with PR antagonist efficacy that can interfere with the transcriptional activity of P4. In the case of single component, there was no single component that increases the transcriptional activity through PRE, but the single component that reduced PRE activity when co-treated with P4 was identified as SK-F3 and SK-F4. %, Reduced to 85% level. SK-F2 and SK-F5 were found to increase P gene transcription activity by P4 by about 160% when co-treated with P4 (FIG. 9) .

All. Flemingia Strobillifera  Uterine growth effect

In order to evaluate the female hormone properties of the extract in the system excluding the action of endogenous estrogen (E2), uterine proliferation was tested in 21-day-old female rats with very low estrogen (estrogen, E2) secretion. Uterine growth results were compared and evaluated as shown in Table 3 below. The ratio of uterine weight to body weight was 0.63 when the corn oil was administered to the vehicle group, and the uterine weight ratio was 1.97 compared to the control group after subcutaneous injection of estrogen (E2) at a dose of 3 μg / kg as a positive control. The uterine ratio was about three times higher. The uterine weight ratio was 1.88 when the Flemingia strobiviera extract was injected subcutaneously into immature rats at 300 mg / kg dose. there was. Flemingia Strobiviera Extracts were found to induce uterine tissue proliferation. In addition, weight gain in rats was not observed during drug administration, and significantly different values from Flemingia strobillifera and vehicle groups were indicated as *** (* ** P <0.001).

Table 3 Flemingia Uterine Proliferation Effect by Strobillifera Extract

la. Flemingia Strobillifera  In uterine tissue pS2  Gene activity induction

Flemingia strobilifera extract was treated with a concentration of 10 ⁻⁷ g / ml to 10 ⁻⁵ g / ml, and the expression of pS2 gene in MCF7 cells was confirmed. In the case of 17β-ES used as a positive control, the expression of pS2 gene was increased by 1.7 times compared with the vehicle group, and genistein increased the expression of pS2 gene by 1.5 times. In the case of the Flemingia strobilifera extract, the expression of the pS2 gene was confirmed to be increased more than two times at all concentrations (FIG. 10).

Vehicles (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), Flemingia strobiviera in immature female rats at 21 days of age The expression of pS2 gene was confirmed in rat uterine tissues obtained after subcutaneous injection of (300 mg / kg) extract at 24 hours interval for 3 days. As a result, it was confirmed that the expression of pS2 gene was increased by about 14 times when the estrogen was administered compared with the vehicle, and about 41 times increased when the Flemingia strobiviera extract was administered. These experimental results confirmed the estrogen activity in the uterine tissue of Flemingia strobiviera extract and confirmed that it induces the expression of the pS2 gene (FIG. 11) .

hemp. Flemingia Strobillifera  In uterine tissue ERα  Inhibit protein expression

Vehicles (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), Flemingia strobiviera in immature female rats at 21 days of age Changes in protein expression of estrogen receptor alpha (ERα), progesterone receptor A (PRA) and progesterone receptor B (PRB) in rat uterine tissues obtained after subcutaneous injection of (300 mg / kg) extract at 24 hour intervals for 3 days. It was confirmed. Experimental results show that ERα protein expression decreases when estrogen is administered compared to vehicle and Flemingia strobiviera The administration of the extract also reduced the expression of protein. Protein expression of PRA and PRB, on the other hand, was compared to Flemingia strobiviera No change could be confirmed when the extract was administered. Through these experimental results, it was confirmed that Flemingia strobiviera extract inhibits the estrogen receptor protein expression in the uterine tissue (FIG. 12) .

bar. Of human and mouse neurons neuroglobin  ( Ngb promoter activity mRNA  Increased Gene Expression and Rat Brain Tissue Ngb  Protein increase

For the effects on the Ngb promoter activity of human neurons SKNSH, Flemingia strobilifera extracts were treated with SKNSH cells at concentrations of 10 ^-6 g / ml ~ 5x10 ^-5 g / ml, followed by luciferase assay to determine Ngb promoter activity by extracts. It was evaluated quantitatively. In treatment with positive control genistein, Ngb promoter activity was increased by 1.4-fold compared to vehicle group, and 17β-ES also increased Ngb promoter activity by 1.3-fold. Treatment with Flemingia strobilifera extract significantly increased Ngb promoter activity by 1.5-1.7 fold at all concentrations (*** p <0.001) (FIG. 13) . Flemingia strobilifera extract was treated with a concentration of 5x10 ^-6 g / ml ~ 10 ^-5 g / ml and the changes of Ngb gene expression in SKNSH cells were confirmed. In the case of 17β-ES used as a positive control, the expression of Ngb gene was increased by 1.6 times compared with the vehicle group. In the case of Flemingia strobilifera extract, the expression of Ngb gene was found to be increased by 1.2 to 1.3 times (FIG. 14).

In order to evaluate the effect on Ngb promoter activity of mouse neuron N2a cells, Flemingia strobilifera extract was treated with N2a cells at a concentration of 10 ^-5 g / ml ~ 10 ^-4 g / ml, followed by luciferase assay. Quantitative evaluation of promoter activity was made. Treatment with the positive control genistein increased the activity of the Ngb promoter by 1.7 times compared to the vehicle group, and 17β-ES did not increase the Ngb promoter activity. Treatment with Flemingia strobilifera extract increased the Ngb promoter activity by about 1.7-2.5 fold at all concentrations (*** p <0.001). In particular, the highest Ngb promoter activity in the 5x10 ^-5 g / ml concentration treatment group (Fig. 15) .

Ah. Human hematopoietic  prostaglandin D synthase (HPDGs)  Anti-inflammatory activity

Flemingia strobilifera . Of extract (FSE) and separating the identified 5 species in the extract and fractions - when the anti-inflammatory active effect on the low molecular weight single compound of (SK-F1 F5) hayeoteul evaluated through the inhibitory effect of human hematopoietic prostaglandin D synthase (HPDGs), Flemingia HPDGs inhibitory effect of the extract (FSE) of strobilifera are very high as 75.3%, the single component of the five kinds of (SK-F1 - F5) of the SK-F1, F5 was 23.3%, respectively HPDGs inhibitory effect, HPGDs inhibition to 21.3% effective Had some significance. The remaining three single components, SK-F2, F3, and F4, had 1.1%, 0%, and 2.2% of HPDGs inhibitory effects, respectively, and did not have HPDGs inhibitory effects.

character. Through acute toxicity testing Safety evaluation

Flemingia Single dose acute toxicity experiments were performed to evaluate the in vivo toxicity of strobilifera extract. Female mice (10 weeks old) were treated with vehicle (1% CMC saline) and Flemingia strobilifera extract at 250 mg / kg, 500 mg / kg, 1000 mg / kg, 1500 mg / kg doses for 2 weeks of mortality, weight change and behavior. When observed, animal movements decreased slightly after administration, but showed normal behavior after 24 hours, and feed and water intake were observed normally. The mortality rate was 0% in all groups, including the vehicle group, during the two-week experiment period. Weight changes between the groups were similar (Table 4) .

(Table 4) Flemingia Evaluation of Acute Toxicity by Strobillifera Extract

2. Flemingia Strobillifera  Bioassay-guided analysis

end. Fraction and Purification of Extracts

In order to investigate the active pharmacologically active ingredient of Flemingia strobiviera extract, methanol extract (12 g) was coated on RP resin (cosmosil 75 C18, 36g) and top loaded to obtain the final 20 fractions (FS1 to FS20). As a result of evaluating the hERα binding capacity of these fractions, fractions FS5, FS6, FS7, FS8 and FS20 showed significant activity. Bioassay-guided analysis of the subfractions allowed us to trace single components with good physiological activity. As a result of hERα binding force analysis of five single components (SK-F1 to SK-F5), the results of Table 5 were obtained.

Table 5 Flemingia Our Billy hERα affinity comparison of five kinds of single-component separated from the extract Blow

FS5 is a mobile phase of methanol and water _{(MeOH: H 2 O, 30} : 70) were divided into a RP-MPLC five small fraction obtained by performing where small fraction FS5-3 each compound by the method of methanol and water and crystallized 3 (83.5 mg) ( III) was isolated.

FS6 was divided into five small fractions by RP-MPLC using methanol and water as a mobile phase (MeOH: H ₂ O, 40: 60). The small fractions FS6-6 and FS6-8 obtained here were crystallized from methanol and water. Compound 1 (55.3 mg) ( I) and compound 2 (46.1 mg) ( II) were separated, respectively.

FS7 isolated Compound 4 (16.4 mg, t _R = 31.57min) ( IV) using HPLC (YMC C18, 5 um, 250 × 20 mm, ACN 45%, 7ml / min, 210nm).

FS8 isolated Compound 5 (4.7 mg, = 41.05min) ( V ) using HPLC (YMC C18, 5 um, 250 × 20 mm, ACN 45% 20min, 55% 35min, 7ml / min, 210nm).

I. Identification of chemical structure of single substance and confirmation of concentration-dependent pharmacological activity of single component

In order to examine chemical structures of the isolated single substances, structural analysis was performed by spectroscopic methods such as NMR and HRESIMS, and the concentration-dependent binding capacity of the human estrogen receptor alpha (hERα) was evaluated. Finally, five single components (SK-F1 to SK-F5) showing effective pharmacological activity were isolated and identified. The five single components are shown in Table 6, and the ER binding force and ERE activity of each compound are expressed as IC ₅₀ (M) and EC ₅₀ (M), respectively. Flemingia Strobillipera This is the first time that the compounds I to V have been discovered from plants as a single component that exhibits female hormone properties.

(Table 6) Flemingia The structure of a single active ingredient present in the straw Billy Blow and estrogen (estrogen) Efficacy

(1) Compound (I)

Cajanin

C ₁₆ H ₁₂ O ₆ (300.26)

HRESIMS: m / z 301.0707 [M + H] ⁺ (calcd: 301.0712)

¹ H NMR (400 MHz, DMSO-d6): δ 8.24 (1H, s, H-2), 6.41 (1H, d, J = 2.0 Hz, H-6), 6.65 (1H, d, J = 2.1 Hz , H-8), 6.37 (1H, d, J = 2.1 Hz, H-3 '), 6.28 dd (1H, dd, J = 8.3, 2.1 Hz, H-5'), 6.99 (1H, d, J = 8.3 Hz, H-6 '), 3.87 (3H, s, OCH ₃ ) ppm.

¹³ C NMR (100 MHz, DMSO-d6): δ 156.2 (C-2), 121.1 (C-3), 181.0 (C-4), 162.1 (C-5), 98.3 (C-6), 165.5 ( C-7), 92.8 (C-8), 158.0 (C-9), 105.9 (C-10), 108.2 (C-1 '), 156.9 (C-2'), 103.0 (C-3 '), 159.1 (C-4 '), 106.7 (C-5'), 132.6 (C-6 '), 56.5 (OCH ₃ ) ppm.

(2) Compound (II)

2-Hydroxygenistein

C ₁₅ H ₁₀ O ₆ (286.24)

HRESIMS: m / z 287.0554 [M + H] ⁺ (calcd: 287.0556)

¹ H NMR (400 MHz, CD ₃ OD): δ 8.01 (1H, s, H-2), 6.23 (1H, d, J = 2.2 Hz, H-6), 6.36 (1H, d, J = 2.2 Hz , H-8), 6.39 (1H, d, J = 2.3 Hz, H-3 '), 6.37 (1H, overlapped, H-5'), 7.04 (1H, d, J = 8.2 Hz, H-6 ' ) ppm.

¹³ C NMR (100 MHz, CD ₃ OD): δ 156.9 (C-2), 122.7 (C-3), 182.8 (C-4), 163.8 (C-5), 100.3 (C-6), 166.1 ( C-7), 94.9 (C-8), 159.9 (C-9), 106.3 (C-10), 110.9 (C-1 '), 158.0 (C-2'), 104.4 (C-3 '), 160.4 (C-4 '), 108.2 (C-5'), 133.3 (C-6 ') ppm.

(3) Compound (III)

Pisatin

C ₁₇ H ₁₄ O ₆ (314.29)

HRESIMS: m / z 313.0716 [M + H] ^- (calcd: 313.0712)

¹ H NMR (400 MHz, CD ₃ OD): δ 7.33 (1H, d, J = 8.7 Hz, H-1), 6.61 (1H, dd, J = 8.6, 2.5 Hz, H-2), 6.42 ( 1H, d, J = 2.5 Hz, H-4), 4.11 (1H, dd, J = 11.5, 0.8 Hz, H-6), 3.95 (1H, d, J = 11.5 Hz, H-6), 6.82 ( 1H, s, H-7), 6.35 (1H, s, H-10), 5.20 (1H, s, H-11b), 3.74 (3H, s, OCH ₃ ), 5.90 (1H, d, J = 1.1 Hz, OCH ₂ O), 5.86 (1H, d, J = 1.1 Hz, OCH ₂ O) ppm.

¹³ C NMR (100 MHz, CD ₃ OD): δ 133.2 (C-1), 110.3 (C-2), 162.6 (C-3), 102.5 (C-4), 157.5 (C-4a), 70.8 ( C-6), 77.8 (C-6a), 121.6 (C-6b), 104.5 (C-7), 143.7 (C-8), 151.0 (C-9), 94.7 (C-10), 155.9 (C -10a), 86.1 (C-11a), 114.3 (C-11b), 55.9 (OCH ₃ ), 102.9 (OCH ₂ O) ppm.

(4) Compound (IV)

2 ', 3', 4 ', 6'-tetramethoxychalcone

C ₁₉ H ₂₀ O ₅ (328.36)

HRESIMS: m / z 329.1384 [M + H] ⁺ (calcd: 319.1389)

¹ H NMR (400 MHz, CD ₃ OD): δ 7.58 (2H, m, H-2,6), 7.4 (3H, overlapped, H-3,4,5), 6.97 (1H, d, J = 16.1 Hz, H-α), 7.32 (1H, d, J = 16.1 Hz, H-β), 6.37 (1H, s, H-5 '), 3.81 (3H, s, OCH ₃ atC-2'), 3.79 (3H, s, OCH ₃ atC-3 '), 3.93 (3H, s, OCH ₃ atC-4' or C-6 '), 3.78 (3H, s, OCH ₃ atC-4' or C-6 ') ppm.

¹³ C NMR (100 MHz, CD ₃ OD): δ 136.1 (C-1), 129.7 (C-2,6), 130.2 (C-3,5), 131.9 (C-4), 129.9 (C-α ), 147.3 (C-β), 196.0 (C = O), 117.1 (C-1 '), 152.9 (C-2'), 137.3 (C-3 '), 157.0 (C-4' or C-6 '), 94.1 (C-5'), 154.9 (C-4 'or C-6'), 62.4 (OCH ₃ atC-2 '), 61.6 (OCH ₃ atC-3'), 56.9 (OCH ₃ atC- 4 'or C-6'), 56.8 (OCH ₃ atC-4 'or C-6') ppm.

(5) Compound (Ⅴ)

Genistein

C ₁₅ H ₁₀ O ₅ (270.24)

HRESIMS: m / z 271.0615 [M + H] ⁺ (calcd: 271.0606)

¹ H NMR (400 MHz, CD ₃ OD): δ 8.04 (1H, s, H-2), 6.21 (1H, d, J = 2.2 Hz, H-6), 6.33 (1H, d, J = 2.2 Hz , H-8), 7.36 (2H, d, J = 8.7 Hz, H-2 ', 6'), 6.84 (2H, d, J = 8.7 Hz, H-3 ', 5') ppm.

¹³ C NMR (100 MHz, CD ₃ OD): δ 154.9 (C-2), 124.9 (C-3), 182.4 (C-4), 164.0 (C-5), 100.2 (C-6), 166.1 ( C-7), 94.9 (C-8), 159.8 (C-9), 106.4 (C-10), 123.4 (C-1 '), 131.5 (C-2', 6 '), 116.4 (C-3 ', 5'), 159.0 (C-4 ') ppm.

[ Example ]

Formulation example  One. Powder  Produce

Compound (II) 1 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

Compound (II) 1 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3. Manufacture of capsule

Compound (II) 1 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of Injectables

Compound (II) 1 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

Compound (II) 1 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, and lemon flavor is added, the above components are mixed, and then purified water is added to adjust the total amount to 100 ml, and then sterilized by filling in a brown bottle. do.

Flemingia strobiviera extract of the present invention, its active fraction and female hormone substances derived from natural products, can be used in the field of medicines and functional foods as an effective substance for the treatment and prevention of female cancer and menopausal symptoms.

Claims (11)

1. Flemingia Flemingia strobilifera ) extract, its active fraction, the compounds represented by the formulas (I) to (IV) below, and any one or more selected from the group consisting of pharmaceutically acceptable salts thereof, female cancer and menopause Pharmaceutical compositions for the prevention and treatment of symptoms.

   (Ⅰ)

   

   (Ⅱ)

   

   (Ⅲ)

   

   (Ⅳ)

   
2. The pharmaceutical composition of claim 1, further comprising a compound represented by the following formula (V) or a pharmaceutically acceptable salt thereof.

   (V)

   
3. The pharmaceutical composition of claim 1, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer.
4. The pharmaceutical composition of claim 1, wherein the menopausal symptoms include any one of hot flashes, hyperlipidemia, decreased brain mental function, osteoporosis, venous thrombosis, and atrophic vaginitis.
5. Flemingia Flemingia strobilifera ) extracts, active fractions thereof, compounds represented by the formulas (I) to (IV) below, and any one or more selected from the group consisting of salts acceptable for foods thereof, female cancer and menopausal symptoms Functional food composition for the prevention and improvement of.

   (Ⅰ)

   

   (Ⅱ)

   

   (Ⅲ)

   

   (Ⅳ)

   
6. The functional food composition according to claim 5, further comprising a compound represented by the following formula (V) or a food acceptable salt thereof.

   (V)

   
7. The functional food composition of claim 5, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer.
8. The functional food composition of claim 5, wherein the menopausal symptoms include any one of hot flashes, hyperlipidemia, cerebral psychiatric function, osteoporosis, venous thrombosis, and atrophic vaginitis.
9. The functional food composition of claim 5, wherein the food has any one of tablets, capsules, powders, granules, liquids, and pills.
10. The functional food composition of claim 5, wherein the food has any one of a beverage, a powdered beverage, a solid, a chewing gum, a tea, a vitamin complex, and a food additive.
11. The functional food composition of claim 9 or 10, wherein the food is a health functional food.

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