KR101580716B1 - Pharmaceutical compositions for prevention and treatment of cancer and menopausal symptom of women containing isolated compound from Larrea nitida or the pharmaceutically acceptable salts as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms comprising, as an active ingredient, single components isolated from Larianitida extract or a pharmaceutically acceptable salt thereof. In the present invention, there are provided single effective components separated and identified from Larianitida extract, a pharmaceutical composition containing the same, and a health functional food composition. The present invention is the first to confirm that the single components derived from larienitidia exhibit female hormone resistance. The female hormone substances provided in the present invention are used for the treatment and prevention of female cancer and menopausal symptoms, Can be used in the field of functional foods.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for prevention and treatment of female cancer and menopausal symptom, which comprises, as an active ingredient, a compound isolated from Larianitida extract or a pharmaceutically acceptable salt thereof. women containing isolated compound from Larrea nitida or the pharmaceutically acceptable salts as an active ingredient}

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

Cancer of women such as endometrial cancer and breast cancer and diseases such as menopausal syndrome are associated with female hormones such as estrogen excess and decrease, estrogen and progesterone imbalance.

Endometrial cancer

In Korean women, endometrial cancer and ovarian cancer have increased about 5 times since 1991, and the incidence of such female hormone cancer is expected to continue to increase in the future. The risk factors for abnormal proliferation of the endometrium are thought to be caused by prolonged exposure to estrogen without sufficient antagonism of progesterone, overweight, and westernized life. The primary treatment for endometrial cancer is surgery, and female hormone therapy is effective in patients who are all positive for estrogen or progesterone receptors after surgery and chemotherapy. Excessive estrogen exposure, unbalanced with progesterone as a major cause of endometrial cancer, is known to increase endometrial cancer more than 8-fold, and megestrol (progesterone receptor agonist) megestrol Widely used. However, side effects of megastrol are hypertension, thrombophlebitis, and weight gain. Therefore, if a substance capable of balancing progesterone and estrogen efficacy from natural products is discovered, it will be a therapeutic agent for endometrial cancer with reduced side effects and excellent efficacy.

Breast cancer

As with endometrial cancer, estrogen-overexpressing breast cancer has also increased in Korean women over the past decade and is currently the highest cancer in women. As a cause of increased breast cancer incidence, high fat, high calorie westernized diet and obesity, late marriage and low birth rate, avoidance of lactation, estrogen overexposure is pointed out as the main cause. In the western region, the incidence of breast cancer is more than three times higher than that of Korean women, and the demand for breast cancer treatment is very high worldwide. Breast cancer therapy is first performed, followed by radiation therapy, chemotherapy, and hormone therapy to prevent recurrence. Although hormone therapy is effective in lowering the recurrence rate, it is a long-term treatment over a period of years, which is followed by resistance. In the case of tamoxifen, the first-line treatment, many patients with metastatic cancer have been shown to have a major problem with endometrial cancer induction and cellular resistance after long-term administration. Anastazole, Letrazole, and Exemestane have been used as inhibitors of aromatase, a second-line antihormonal drug that has improved the side effects and recurrence rate. However, its use is limited to women after menopause. An important aspect in the development of therapeutic agents for breast cancer is to target molecules related to cancer in order to reduce toxicity, and to minimize the occurrence of drug resistance to prevent long-term use and recurrence. As ER / PR positive patients account for two-thirds of all breast cancer patients, ER / PR is the best molecular target. The development of selective estrogen receptor modulators (SERMs) and selective progesterone receptor modulators (SPRMs), which minimize ER and PR expression while minimizing side effects and drug resistance, It is a core goal of the development of therapeutic drugs.

Menopausal syndrome

The rapid decrease in postmenopausal estrogen causes symptoms such as facial flushing, osteoporosis worsening, hyperlipidemia, mental instability and sudden changes in emotions, and vaginal dryness. For postmenopausal symptoms, administration of estrogen alone or an estrogen / progesterone combination is the only treatment that 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 with ER, such as breast, uterus, and ovary. According to the Women's Health Initiative (WHI) study, Premarin and Prempro, one of the most commonly prescribed HRT formulations in North America, are associated with increased breast cancer and cardiovascular disease outbreaks. In addition, various small-scale clinical results suggest that the use of HRT, including increased uterine proliferation in postmenopausal women taking HRT, is closely related to hyperplasia and carcinogenesis.

As a carcinogenic mechanism related to HRT, excessive cell proliferation and accumulation of DNA mutation due to E2 / ER interaction are proposed. Genotoxicity caused by reactive metabolites occurring in the in vivo drug metabolism of estrogen is also suggested as its carcinogenic mechanism . This pharmacological effect of tissue-specific estrogens has been shown to be due to the variety of molecular mechanisms and associated transcription factors that interact with estrogen and ER in specific tissues / cells (Jordan, 2007). For example, tamoxifen, which is used as a treatment for breast cancer, 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 for postmenopausal cancer or a cancer treatment for ER involved in the carcinogenic mechanism of ER, such as breast cancer or endometrial cancer, may be used to select a substance capable of having a tissue-specific differentiation effect on ER, namely, a selective female hormone receptor modulator (SERMs) Development is the ultimate goal of therapeutic development.

On the other hand, pomegranate extract, soybean extract, isoflavone preparation, and evening primrose oil have been used as health foods for improvement of women's menopausal symptoms and prevention / treatment of osteoporosis. These products made from vegetable raw materials are rapidly expanding in recognition of their safety to consumers. However, the recognition that it is safe from being vegetable is not scientifically grounded, and these products are made up of complex extracts made up of various kinds of chemical components. In the case of health supplements, there are many cases where they are not subjected to strict toxicity tests. none. The side effects of these products can not be monitored by the government. In addition, it is difficult to judge whether its efficacy has a lack of scientific basis for pharmacological efficacy and strict and precise quality control regulations. Therefore, there is a demand for safer and more effective substances derived from natural materials, which can overcome the disadvantages of existing plant extracts.

Pharmacological actions such as anti-inflammation, anti-bacterial and antiviral effects of L. tridentata or L. divaricata shrub have been reported in Larrea plants (Heinrich M. Curr Topics Med Chem 2003; 3: 141-154; Gagnier JJ et al. Am J Med 2006; 119: 1-11). In addition, there have been reports of inhibiting the tumor cell colonization formation and inhibiting the cancer cell proliferation of Larix spp. (Garreau B et al. Biochim Biophys Acta 1991; 1094: 339-45; Anesini C et al. Phytomedicine 2001; 8: -7). The Larix plants studied so far contain a large amount of lignan components and also contain cinnamic acid and its derivatives. A representative component of the known Larix sp. Is the lignan nordihydroguaiaretic acid (NDGA). NDGA is a powerful antioxidant and various physiological activities are being studied. It has been reported that it inhibits tumor cell proliferation through accumulation of PKC (protein kinase C) and cAMP (cyclic adenosine monophosphate) accumulation 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, anticancer effects of receptor tyrosine kinase (RTK) and insulin-like growth factor receptor (IGF-1R) inhibitors have been reported (Youngren JF et al., Breast Cancer Res Treat 2005; 94: 37-46). NDGA has also been shown to have estrogen receptor binding and transcriptional activity in estrogenic activity evaluation (Fujimoto N et al. Life Sci 2004; 74: 1417-25). However, NDGA contents and related physiological activities are only confirmed in some plants of Laria genus. Various physiological activities and related active substances in various plants of Laria genus have not been disclosed yet.

Korean Patent Publication No. 10-0419121 Korean Patent Publication No. 10-0542479 Korean Patent Publication No. 10-2004-0101694 Korean Patent Publication No. 10-0557006 Korean Patent Publication No. 10-1141194 Korean Patent Publication No. 10-1177508

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. 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. Fujimoto, N., Kohta, R., and Kitamura, S. (2004). Estrogenic activity of an antioxidant, nordihydroguaiaretic acid (NDGA). Life Sci 74, 1417-1425. 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 e 801-811. 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. 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. Jordan, V.C. (2007). Chemoprevention of breast cancer with selective oestrogen-receptor modulators. Nat Rev Cancer 7, 46-53. E. Pavano, M., Fones, E., Oksenberg, D., Garcia, M., Hernandez, C., Cordano, G., Munoz, S., Mancilla, J., Guerrero, A., and Ferreira, J. (1994). Inhibition of tumor cell respiration and growth by nordihydroguaiaretic acid. Biochem Pharmacol 48, 1935-1942. Youngren, J. F., Gable, K., Penaranda, C., Maddux, B. A., Zavodovskaya, M., Lobo, M., Campbell, M., Kerner, J., and Goldfine, I. D. (2005). IGF-1 and c-erbB2 / HER2 / neu receptors and suppresses growth of breast cancer cells. Breast Cancer Res Treat 94, 37-46.

DISCLOSURE OF THE INVENTION The present invention seeks to provide novel natural medicines and health functional foods for the prevention and treatment of female cancer and menopausal symptoms by isolating and identifying single components showing female hormonality from Laria nitida .

The development of therapeutic or postmenopausal treatments for cancers in which ER is implicated in the oncogenic mechanism of ER, such as breast cancer or endometrial cancer, can be used to select substances that have a tissue-specific differentiation effect on ER, namely selective female hormone receptor modulators (SERMs) Development is the ultimate goal. One of the important objects of the present invention is to provide a plant-derived material capable of improving side effects of conventional endometrial cancer treatment agents and balancing estrogen efficacy in the body.

In the present invention,

A pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms, comprising as an active ingredient at least one compound selected from the group consisting of compounds represented by the following formulas (I) to (IV) and pharmaceutically acceptable salts thereof: A composition is provided. The pharmaceutical composition of the present invention may further comprise a compound represented by the following formula (V) or a pharmaceutically acceptable salt thereof.

Further, in the present invention,

A food composition for prevention and improvement of female cancer and menopausal symptoms, comprising as an active ingredient at least one member selected from the group consisting of compounds represented by the following formulas (I) to (IV) and acceptable salts thereof / RTI > The food composition of the present invention may further comprise a compound represented by the following formula (V) or a pharmaceutically acceptable salt thereof.

(I)

(II)

(III)

(IV)

(V)

It has been found in the present invention that the single components of the above formulas (I) to (IV) derived from larienitida exhibit female hormone resistance.

The present invention relates to a process for the preparation of < RTI ID = nitida ) The present invention provides a new plant-derived single component pharmaceutical and health functional food useful for the prevention and treatment of female cancer and menopausal symptoms by isolating and identifying single components representing female hormone in plants. In particular, the present invention is the first to confirm that single components derived from larienitidia exhibit female hormone resistance. The pharmaceutical composition of the present invention containing these components occurs at the highest frequency among female cancers, and its incidence May be useful for the treatment of breast cancer and endometrial cancer, which have a similar pathogenesis.

Figure 1 shows the results of experiments on the estrogen receptor-dependent MCF-7 breast cancer cell proliferation effect of Larianitida extract. Significantly different values for the vehicle group were denoted by ***, and other values than the ES group were denoted by +++ (*** <0.001, +++ <0.001).
FIG. 2 is a cross- vitro system It is the result of measuring the competitive binding force of pure hER (recombinant human estrogen receptor) of extracts of Lauriernidae () and [ ³ H] 17β-ES (●).
Figure 3 shows the results of an experiment in which larienitidase changes the ERE- gene activity in MCF-7 cells transfected with an estrogen responsive element (ERE). , And the other values are shown as +++ (*** < 0.001, +++ < 0.001).
FIG. 4 shows the results of analysis of the concentration-dependent ER binding capacity of the three fractions (1-8) of the extract of Larianitida.
FIG. 5 shows the results of analysis of the concentration-dependent ER binding capacity of the three fractions (9-16) of the extract of Larianitida.
FIG. 6 shows the result of analyzing the concentration-dependent PR binding force of the fractions (8-12) of the extract of Larianitida.

In the present invention, the therapeutic efficacy, pharmacological metabolism and safety of female hormone efficacious individuals isolated from various plants including Larix genus are investigated to prevent female cancer such as endometrial cancer, breast cancer, and menopausal symptoms, A novel pharmaceutical composition or food composition effective for treatment.

The term &quot; female cancer &quot; in the present invention is meant to include all endometrial cancer, breast cancer, ovarian cancer, and other female genital organs in which female hormones such as estrogen and progesterone directly or indirectly participate in the carcinogenesis.

In the present invention, &quot; menopausal symptoms &quot; is meant to include both facial flushing, hyperlipidemia, osteoporosis, venous thrombosis, and atrophic vaginitis, which occur in menopausal women.

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

(I)

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

(II)

Norisoguaiacin

(III)

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

(IV)

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

In addition, NDGA (nordihydroguaiaretic acid), which is conventionally known as one of the representative components of the genus Larix, is also recognized as a compound (V) which is a single active ingredient having estrogen potency in the present invention. NDGA, a lignan component, is a powerful antioxidant and various physiological activities have been studied. In the past, the inhibitory effects of PKC (Protein kinase C) and cAMP (cyclic adenosine monophosphate) accumulation and calcium infusion inhibition on tumor cell proliferation inhibition (Erashi M et Receptor tyrosine kinase (RTK), insulin-like growth factor (IGF-1R), insulin (IGF-1R) (Youngren JF et al., Breast Cancer Res Treat 2005; 94: 37-46). In the evaluation of estrogenic activity, the binding force to the estrogen receptor and the transcriptional activity (Fujimoto N et al. Life Sci 2004; 74: 1417-25). In the present invention, NDGA was identified as a single component having estrogen potency, which was isolated and identified from Larianitida.

(V)

Nordihydroguaiaretic acid (NDGA) &lt; RTI ID = 0.0 &gt;

Larrea nitida ( Larrea nitida ) is a plant of southern gray legged and evergreen shrub that lives mainly in Argentina. In South America, it is known that folk remedies are mainly used for dyspepsia, body odor suppression, menstrual control, but the efficacy has not yet been scientifically proven. Larianitida used in the experiment in the present invention was distributed from the overseas biomaterial hub center.

In the present invention, the process of isolating and identifying the components (I) to (IV) effective for the prevention and treatment of female cancer and menopausal symptoms from lariatidia is as follows.

One. Laria Nitida Female hormone  Pharmacological efficacy experiment

Proliferation of estrogen receptor-dependent MCF-7 breast cancer cells, competitive inhibition of ER-binding to ER binding, transcriptional analysis of MCF-7 cells through MCF-7 estrogen response gene, uterine proliferation effect And acute toxicity tests were conducted.

2. Laria Nitida  Isolation of active ingredient and confirmation of pharmacological activity

The isolated fractions of Larianitida extract and the pharmacological activities of the active ingredients were examined to confirm that more than 11 kinds of single components were isolated and identified. Among them, five pharmacological activities were excellent. Four of these compounds are the compounds represented by the above formulas (I) to (IV), confirming the first female hormone pharmacological activity in the present invention. The remaining compound, the compound of formula (V), was NDGA in which binding and transcriptional activity to estrogen receptor was confirmed in previous studies.

The pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms of the present invention is characterized in that any one or more selected from the group consisting of the compounds represented by the above-mentioned formulas (I) to (IV) and the pharmaceutically acceptable salts thereof is effective As an ingredient. The pharmaceutical composition of the present invention may further comprise a compound represented by the above formula (V) or a pharmaceutically acceptable salt thereof.

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

The pharmaceutical composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . Examples of 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, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose Sucrose), lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Witepsol, macrogol, Tween 61, cacao paper, laurin, glycerogelatin and the like may be used as a base for suppositories.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, in order to obtain the desired effect, it is preferable that the compound is administered at a dose of 0.0001 to 1000 mg / kg per day based on the compound represented by the above formulas (I) to (IV). The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injection.

The definitions of the excipients, binders, disintegrants, lubricants, mating agents, flavoring agents, etc. of the present invention are described in documents known in the art and include the same or similar functions.

The food composition for preventing and ameliorating female cancer and menopausal symptoms according to the present invention may contain at least one selected from the group consisting of the compounds represented by the above-mentioned formulas (I) to (IV) . The food composition of the present invention may further comprise a compound represented by the above general formula (V) or a salt thereof acceptable as a food thereof.

The food composition contains 0.0001 to 20% by weight of the compound represented by the above formulas (I) to (IV) in the total weight. Such foods particularly include health functional foods. As used herein, the term " health functional food "means food prepared and processed using raw materials or ingredients having useful functions in the human body. The term" functional " And the like, for the purpose of obtaining a beneficial effect for health use such as exercise. The health functional food may be in the form of tablet, capsule, powder, granule, liquid, or ring.

In addition, the food composition of the present invention may be a food composition in which a functional ingredient is added to various foods or beverages. The food may be in the form of any one of, for example, a beverage, a powdered beverage, a solid, a chewing gum, a tea, a vitamin complex, or a food additive.

The food composition of the present invention is not particularly limited as long as it contains any one of the above-mentioned components (I) to (IV) as an essential ingredient and is not limited to any of various flavors or natural carbohydrates And 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 polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, A salt thereof, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like. In addition, the food composition of the present invention may include natural fruit juice and fruit juice beverage and flesh for the production of vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not critical, but is preferably selected from the range of 0 to about 20 weight percent 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 embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these embodiments.

[ Experimental Example ]

Experimental Method

1. Female hormone receptor binding test: Estrogen receptor ( ER ) / Progesterone receptor ( PR )

Recombinant human ER (hER) and PR were purchased from Invitrogen. 750mol of each receptor protein was diluted with binding buffer to 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 moles, [2,4,6,7- ³ H] estradiol (10 nM), and a certain concentration of test substance (dissolved in DMSO) were placed in a microcentriguge tube to a final reaction volume of 100 ml. After incubation at 25 ° C for 3 hours, unreacted free [2,4,6,7- ³ H] estradiol was added to charcoal solution (0.5% charcoal, 10 mM Tris / pH 8.0, 1 mg / And then centrifuged at 10,000 rpm for 5 minutes. To the supernatant, 3 ml of Ultima Gold scintillation cocktail was added and the radioactivity remaining in the sample was measured using a liquid scintillation counter. 10 nM 17? -ES was used to measure non-specific binding. As a comparative control drug, 10 nM 17? -ES was used. In each experiment, several concentrations were tested for the same test substance to obtain a curve for the concentration-radioactivity count and the IC ₅₀ value by the degree of interference with the receptor binding ability of radioactive 17β-ES. IC ₅₀ values were calculated using Prism 3.0 Software. Progesterone binding activity also proceeded in a manner similar to that described above except that PR was used instead of ER.

2. Secondary Screening: MCF -7 cell proliferation test (E- SCREEN Assay )

This test, developed by Soto et al., Uses the principle that the test substance activates ER signal transduction and thus over-proliferates ER-positive breast cancer cells (MCF-7: BUS). In general, the relationship between the estrogenicity of the test substance and the degree of cell proliferation of MCF-7: BUS is used, and the degree of cell proliferation of 17β-ES, which is the most potent ER ligand, The estrogenicity of the test substance can be evaluated by comparison. In the present invention, the method introduced by Soto was modified and used. The cell culture medium used in this study was phenol-red free media (CD-DMEM) containing charcoal treated serum to preclude extrinsic estrogenic effects. MCF-7 cells were seeded on a 12-well plate at 3 × 10 ⁴ cells / well. After 24 hours, the medium was removed and replaced with phenol red free-DMEM medium (CD-DMEM) supplemented with 5% charcoal dextran treated FBS containing test substance at a constant concentration. The CD-DMEM medium containing the test substance was prepared by diluting the stock solution of the test substance in DMSO at a concentration of 500 to 1000 times immediately before the experiment. In the control group, only DMSO (0.5-0.1%) CD-DMEM was added, and the positive control group was treated with 100 pM 17? -ES. The final concentration of solvent in all experimental systems was 0.5% (v / v) or less. MTT assay was performed for the measurement of viable cell counts at 37 ℃ and 5% CO ₂ incubator for 18 ~ 36 hours. After culturing for 4 hours, MTT [3- (4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide, Sigma] was added to the culture solution to a concentration of 1 mg / And lysed the purple MTT metabolite (crystalline) produced by living cells. The amount of MTT metabolite dissolved in DMSO was measured at 540 nm using an ELISA Plate Reader (UVmax, USA). Since the absorbance is known to be proportional to the number of living cells, the degree of cell proliferation by the test substance can be quantitatively evaluated.

3. Gene Reporter Assay: Estrogen ( ERE ) Or progesterone ( PRE ) Reactive gene transcription experiment

For the ERE-luciferase assay, MCF-7 cells, known to have a high ER content, were used for the PRE-luciferase assay. T47D cell line of human breast cancer cells, which has an intrinsically high expression level of PR, was used. Both cell lines were purchased from the American Tissue Culture Collection (USA). Twenty-four hours before seeding, cells were cultured in CD-DMEM medium, and cells that reached about 90% confluency were seeded in a 12-well plate at a concentration of about 5 × 10 ⁵ / well. All subsequent tests were performed in CD-DMEM medium. After incubation for 24 hours, the luciferase gene containing ERE or PRE-luciferase plasmid progesterone responsive elements (PRE) was transfected using Lipofectamine 2000 reagent (Invitrogen, USA). In the ERE-luciferase assay, 17β-ES (1 nM) was used as a positive control and ICI-182,780 (1 mM) as an antagonist control. Various concentrations of test substances (stock solution dissolved in DMSO Cell culture was terminated after 24 hours of incubation, and water-soluble cell extracts were obtained using Passive Lysis Buffer (Promega, USA). The luciferase activity was measured by Luciferase Assay System (Promega; luciferase substrate (PR agonist, 10 nM) and mifepristone (PR antagonist, 1 mM) were included as positive and inhibitor control groups, respectively, in the PRE-luciferase assay. The activity of the gene indicated by the test substance is determined by the activity of the positive control (17β-ES or progesterone) at 100% It was added.

4. Intracellular  Evaluation of effects on transcriptional activity of endogenous hormone reactive target genes

For the measurement of estrogen-responsive genes, MCF-7 was cultured in CD-DMEM (phenol red-free) for 2 days and 17β-estradiol (10 nM), ICI (ER antagonist, 1 mM) Concentration of the test substance was treated in the cell culture solution. After 24 hours of drug treatment according to the conventional method, the cells were trypsinized, separated from the culture vessel surface, and mRNA was extracted from the cell mass using Qiagen RNeasy mini kit. cDNA was obtained by reverse transcription of a certain amount of mRNA (5 mg) using iScript cDNA synthesis kit (Bio-Rad). Real-time PCR was performed using a pair of primers capable of recognizing the cDNA and cDNA of the target gene and a PCR SYBR green kit (Qiagen) reagent to quantitatively measure the expression level of the gene. In order to compensate for the technical mistakes of the various stages of the reaction, the expression level of the housekeeping gene, GAPDH, was measured at the same time, and the ratio of the amount of expression of the gene and the expression level of the target gene was used for the final quantitative analysis. The primers recognizing GAPDH are forward 5'-CTCTCTGCTCCTCCTGTTCGAC; And reverse 5'-TGAGCGATGTGGCTCGGCT.

Expression of transforming growth factor (TGF) -a and trefoil factor 2 (or pS2) as estrogen target genes was measured. For real-time RT PCR, primers that recognize TGF-a are forward 5'-GTTTTTGGTGCAGGAGGACAA; And reverse 5'-CACAGCGTGCACCAACGT. The primers recognizing pS2 were 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 as a logarithm, and the threshold cycle (C _T ) value is mathematically obtained. Value obtained by the value substituted 2-DDC _T represents the relative expression level of a particular gene. The value obtained by dividing the expression level of the specific gene by the expression level of the house keeping gene was selected as the final value for the expression level of the gene, and the relative value of the ER antagonist or agent treated value was compared with that of the test substance. The level of activity of the PR target gene by the test substance was similar to that of the expression level of the ER target gene, but the expression level of alkaline phosphatase and fatty acid synthase as a target gene of PR was searched. For this test, the ER / PR-positive human breast adenocarcinoma cell line T47D was used.

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

Uterotropic assay is a method of indirectly assessing estrogenicity by measuring estrogen-induced increase in uterine tissue mass. The effect of the test substance on the immature ovary was evaluated by comparing the effect of the test substance on the immature ovary with the control group for 3 days in the female rats on the 21th day after birth for 3 days. Immature female rats were used for the experiments. The subjects were selected as the average body weight during the refinement period. Animal identification was done by tail marking and tagging by box. In the positive control group, 17β-ES was suspended in corn oil to a concentration of 30 μg / 3 ml and then diluted with corn oil. 17β-ES was intraperitoneally injected (ip) for three days at three doses of 0.3, 3, and 30 μg / kg. The dose was 0.03 ml per 10 g of immature rat. The solution preparation of the material was carried out on the day of administration. The items to be tested in this test are weight and uterine weight. The body weight was measured immediately before administration and before the autopsy for all animals. The uterine weight was sacrificed by cervical dislocation 25 days after the last administration and the uterus was carefully removed to remove fat and fiber tissue The uterine weight of each rat was precisely measured using a Mettler microbalance.

6. Analysis of active ingredients on plant extract fractions

By separating the active fractions from crude extracts, active plant metabolites were identified and identified as the major single component of pharmacological effects. Specific experimental methods are as follows.

(1) Extraction and Separation: MeOH extracts from each plant were subjected to Si gel column chromatography to obtain several fractions.

(2) Bioassay-guided fractionation: The hormone receptor potency and antagonism in the cultured cells of each fraction was performed as a standard test, and fractions showing activity were selected.

(3) Separation and single component separation of active fractions Purification: Several active fractions were obtained by secreting several fractions through Si gel column chromatography, MPLC, Sephadex LH-20, HPLC and LC-MS Fractions containing plant metabolites were subdivided and purified.

(4) Analysis and identification of chemical structure of single components: Single components were separated on a chromatogram by analytical methods such as UPLC, LC-MS / MS, IR analysis and NMR analysis ( ¹ H, ¹³ C, DEPT, COZY, HSQC, HMBC, NOESY, ROESY), CD, specific rotation, and UV. Using existing DB of medicinal plants, it was confirmed whether the finally obtained substance was new substance or known substance.

result

One. Laria Nitida Female hormone  Pharmacological efficacy

end. Cell proliferation effect

The results are shown in Fig. 1. The results are shown in Fig. 1. The results are shown in Fig. As a positive control, MCF-7 cell proliferation was increased 4.3-fold when treated with 17β-ES alone, and larienitidase treatment with 10 ^-5 g / ㎖ resulted in a 1.2-fold increase over the vehicle treated with 0.1% . However, (At 10 ^-7 to 10 ^-5 g / ml concentration) and 17β-ES, the cell proliferation was inhibited (at each concentration, 1.3, 1.5 and 2.7 times inhibition was observed compared to 17β-ES). When the ER antagonist ICI 182,780 was treated together, the cell proliferation of larienitidia was completely blocked, confirming that the MCF-7 cell proliferation of this experiment was mediated by the ER mediator.

I. ER  For binding Laria Nitida  Competitive Inhibition of Extracts

In vitro system The competitive binding capacity of the extracts of laranitida and [ ³ H] 17β-ES against the 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 values} for 17β-ES (●) were 1.16 × 10 ^-9 g / ml (hERα) and 2.49 × 10 ^-9 g / ml (hERβ), respectively. A kind of phytoestrogen, (?) Had an IC ₅₀ of 1.23 × 10 ^-7 g / ml (hERα) and 9.49 × 10 ^-8 g / ml (hERβ). These results indicate that the laranitida extract acts in a concentration-dependent manner on the [ ³ H] 17β-ES cleavage bond, and the RBA compared to the endogenous 17β-ES is about 100 times lower (0.943% for hERα, 2.641% for hER [beta]) (Fig. 2).

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

All. Laria Nitida MCF -7 Through estrogen response genes MCF -7 Intracellular  Enterprise analysis

The effect of larienitidia on the activity of ERE- gene in MCF-7 cells transfected with estrogen responsive element (ERE) was tested and the results are shown in Fig. At the concentration of 10 ^-6 g / ml alone, the ERE gene activity was doubled compared to the vehicle when larienitida was treated alone, (10 ^-8 to 10 ^-6 g / ㎖) was treated with 17β-ES in the same manner as 17β-ES alone, larienitidase did not affect ERE gene activity of 17β-ES And it was found. In the present study using MCF-7 cells, it was confirmed that ERE gene activity by 17β-ES was blocked when co-administered with ICI, an ER antagonist. These results suggest that there is no interaction between larienitida extract and 17β-ES during gene transcription.

la. Laria Nitida  Uterine proliferation effect

In order to evaluate the female hormone of the extract in the system excluding the action of endogenous estrogen, the female uterus proliferation was examined by using female rat of 21 days of immature age with very low estrogen secretion. The uterus proliferation test results were compared and evaluated as shown in Table 2 below. The weight ratio of uterus was 4.44 as compared with the control group when corn oil was administered to the vehicle group. The ratio of uterine weight was 8.18 as a positive control group and 17β-ES (3 μg / kg) The ratio was increased by 1.8 times. When the laranitida extract was administered to immature rats at 100, 500 ㎎ / ㎏ sc, the uterine weight ratio was 1.64, which was 0.36 times lower than that of the control group treated with corn oil alone there was. The uterus proliferation test showed that the laranitida extract inhibited the uterine proliferation of the uterus. In addition, the weight gain of the rats was observed during the administration of the drug, Of the total population.

Values significantly different from the vehicle group were marked with *** (*** P <0.001).

hemp. Acute Toxicity Experiment

The concentration-dependent acute toxicity of the laranitida extract was tested. Larianitida The acute toxicity of the extract was evaluated by sc injection in mice at 50, 100, 250, 500 mg / kg for 3 days. 50, 100, 250 mg / kg and the maximum dose of 500 mg / kg were continuously administered for 3 days and observed for 4 days. The maximum dose of 500 ㎎ / ㎏ did not show any physiological abnormalities, and the death rate was 0% because there were no dead animals. Therefore, LD ₅₀ for acute toxicity was estimated to be more than 500 ㎎ / ㎏ as a safe plant extract. The results are shown in Table 3.

2. Laria Nitida  Physiologically active chemical components ( bioassay - guided 분석 ) analysis

end. Fractionation and separation of the extract

Pharmacological Activity of Larianitida Extracts The methanol extracts (12 g) were coated on RP resin (cosmosil 75 C18, 36 g) and top loaded in order to investigate the active ingredients. The final 16 fractions (LC1 to LC16 ). The fractions LC8, 9, 10, 11, and 12 showed significant activity when the binding force of ER and PR of these fractions was evaluated. Bioassay-guided analysis of the three fractions led to a single component with excellent physiological activity. Concentration-dependent ER binding analysis of the three fractions (1-16) showed that ER fractions of fractions 8, 9, 10, 11, and 12 were excellent. The results are shown in FIG. 4, FIG. 5, and Table 4. Therefore, we examined their binding ability to PR, which is another female hormone receptor. As a result, the following three fractions showed excellent PR binding force, and the results are shown in FIG. 6 and Table 5. Therefore, chemical components were identified with particular emphasis on three fractions 8-12.

LC8 was divided into 14 small fractions by performing RP-MPLC with methanol and water as mobile phase (MeOH: H ₂ O, 25:75 to 100: 0), and the small fraction LC8-7 obtained was analyzed by HPLC (Phenomenex Luna C18, The compound 1 (1.8 mg, t _R = 22.09 min) and the compound I (8.6 mg, t _{R =} 31.25 min) were separated by the same column and solvent conditions and the compound V It was purified to _{6.8 mg, t R = 38.04 min} ).

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 was divided into 16 small fractions by performing RP-MPLC using methanol and water as mobile phase (MeOH: H ₂ O, 40:60 to 100: 0). LC11-6 was analyzed by HPLC (Phenomenex Luna C ₁₈ , ACN 45 %) carried by the _{compound ⅲ (7.7 mg, t R} = 42.06 min) the tablets were the same column and the coming ⅳ from LC11-10 compound in solvent conditions _{(5.4 mg, t R = 50.92} min) and compound 8 (14.5 mg, t _R = 84,73 min). In addition, compound 11 (1.4 mg, t _R = 45.50 min) and compound 10 (1.7 mg, t _R = 54.15 min) were subjected to HPLC (Phenomenex Luna C18, gradient ACN 45%, 55% , compound 11 1.6 mg, t _R = 55.98 min) and compound 12 (5.1 mg, t _R = 78.00 min).

I. Identification of the chemical structure of a single substance and identification of concentration-dependent pharmacological activity of a single component

The structural analysis of the isolated single substances was carried out by spectroscopic methods such as NMR and HRESIMS for chemical structure identification and the concentration dependent binding force of ER and PR of these monomers was evaluated. Finally, five kinds of single components (Compounds I to V) showing effective pharmacological activity were isolated and identified. Table 5 shows the five single components. Larianitida The present invention is the first to find compounds I to IV as a single component showing female hormone resistance from plants.

(1) Compound (I)

3 ', 6-Di-O-demethylisoguaniacin

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-6), 2.79 (1H, dd, J = 16.2, 5.4 Hz, H-7'a), 6.23 (1H, s, 2.37 (1H, dd, 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 (100 MHz, CD ₃ OD):? 140.8 (C-1), 117.4 (C-2), 145.2 (C-3), 144.3 C-6), 51.6 (C-7), 42.4 (C-8), 16.3 (C-9), 128.7 144.6 (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-6 '), 2.82 (1H, dd, J = 16.4, 5.4 Hz, H-7'a), 6.22 (1H, s, 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 (100 MHz, CD ₃ OD):? 140.6 (C-1), 115.8 (C-2), 148.8 (C-3), 145.7 C-6), 56.5 (OMe), 51.8 (C-7), 42.4 (C-8), 16.3 (C-4 '), 144.4 (C-5'), 116.2 (C-6 '), 36.3 ').

(3) Compound (III)

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

C ₁₈ H ₂₂ O ₃ (m / z 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 (100 MHz, CD ₃ OD):? 134.9 (C-1), 117.3 (C-2), 146.1 (C-3), 144.2 C-6), 39.8 (C-7), 40.8 (C-8), 16.6 (C-9), 134.1 ', 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) benzene-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 Overlapped, H-7b, 7'b), 1.70 (1H, m, H-6), 3.81 (3H, s, OMe), 2.66 , 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 (100 MHz, CD ₃ OD):? 136.3 (C-1), 117.2 (C-2), 147.4 (C-3), 147.2 C-6), 39.7 (C-7,7 '), 40.6 (C-8,8 &apos;), 16.7 , 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 (m / z 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 (100 MHz, CD ₃ OD):? 135.0 (C-1,1 '), 117.3 (C-2,2'), 146.1 ), 116.2 (C-5,5 &apos;), 121.6 (C-6,6 &apos;), 39.8 ').

[ Example ]

Formulation example  One. Sanje  Produce

Compound (II) 1 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

Compound (II) 1 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

Compound (II) 1 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

Compound (II) 1 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _{4 12} H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

Compound (II) 1 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual preparation method of the liquid preparation and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto to adjust the total volume to 100 ml. The mixture was filled in a brown bottle and sterilized to prepare a liquid preparation do.

The female hormone substances of the present invention derived from natural products are effective substances for the treatment and prevention of female cancer and menopausal symptoms and can be used in medicine and health functional foods.

Claims (10)

A pharmaceutical composition for preventing and treating menopausal symptoms, which comprises at least one compound selected from the group consisting of compounds represented by the following formulas (I) to (IV) and pharmaceutically acceptable salts thereof as an active ingredient showing female hormone resistance A pharmaceutical composition.
(I)

(II)

(III)

(IV)

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

delete The pharmaceutical composition according to claim 1, wherein the menopausal symptom includes any one of facial flushing, hyperlipidemia, osteoporosis, venous thrombosis and atrophic vaginitis. A composition for preventing and improving menopausal symptoms, which comprises at least one member selected from the group consisting of compounds represented by the following formulas (I) to (IV) and acceptable salts thereof as an active ingredient showing female hormone resistance Food composition.
(I)

(II)

(III)

(IV)

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

delete The food composition according to claim 5, wherein the menopausal symptom includes any one of facial flushing, hyperlipidemia, osteoporosis, venous thrombosis and atrophic vaginitis. The food composition according to claim 5, wherein the food is a health functional food having any one of tablet, capsule, powder, granule, liquid, and ring. 6. The food composition according to 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.

Images