WO2014193067A1 - Pharmaceutical composition containing, as active ingredient, compound separated from laria nitida extract or pharmaceutically acceptable salt thereof for preventing and treating female cancers and menopausal symptoms - Google Patents

Abstract

The present invention relates to pharmaceutical compositions containing, as active ingredients, single components separated from a Laria nitida extract or pharmaceutically acceptable salts thereof, for preventing and treating female cancers and menopausal symptoms. The present invention provides single components separated and identified from a Laria nitida extract and pharmaceutical compositions and functional health compositions containing the same. The present invention first verified that the single components derived from Laria nitida exhibit female hormone characteristics. The female hormonal substances provided in the present invention can be used in fields of medicines and functional health foods to treat and prevent female cancers and menopausal symptoms.

Description

A pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms, containing as an active ingredient a compound isolated from Laria nitida extract or a pharmaceutically acceptable salt thereof.

The present invention relates to a pharmaceutical composition for the prophylaxis and treatment of female cancer and menopausal symptoms containing the compound or pharmaceutically acceptable salt thereof isolated from Laria nitida extract as an active ingredient.

Women's cancers such as endometrial and breast cancers 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 for 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 more than eight-fold, suggesting that megestrol, Megage ™, a progesterone receptor agonist. It is widely used. However, side effects of megestrol include high blood pressure, thrombophlebitis, and weight gain. Therefore, if a substance that can balance the efficacy of progesterone and estrogen from natural products is discovered, it may be an effective drug for endometrial cancer 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 the high fat and high calorie westernized diet and obesity, late marriage and low 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 cell resistance after long-term administration have been pointed out as a major problem 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, the development of selective estrogen receptor modulators (SERMs) and selective progesterone receptor modulators (SPRMs) with molecular targeting of ER / PR while minimizing side effects and drug resistance expression have been associated with breast cancer. This is a key goal in the development of therapeutics.

Menopause syndrome

The rapid decrease in estrogen after menopause causes symptoms such as hot flashes, worsening of osteoporosis, hyperlipidemia, mental instability and sudden emotional changes, and vaginal dryness. For these postmenopausal symptoms, administration of estrogen alone or a combination of estrogen / progesterone is almost the only treatment, which 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, excess cell proliferation due to E2 / ER interaction and accumulation of DNA mutations have been suggested, and genotoxicity by reactive metabolites occurring during in vivo drug metabolism is also suggested as a carcinogenic mechanism. It is. The pharmacological effects of these tissue-specific estrogens have been known to be due to a variety of molecular mechanisms that interact with 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.

Plants of the genus Larrea have been reported to have anti-inflammatory, antibacterial and antiviral effects of L. tridentata or L. divaricata shrubs (Heinrich). M. Curr Topics Med Chem 2003; 3: 141-154; Gagnier JJ et al. Am J Med 2006; 119: 1-11). There has also been a study on the effects of inhibiting tumor cell colonization and cancer cell proliferation of the genus Laria (Garreau B et al. Biochim Biophys Acta 1991; 1094: 339-45; Anesini C et al. Phytomedicine 2001; 8: 1 -7). Plants of the genus Laria so far studied contain many lignan-based components, as well as cinnamic acid and its derivatives. A representative component of a known plant of the genus Laria is lignan nordihydroguaiaretic acid (NDGA). NDGA is a potent antioxidant and various biological activities are being studied. Reportedly, tumor cell proliferation inhibitory effect is observed through accumulation of protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP) and inhibition of calcium influx (Erashi M et al., Oncology 1995; 52: 150-155; Pavani M et al. Biochemical Pharmacology 1994; 48: 1935-1942). In addition, anti-cancer effects have been reported through the inhibition of activities such as receptor tyrosine kinase (RTK) and insulin-like growth factor receptor (IGF-1R) (Youngren JF et al. Breast Cancer Res Treat). 2005; 94: 37-46). In addition, NDGA was found to have binding and transcriptional activity to estrogen receptor in evaluation of estrogen activity (Fujimoto N et al. Life Sci 2004; 74: 1417-25). However, NDGA content and related biological activities have been identified only in some plants of the genus Laria, and various biological activities and related substances appearing in various plants of the genus Laria are still unknown.

Prior art literature

[Patent Documents]

(Patent Document 1) Republic of Korea Registered Patent Publication 10-0419121

(Patent Document 2) Republic of Korea Patent Registration 10-0542479

(Patent Document 3) Korean Unexamined Patent Publication 10-2004-0101694

(Patent Document 4) Republic of Korea Patent Registration 10-0557006

(Patent Document 5) Republic of Korea Patent Publication 10-1141194

(Patent Document 6) Republic of Korea Patent Registration 10-1177508

[Non-Patent Documents]

(Non-Patent Document 1) Anesini, C., Ferraro, G., Lopez, P., and Borda, E. (2001). Different intracellular signals coupled to the antiproliferative action of aqueous crude extract from Larrea divaricata Cav. and nor-dihydroguaiaretic acid on a lymphoma cell line. Phytomedicine 8, 1-7.

(Non-Patent Document 2) Earashi, M., Noguchi, M., Kinoshita, K., and Tanaka, M. (1995). Effects of eicosanoid synthesis inhibitors on the in vitro growth and prostaglandin E and leukotriene B secretion of a human breast cancer cell line. Oncology 52, 150-155.

(Non-Patent Document 3) Fujimoto, N., Kohta, R., and Kitamura, S. (2004). Estrogenic activity of an antioxidant, nordihydroguaiaretic acid (NDGA). Life Sci 74, 1417-1425.

(Non-Patent Document 4) Gagnier, J.J., DeMelo, J., Boon, H., Rochon, P., and Bombardier, C. (2006). Quality of reporting of randomized controlled trials of herbal medicine interventions. Am J Med 119, 800 e801-811.

(Non-Patent Document 5) Garreau, B., Vallette, G., Adlercreutz, H., Wahala, K., Makela, T., Benassayag, C., and Nunez, E.A. (1991). Phytoestrogens: new ligands for rat and human alpha-fetoprotein. Biochim Biophys Acta 1094, 339-345.

(Non-Patent Document 6) Heinrich, M. (2003). Ethnobotany and natural products: the search for new molecules, new treatments of old diseases or a better understanding of indigenous cultures? Curr Top Med Chem 3, 141-154.

(Non-Patent Document 7) Jordan, V.C. (2007). Chemoprevention of breast cancer with selective oestrogen-receptor modulators. Nat Rev Cancer 7, 46-53.

(Non-Patent Document 8) Pavani, M., Fones, E., Oksenberg, D., Garcia, M., Hernandez, C., Cordano, G., Munoz, S., Mancilla, J., Guerrero, A. , and Ferreira, J. (1994). Inhibition of tumoral cell respiration and growth by nordihydroguaiaretic acid. Biochem Pharmacol 48, 1935-1942.

(Non-Patent Document 9) Youngren, J.F., Gable, K., Penaranda, C., Maddux, B.A., Zavodovskaya, M., Lobo, M., Campbell, M., Kerner, J., and Goldfine, I.D. (2005). Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 and c-erbB2 / HER2 / neu receptors and suppresses growth in breast cancer cells. Breast Cancer Res Treat 94, 37-46.

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 by isolating and identifying a single component showing female hormones from Larrea nitida .

The development of drugs for treating ER or postmenopausal symptoms such as breast cancer or endometrial cancer, such as breast cancer or endometrial cancer, has been described as a substance capable of having a tissue-specific differentiation effect on ER, that is, selective female hormone receptor modulators (SERMs). Developing 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,

A pharmaceutical for the prevention and treatment of female cancers and menopausal symptoms, containing any one or more selected from the group consisting of compounds represented by the following formulas (I) to (IV) and pharmaceutically acceptable salts thereof as an active ingredient A composition is provided. 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,

Food composition for the prevention and improvement of female cancer and menopausal symptoms containing any one or more selected from the group consisting of a compound represented by the formula (I) to (IV) and a food acceptable salt thereof as an active ingredient This is provided. The food composition of the present invention may further include a compound represented by the following formula (V) or a food acceptable salt thereof.

(Ⅰ)

(Ⅱ)

(Ⅲ)

(Ⅳ)

(Ⅴ)

It was first discovered in the present invention that the single components of the above formulas (I) to (IV) derived from Laria nitida exhibit female hormones.

The present invention, Larrea nitida By identifying and identifying single components that show female hormones in plants, we provide new plant metabolism-derived single-component medicines and dietary supplements useful for the prevention and treatment of female cancer and menopausal symptoms. In particular, the present invention first confirmed that single components derived from Laria nitida exhibit female hormones, and the pharmaceutical composition of the present invention containing these components occurs with the highest frequency among female cancers and its incidence rate. This may be useful for increasing breast cancer and endometrial cancer having a similar pathogenesis.

1 is a test result of the estrogen receptor-dependent MCF-7 breast cancer cell proliferation effect of Laria nitida extract. Significantly different values from the vehicle group are indicated with ***, of which ES and other values are indicated with +++ (*** <0.001, +++ <0.001).

2 is in an in vitro system Competitive binding of laria nitida (◆) extract and [ ³ H] 17β-ES (●) to pure hER (recombinant human estrogen receptor) was measured.

FIG. 3 shows the results of experiments on whether or not Raria nitida alters ERE-gene activity in MCF-7 cells transfected with Estrogen responsive element (ERE) (significantly different values from vehicle group are indicated by ***). In other words, the ES group and other values are indicated by +++ (*** <0.001, +++ <0.001).

Figure 4 is a result of analyzing the concentration-dependent ER binding force of the fraction (No. 1-8) of the extract of Laria nitida.

5 is a result of analyzing the concentration-dependent ER binding force of the subfractions (No. 9-16) of the extract of Laria nitida.

Figure 6 is the result of analyzing the concentration-dependent PR binding force of the fraction (8-12) of the extract of Laria nitida.

In the present invention, by identifying the therapeutic efficacy, pharmacological metabolism and safety of female cancer of female hormone-like active ingredients isolated from various plants, including the genus Laria, preventing cancer and menopausal symptoms, such as endometrial cancer, breast cancer and Provided are new pharmaceutical compositions or food compositions effective for treatment.

In the present invention, "female cancer" is meant to include both 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, osteoporosis, venous thrombosis and atrophic vaginitis appearing in menopausal women.

In the present invention, four single components isolated from Larrea nitida , which exhibit female hormones, are represented by the following formulas (I) to (IV).

(Ⅰ)

3 ', 6-di-O-demethylisoguaiacin (3', 6-di-O-demethylisoguaiacin)

(Ⅱ)

Norisoguaiacin

(Ⅲ)

4- (4- (4-hydroxyphenyl) -2,3-dimethylbutyl) benzene-1,2-diol (4- (4- (4-hydroxyphenyl) -2,3-dimethylbutyl) benzene-1,2 -diol)

(Ⅳ)

4- (4- (3-hydroxy-4-methoxyphenyl) -2,3-dimethylbutyl) benzene-1,2-diol (4- (4- (3-hydroxy-4-methoxyphenyl) -2, 3-dimethylbutyl) benzene-1,2-diol)

In addition, NDGA (nordihydroguaiaretic acid), which is conventionally known as one of the representative components of the plant of the genus Laria, has also been identified as a compound (V) which is a single active ingredient having estrogen efficacy in the present invention. NDGA, a lignan-based component, is a potent antioxidant and has been studied for various physiological activities. In the past, tumor cell proliferation inhibitory effect was inhibited by accumulation of protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP) and inhibition of calcium influx (Erashi M et. al., Oncology 1995; 52: 150-155; Pavani M et al. Biochemical Pharmacology 1994; 48: 1935-1942), receptor tyrosine kinase (RTK; Receptor tyrosine kinase), insulin-like growth factor (IGF-1R; Insulin anti-cancer effects through inhibition of activities such as -like growth factor receptors (Youngren JF et al. Breast Cancer Res Treat 2005; 94: 37-46), and the binding and transcriptional activity of estrogen receptors in estrogen activity evaluation (Fujimoto N et al. Life Sci 2004; 74: 1417-25). In the present invention, NDGA has been identified as one of the single components having estrogen efficacy, isolated from Laria nitida.

(Ⅴ)

Nordihydroguaiaretic acid (NDGA)

Larrea nitida is a plant of perennial and evergreen shrubs, mainly in Argentina. Folk remedies in South America are known to be used mainly for indigestion, body odor control, and menstrual control, but their efficacy has not been scientifically identified. Laria nittida used in the experiment in the present invention was sold from the overseas biological material hub center.

In the present invention, the process of confirming the effectiveness of Laria nitida and separating and identifying the components of the above (I) to (IV) from Laria nitida, which is effective for the prevention and treatment of female cancer and menopausal symptoms, is as follows.

1.Laboratory Pharmacological Validation of Laria nittida

Proliferation of Estrogen Receptor-Dependent MCF-7 Breast Cancer Cells against Laria Nitida Extract, Competitive Inhibition of Laria Nitida Extract Against ER Binding, Analysis of MCF-7 Intracellular Transcription with MCF-7 Estrogen Responsive Gene, Proliferation Effect And acute toxicity test.

2. Isolation and Pharmacological Activity of Active Ingredients of Laria nitida

The pharmacological activity of the isolated fraction and the active ingredient of Laria nitida extract was tested to isolate and confirm 11 or more single components, and among these, 5 pharmacological activities were confirmed to be excellent. Four of these are compounds represented by the above formulas (I) to (IV) to confirm female hormone pharmacological activity for the first time in the present invention. The remaining one compound of formula (V) was NDGA, 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 effective at least one selected from the group consisting of the compounds represented by the formula (I) to (IV) and pharmaceutically acceptable salts thereof Contains as an 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.

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

The food composition comprises 0.0001 to 20% by weight of the compound represented by the formula (I) to (IV) in the total weight. The food especially includes a dietary supplement. "Health functional food" as defined in the present invention means a food prepared and processed using raw materials or ingredients having a useful functionality to the human body, "functional" is to control nutrients or physiology for the structure and function of the human body Ingestion is intended for the purpose of obtaining a beneficial effect on health uses such as a pharmaceutical action. The health functional food may have any one form of tablets, capsules, powders, granules, liquids, and pills.

In addition, the food composition of the present invention may be a 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.

The food composition of the present invention is an essential ingredient, except for containing any one of the components (I) to (IV), there are no special restrictions, and various flavors or natural carbohydrates, such as ordinary food or drink It may further contain various components. 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 food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the 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 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.Hormone Receptor Binding Experiment: Estrogen Receptor (ER) / Progesterone Receptor (PR)

Recombinant human ER (hER) and PR were purchased from Invitrogen. 750 mol of each receptor protein was diluted with binding buffer and used as 10 nM. The composition of the binding buffer is 10 mM Tris / pH 7.5, 10% glycerol, 1 mM DTT and 1 mg / ml BSA. ^{ER 750 mol, [2,4,6,7- 3 H} ] into the estradiol (10nM), the test substance in a concentration (dissolved in DMSO) in microcentriguge tube was such that the final 100 ml reaction capacity. Unreacted after cultured at 25 ° C 3 sigan free [2,4,6,7- ³ H] estradiol is charcoal solution (0.5% charcoal, 10mM Tris / pH 8.0, 1mg / ml) was added and allowed to react 15 minutes After centrifugation for 5 minutes at 10,000 rpm was removed. 3 ml of Ultima Gold scintillation cocktail was added to the supernatant, and the radioactivity remaining in the sample was measured using a liquid scintillation counter. 10 nM 17β-ES was used to measure nonspecific binding. 10 nM 17β-ES was used as a comparative control. In each experiment, several concentration values of the same test substance were tested together to obtain a curve for the concentration-radioactivity count and to calculate the IC ₅₀ value by interfering with the receptor binding capacity of radioactive 17β-ES. IC ₅₀ values were calculated using Prism 3.0 Software. Adhesion of progesterone was also proceeded in a similar manner as above but using PR instead of ER.

2. Secondary Screening: MCF-7 Cell Proliferation Test (E-SCREEN Assay)

The assay, developed by Soto et al., Uses the principle that ER-positive breast adenocarcinoma cells (MCF-7: BUS) overproliferate by enabling test substances to activate ER signaling. Normally, the relationship between the estrogen of the test substance and the cell proliferation of MCF-7: BUS is used, and the cell proliferation of 17β-ES, which is the strongest ER ligand and has the largest cell proliferation effect, is relatively similar to that of the test substance. By comparison, the estrogen properties of the test substance can be evaluated. In the present invention, the method introduced by Soto was modified and used. The cell culture medium used in this test was phenol-red free media (CD-DMEM) containing charcoal treated serum to thoroughly exclude external estrogen effects. MCF-7 cells were seeded to 3 × 10 ⁴ cells / well in a 12-well plate. After 24 hours, the medium was removed and replaced with phenol red free-DMEM medium (CD-DMEM) containing 5% charcoal dextran treated FBS containing a certain concentration of test substance. CD-DMEM medium containing the test substance was prepared by diluting a concentration of test solution stock solution dissolved in DMSO 500-1000 times in the medium immediately before the experiment. The control group was added only DMSO (0.5-0.1%) CD-DMEM, the positive control was treated with 100 pM 17β-ES. The final concentration of solvent in all experimental systems was 0.5% (v / v) or less. MTT assay was performed after 18-36 hours in 37 ℃, 5% CO ₂ incubator to measure the number of living cells. MTT [3- (4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide, Sigma] was treated to 1 mg / ml in culture and cultured for 4 hours, then the medium was removed. While lysis was performed, the purple MTT metabolite (crystalline) produced by living cells was dissolved. The amount of MTT metabolite dissolved in DMSO was measured at 540 nm by ELISA Plate Reader (UVmax, USA). Since absorbance is known to be proportional to the number of living cells, it is possible to quantitatively evaluate the degree of cell proliferation by the test substance.

3. Gene Reporter Evaluation Test: Estrogen (ERE) or Progesterone (PRE) Reactive Gene Transcription

MCF-7 cells known to have high ER content for ERE-luciferase assay and human breast cancer T47D cell line with intrinsically high PR expression were used for PRE-luciferase assay. Both cell lines were purchased from the American Tissue Culture Collection (USA). Twenty four hours before seeding, the cells were incubated in CD-DMEM medium, 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 reporter (luciferase) gene including ERE or PRE-luciferase plasmid progesterone responsive elements (PRE) was transfected with Lipofectamine 2000 reagent (Invitrogen, USA). In the case of the ERE-luciferase assay, 17β-ES (1 nM) was treated as a positive control group, and ICI-182,780 (1 mM) as an antagonist control group, and various other concentrations of test substance (stock solution dissolved in DMSO were diluted in the medium. After 24 hours of incubation, cell culture was terminated and water-soluble cell extracts were obtained using Passive Lysis Buffer (Promega, USA.) The activity of luciferase present in the Luciferase Assay System (Promega; substrate of luciferase) Quantitative measurement using a Luminometer and a pre-luciferase assay, including progesterone (PR agonist; 10 nM) and mifepristone (PR antagonist; 1 mM) as positive and inhibitor control treatment groups, respectively. The gene activity represented by the test substance was defined as 100% of the activity of the positive control (17β-ES or progesterone), indicating the relative activity, and the final comparison. It was added.

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

MCF-7 was incubated in phenol red-free (CD-DMEM) for 2 days to measure estrogen-responsive genes. When cells were 90% confluency, 17β-estradiol (10 nM), ICI (ER antagonist, 1 mM), constant Concentration test material was treated with the cell culture solution. After 24 hours of drug treatment, cells were trypsinized and separated from the culture vessel surface, and mRNA was extracted from the cell masses using a Qiagen RNeasy mini kit. cDNA was obtained by reverse transcription of a predetermined amount of mRNA (5 mg) using an iScript cDNA synthesis kit (Bio-Rad). The expression level of the gene was quantitatively measured by real-time PCR using cDNA, a pair of primers capable of recognizing the cDNA of the target gene, and a 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.

Expression levels of Transforming growth factor (TGF) -a and Trefoil factor 2 (or pS2) were measured as target genes for estrogen. Primers that recognize TGF-a for real-time RT PCR reactions are forward 5'-GTTTTTGGTGCAGGAGGACAA; And reverse 5'-CACAGCGTGCACCAACGT. Primers that recognize pS2 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. The activity level of the PR target gene by the test material was similar to that of the ER target gene expression, but the expression levels of alkaline phosphatase and fatty acid synthase were detected as target genes of PR. In addition, T47D, an ER / PR-positive human breast adenocarcinoma cell line, was used for this test.

5. In vivo ( in vivo ) Female hormone evaluation: Uterotrophic assay

Uterotropic assay is an indirect assessment of estrogenicity 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 immature ovary when intraperitoneally administered to female rats at 21 days of age. Immature female rats were used for the experiment. During the period of acclimatization, 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. The positive control was used to homogeneously suspend 17β-ES in corn oil to 30 μg / 3 ml and then dilute it with corn oil step by step. 17β-ES was intraperitoneally injected (i.p.) for three days at three doses of 0.3, 3 and 30 μg / kg. The dosage was 0.03 ml per 10 g body weight of immature let and solution preparation of the material 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 completely drying the water on the room, the weight of the uterus obtained from each rat was accurately measured using Mettler microbalance.

6. 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: Several fractions were obtained by performing Si gel column chromatography on MeOH extract of each plant.

(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 of active fraction: The active fraction is obtained by obtaining several fractions through Si gel column chromatography, MPLC, Sephadex LH-20, HPLC, LC-MS analysis. Fractions containing plant metabolites were subdivided and purified.

(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, COSY, HSQC, HMBC, NOESY, ROESY), CD, specific rotation, UV and other structural crystallographic methods were used. Existing medicinal plant DB was used to confirm whether the final substance was new or known.

result

1. Female hormonal pharmacological efficacy of Laria nittida

end. Cell proliferation effect

We tested how Laria nitida extract increased the proliferation of estrogen receptor dependent MCF-7 breast cancer cells, and the results are shown in FIG. MCF-7 cell proliferation was increased 4.3 times when only 17β-ES was treated as a positive control, and 1.2 times higher than vehicle group treated with 0.1% ethanol only after treatment with 10 ^-5 g / mL Laria nitida. It became. But Laria Nitti (10 ^-7 -10 ^-5 g / ml concentration) and 17β-ES in combination treatment inhibited cell proliferation (1.3, 1.5 and 2.7-fold inhibition, compared to 17β-ES at each concentration). Treatment with CI 182,780, an ER antagonist, confirmed that MCF-7 cell proliferation in this experiment was ER-mediated through the complete blockade of Laria nitida cell proliferation.

I. Competitive Inhibition of Laria nitida Extract on ER Binding

In vitro system Competitive binding of laria nitida extract and [ ³ H] 17β-ES to pure recombinant human estrogen receptor (hER) was measured. The results are shown in FIG. 2 and Table 1. In competitive binding assays, the IC ₅₀ of 17β-ES (•) was 1.16 × 10 ⁻⁹ g / mL (hERα) and 2.49 × 10 ⁻⁹ g / mL (hERβ), respectively. Laria nittida, a kind of phytoestrogen IC ₅₀ of (◆) was 1.23 × 10 ⁻⁷ g / mL (hERα) and 9.49 × 10 ⁻⁸ g / mL (hERβ). From these results, it was confirmed that Laria nitida extract acts concentration-dependently on [ ³ H] 17β-ES separation binding. RBA compared with endogenous 17β-ES was about 100 times lower (0.943% for hERα, 2.641% for hERβ) was shown (Fig. 2).

Table 1

RBA = [Ki _(17β-ES) / Ki _{( Larreanitida )} ] × 100.

All. Analysis of MCF-7 Intracellular Transcription Through Laria Nitida's MCF-7 Estrogen Response Gene

In the MCF-7 cells transfected with Estrogen responsive element (ERE), it was tested whether Laria nitida alters the activity of ERE-gene. When treated with laria nitida alone, the ERE gene activity was doubled compared to vehicle at a concentration of 10 ^-6 g / ml. (10 ^-8 -10 ^-6 g / mL) when combined with 17β-ES showed high gene activity similar to 17β-ES alone treatment, so that rianitida did not affect the ERE gene activity of 17β-ES And it was found. In this experiment using MCF-7 cells, ERE gene activity by 17β-ES was confirmed to be blocked when co-administered with ICI, an ER antagonist. The results showed that there was no interaction between Laria nitida extract and 17β-ES during gene transcription.

la. Proliferative effect of Laria nittida

In order to evaluate the female hormone resistance of the extract in the system excluding endogenous estrogen, uterine proliferation was tested in 21-day-old female rats with very low estrogen secretion. Uterine growth results were compared and evaluated as shown in Table 2 below. Uterine weight ratio (weight to body weight) was 4.44 when corn oil was administered to the vehicle group, and 17β-ES (3 ㎍ / ㎏) sc as a positive control resulted in a uterine weight ratio of 8.18 compared to the control group. The ratio increased 1.8 times. The uterine weight ratio was 1.64 when laria nitida extract was administered to sc immature rats at 100 and 500 mg / kg, which is a 0.36-fold reduction in uterine weight compared to the control group administered only corn oil. there was. In this study, it was confirmed that Laria nitida extract inhibits the growth of uterine tissues. In addition, weight gain in rats was observed during drug administration, and Laria nitida increased uterine atrophy and appetite through PR. It was predicted that.

TABLE 2

Significantly different values from vehicle group are indicated with *** (*** P <0.001).

hemp. Acute Toxicity Test

The concentration-dependent acute toxicity of Laria nitida extract was tested. Laria Nittida The acute toxicity of the extracts was evaluated by sc injection at 50, 100, 250, 500 mg / kg for 3 days. 50, 100, 250 mg / kg and the maximum dose of 500 mg / kg were administered continuously for 3 days daily and observed for 4 days. At the maximum dose of 500 mg / kg, no physiological abnormalities were observed and the death rate was 0% because no animals died. So Laria Nittida LD ₅₀ for acute toxicity of was evaluated as a high safety plant extract as more than 500 mg / kg, these results are shown in Table 3.

TABLE 3

2. Bioassay-guided analysis of Laria nitida

end. Fraction and Purification of Extracts

In order to follow up the pharmacologically active ingredients of Laria nitida extract, methanol extract (12 g) was coated on RP resin (cosmosil 75 C18, 36g) and top loaded to obtain the final 16 fractions (LC1 to LC16). ) As a result of evaluating the binding strength of ER and PR of these fractions, fractions LC8, 9, 10, 11, 12 showed significant activity. Bioassay-guided analysis of the subfractions allowed us to trace single components with good physiological activity. Concentration-dependent ER binding of subfractions (Nos. 1-16) showed excellent ER binding strengths of fractions 8, 9, 10, 11 and 12. The results are shown in FIGS. 4 and 5 and Table 4. Therefore, the binding force to PR, another female hormone receptor, was examined. As a result, the subfractions showed excellent PR binding force as shown below, and the results are shown in FIGS. 6 and 5. Therefore, the chemical composition was identified by focusing on subfraction 8-12.

Table 4

Table 5

LC8 divided methanol and water into 14 small fractions by RP-MPLC in a mobile phase (MeOH: H ₂ O, 25:75 to 100: 0). The small fraction LC8-7 obtained was HPLC (Phenomenex Luna C18, ACN 40%) was carried out to separate compound 1 (1.8 mg, t _R = 22.09 min) and compound I (8.6 mg, t _{R =} 31.25 min), and then to compound V (small fraction LC8-9 from small fraction LC8-9 under the same column and solvent conditions). 6.8 mg, t _R = 38.04 min) was purified.

LC10 is the _{compound 4 (2.3 mg, t R} = 44.13 min) and _{compound Ⅱ (1.5mg, t R =} 60.40 min) was isolated using HPLC (Phenomenex Luna C18, ACN 40 %).

LC11 divided methanol and water into 16 small fractions by RP-MPLC using mobile phase (MeOH: H ₂ O, 40:60 to 100: 0), of which LC11-6 was HPLC (Phenomenex Luna C ₁₈ , ACN 45). %) Was purified to give compound III (7.7 mg, t _R = 42.06 min), and from compound LC11-10 with compound IV (5.4 mg, t _R = 50.92 min) and compound 8 (14.5 mg, t _R = 84,73 min). LC11-12 was also subjected to HPLC (Phenomenex Luna C18, gradient ACN 45%, 55%, 80%) compound 9 (1.4 mg, t _R = 45.50 min), compound 10 (1.7 mg, t _R = 54.15 min) , compound 11 1.6 mg, t _R = 55.98 min) and compound 12 (5.1 mg, t _R = 78.00 min) were separated.

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

In order to investigate chemical structure of separated single materials, structural analysis was performed by spectroscopic methods such as NMR and HRESIMS, and the concentration-dependent binding strengths of ER and PR were evaluated. Finally, five single components (compounds I to V) showing effective pharmacological activity were isolated and identified. The five single components are shown in Table 6. Laria Nittida This invention is the first time that compounds I-IV have been discovered from plants as a single component exhibiting female hormone properties.

Table 6

(1) Compound (I)

3 ′, 6-Di-O-demethylisoguaiacin

C ₁₈ H ₂₀ O ₄ (mw 300.35)

HRESIMS: m / z 301.1444 [M + H] ⁺ (calcd: 301.1440)

¹ H NMR (400 MHz, CD ₃ OD): δ 6.41 (1H, d, J = 2.0 Hz, H-2), 6.64 (1H, d, J = 8.0 Hz, H-5), 6.37 (1H, dd , J = 8.0, 2.0 Hz, H-6), 3.46 (1H, d, J = 6.5 Hz, H-7), 1.87 (1H, m, H-8), 0.86 (3H, d, J = 6.9 Hz , H-9), 6.23 (1H, s, H-3 '), 6.48 (1H, s, H-6'), 2.79 (1H, dd, J = 16.2, 5.4 Hz, H-7'a), 2.37 (1H, doublet of doublets, J = 16.2, 7.2 Hz, H-7′b), 1.99 (1H, m, H-8 ′), 0.87 (3H, d, J = 6.9 Hz, H-9 ′).

¹³ C NMR (100 MHz, CD ₃ OD): δ 140.8 (C-1), 117.4 (C-2), 145.2 (C-3), 144.3 (C-4), 115.9 (C-5), 121.7 ( C-6), 51.6 (C-7), 42.4 (C-8), 16.3 (C-9), 128.7 (C-1 '), 131.0 (C-2'), 118.3 (C-3 '), 144.6 (C-4 '), 144.4 (C-5'), 116.1 (C-6 '), 36.2 (C-7'), 30.8 (C-8 '), 16.3 (C-9').

(2) Compound (II)

Norisoguaiacin

C ₁₉ H ₂₂ O ₄ (mw 314.38)

HRESIMS: m / z 315.1589 [M + H] ⁺ (calcd: 315.1596)

¹ H NMR (400 MHz, CD ₃ OD): δ 6.57 (1H, d, J = 1.9 Hz, H-2), 6.67 (1H, d, J = 8.1 Hz, H-5), 6.45 (1H, dd , J = 8.1, 1.9 Hz, H-6), 3.52 (1H, d, J = 6.8 Hz, H-7), 1.91 (1H, m, H-8), 0.87 (3H, d, J = 6.9 Hz , H-9), 6.22 (1H, s, H-3 '), 6.49 (1H, s, H-6'), 2.82 (1H, dd, J = 16.4, 5.4 Hz, H-7'a), 2.38 (1H, dd, J = 16.4, 7.3 Hz, H-7'b), 1.99 (1H, m, H-8 '), 0.87 (3H, d, J = 6.9 Hz, H-9'), 3.74 (3H, s, OMe).

¹³ C NMR (100 MHz, CD ₃ OD): δ 140.6 (C-1), 115.8 (C-2), 148.8 (C-3), 145.7 (C-4), 113.8 (C-5), 123.0 ( C-6), 56.5 (OMe), 51.8 (C-7), 42.4 (C-8), 16.3 (C-9), 128.7 (C-1 '), 130.9 (C-2'), 118.2 (C -3 '), 144.7 (C-4'), 144.4 (C-5 '), 116.2 (C-6'), 36.3 (C-7 '), 31.1 (C-8'), 16.3 (C-9 ').

(3) Compound (III)

4- (4- (4-hydroxyphenyl) -2,3-dimethylbutyl) benzene-1,2-diol

C ₁₈ H ₂₂ O ₃ (mw 286.37)

HRESIMS: m / z 287.1650 [M + H] ⁺ (calcd: 287.1647)

¹ H NMR (400 MHz, CD ₃ OD): δ 6.59 (1H, d, J = 1.9 Hz, H-2), 6.66 (1H, d, J = 7.9 Hz, H-5), 6.45 (1H, dd , J = 8.0, 1.9 Hz, H-6), 2.67 (2H, overlapped, H-7a, 7'a), 2.20 (2H, overlapped, H-7b, 7'b), 1.70 (1H, m, H -8), 0.81 (3H, d, J = 7.1 Hz, H-9), 6.93 (2H, d, J = 8.4 Hz, H-2 ', 6'), 6.68 (2H, d, J = 8.5 Hz , H-3 ', 5'), 1.70 (1H, m, H-8 '), 0.80 (3H, d, J = 7.0 Hz, H-9').

¹³ C NMR (100 MHz, CD ₃ OD): δ 134.9 (C-1), 117.3 (C-2), 146.1 (C-3), 144.2 (C-4), 116.3 (C-5), 121.6 ( C-6), 39.8 (C-7), 40.8 (C-8), 16.6 (C-9), 134.1 (C-1 '), 131.1 (C-2', 6 '), 116.1 (C-3 ', 5'), 156.4 (C-4 '), 39.4 (C-7'), 40.7 (C-8 '), 16.8 (C-9').

(4) Compound (IV)

4- (4- (3-hydroxy-4-methoxyphenyl) -2,3-dimethylbutyl) ben zene-1,2-diol

C ₁₉ H ₂₄ O ₄ (mw 316.39)

HRESIMS: m / z 317.1749 [M + H] ⁺ (calcd: 317.1753)

¹ H NMR (400 MHz, CD ₃ OD): δ 6.61 (1H, d, J = 2.0 Hz, H-2), 6.80 (1H, d, J = 8.2 Hz, H-5), 6.61 (1H, overlapped , H-6), 3.81 (3H, s, OMe), 2.66 (2H, overlapped, H-7a, 7'a), 2.18 (2H, overlapped, H-7b, 7'b), 1.70 (1H, m , H-8,8 '), 0.81 (3H, d, J = 6.7 Hz, H-9,9'), 6.58 (1H, overlapped, H-2 '), 6.66 (1H, d, J = 8.0 Hz , H-3 ').

¹³ C NMR (100 MHz, CD ₃ OD): δ 136.3 (C-1), 117.2 (C-2), 147.4 (C-3), 147.2 (C-4), 112.9 (C-5), 121.6 ( C-6), 39.7 (C-7,7 '), 40.6 (C-8,8'), 16.7 (C-9,9 '), 134.9 (C-1'), 117.4 (C-2 ') , 146.1 (C-3), 144.3 (C-4), 116.3 (C-5 '), 121.4 (C-4'), 56.7 (OMe).

(5) Compound (Ⅴ)

Nordihydroguaiaretic acid

C ₁₈ H ₂₂ O ₄ (mw 302.36)

¹ H NMR (400 MHz, CD ₃ OD): δ 6.58 (1H, d, J = 2.0 Hz, H-2,2 '), 6.66 (1H, d, J = 8.0 Hz, H-5,5') , 6.45 (1H, dd, J = 8.0, 2.0 Hz, H-6,6 '), 2.65 (1H, dd, J = 13.4, 5.0 Hz, H-7a, 7'a), 2.37 (1H, dd, J = 13.4, 9.2 Hz, H-7b, 7'b), 1.69 (1H, m, H-8, 8 '), 0.80 (3H, d, J = 6.7 Hz, H-9,9'),

¹³ C NMR (100 MHz, CD ₃ OD): δ 135.0 (C-1,1 '), 117.3 (C-2,2'), 146.1 (C-3,3 '), 144.2 (C-4,4 '), 116.2 (C-5,5'), 121.6 (C-6,6 '), 39.8 (C-7,7'), 40.1 (C-8,8 '), 16.8 (C-9,9 ').

EXAMPLE

Formulation Example 1 Preparation of Powder

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 Tablet

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 Preparation 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 Injection

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 Preparation of Liquid

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 and adjusted to 100 ml. do.

Female hormone substances of the present invention 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 (10)

1. A pharmaceutical for the prevention and treatment of female cancers and menopausal symptoms, containing any one or more selected from the group consisting of compounds represented by the following formulas (I) to (IV) and pharmaceutically acceptable salts thereof as an active ingredient Composition.

   (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, osteoporosis, venous thrombosis, and atrophic vaginitis.
5. Food composition for the prevention and improvement of female cancer and menopausal symptoms containing any one or more selected from the group consisting of a compound represented by the formula (I) to (IV) and a food acceptable salt thereof as an active ingredient .

   (Ⅰ)

   

   (Ⅱ)

   

   (Ⅲ)

   

   (Ⅳ)

   
6. The 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 food composition of claim 5, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer.
8. The food composition of claim 5, wherein the menopausal symptoms include any one of hot flashes, hyperlipidemia, osteoporosis, venous thrombosis, and atrophic vaginitis.
9. The food composition of claim 5, wherein the food is a dietary supplement having any one of tablets, capsules, powders, granules, liquids, and pills.
10. The food composition of claim 5, wherein the food is in the form of a beverage, a powdered beverage, a solid, a chewing gum, a tea, a vitamin complex, or a food additive.

Images