KR20170043232A - Pharmaceutical compositions for prevention and treatment of cancer and menopausal symptom of women containing Prunus cerasoides or its isolated compound as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prophylaxis and treatment of female cancer and menopausal symptoms, comprising an extract of Prunus cerasoides , an active fraction thereof, a single component separated therefrom and a pharmaceutically acceptable salt thereof as an active ingredient ≪ / RTI > The present invention is the first to confirm that the Prunus cerasoids extract, its active fraction and a single component derived therefrom exhibit female hormone resistance. The female hormone substances provided in the present invention are useful as a therapeutic agent for female cancer and menopausal symptoms Can be used in medicine and health functional foods for treatment and prevention.

Description

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms, which comprises as an active ingredient an extract of Prunus cerasoids or a compound isolated therefrom, its isolated compound as an active ingredient}

The present invention relates to a pharmaceutical composition for the prophylaxis and treatment of female cancer and menopausal symptoms comprising Prunus cerasoides extract, an active fraction thereof, a compound isolated therefrom and a pharmaceutically acceptable salt thereof as an active ingredient ≪ / RTI >

Cancer of women such as endometrial cancer and breast cancer and diseases such as menopausal syndrome are associated with female hormones such as estrogen hyperactivity, 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 most common cancer in women. The major causes of breast cancer are high fat, high calorie westernized diet and obesity, late marriage and low birth rate, avoidance of lactation, and estrogen overexposure. 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, deterioration of osteoporosis, hyperlipidemia, depression of the brain, depression of memory and cognitive impairment, changes in mental function, mental instability and rapid emotional changes, . 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.

The role of estrogen in the metabolism of estrogen in the metabolism of carcinogens and their metabolism has been suggested to be related to the carcinogenesis of HRT as well as the accumulation of excess cell proliferation and DNA mutation due to E2 / It is presented as a mechanism. This pharmacological effect of tissue-specific estrogen 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 composed of complex extracts composed of various kinds of chemical components. In the case of health supplements, strict safety tests can not be proved . 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.

Prunus cerasoides ) is a plant of Rosaceae Prunus , called wild Himalayan cherry or sour cherry. It is known to live in Himachal Pradesh province, northern India, the Himalayas, southwestern China, Burma province, and Thailand. Prunus cerasoides Water-soluble extracts of plant limbs have been reported to be used in India for the prevention of abortion (Kirtkar, KR et al. , 1975). Prunus cerasoides Leaf and bark are used for cough, asthma, indigestion and diarrhea. Leaves and flowers have been used for kidney stones (Chopra, RN et al ., 1956; Vaidyaratnam, PS et al ., 1995). Prunus cerasoides is also used as an extinguishing agent and as a remedy for gastric ulcers and heartwood is known to be useful for pitta , burning sensations, sprain and skin bleaching (Vaidyaratnam, PS et al . 1995). Studies have so far found that Prunus cerasoides contain flavonoids, steroids, terpenes, and the like. It contains glucogenkwanin as a flavone series and contains naringenin as flavanone. It contains isoflavonoids such as genistein and prunetin. Steroids and terpenes contain beta-sitosterol and uric acid. Studies on the components identified from Prunus cerasoides have shown that studies on naringenin, prunetin, β-sitosterol, and ursic acid have. Naringenin has been reported to act as an estrogen agonist in the absence of estrogen in breast cancer cell lines and to act as an antagonist at high estrogen levels (Kim S et al ., 2013). It has also been reported that prunetin inhibits cell growth in a concentration-dependent manner in the Syrian hamster embryo (SHE) cell model (Tsutsui T et al ., 2003). Beta-sitosterol (Touillaud MS et al ., 2005), which reduces the risk of ER negative tumors and reduces ER-positive breast cancer risk in postmenopausal women. Ursolic acid has been reported to increase the expression of p53 gene and to inhibit the growth of breast cancer cells and to inhibit the growth of MCF-7 breast cancer cells that express tamoxifen resistance (Gu G et al ., 2012) .

Kirtkar, K. R .; Basu, B.D. Indian Medicinal Plants, M / s Periodicals, New Delhi, 1975, 2, 959.   Chopra, R. N .; Handa, K. L .; Kapoor, L. D .; Singh, T. Ind. J. Agric. Sci., 1956, 26, 415.  Vaidyaratnam, P.S .; Varier, P.S .; Nambiar, V.P.K. Indian Medicinal Plants, Orient Longman, 1995, IV.   Kim S, Park TI; Naringenin: a partial agonist on estrogen receptor in T47D-KBluc breast cancer cells; Int J Clin Exp Med. Oct 25; 6 (10): 890-9.   Tsutsui T, Tamura Y, Yagi E, Someya H, Hori I, Metzler M, Barrett JC .; Cell-transforming activity and mutagenicity of 5 phytoestrogens in cultured mammalian cells .; Int J Cancer. 2003 Jun 20; 105 (3): 312-20.   Touillaud MS, Pillow PC, Jakovljevic J, Bondy ML, Singletary SE, Li D, Chang S .; Effect of dietary intake of phytoestrogens on estrogen receptor status in premenopausal women with breast cancer. ; Nutr Cancer. 2005; 51 (2): 162-9.  Gu G, Barone I, Gelsomino L, Giordano C, Bonofiglio D, Statti G, Menichini F, Catalano S, AndoS .; Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα / Sp1-mediated p53 activation .; J Cell Physiol. 2012 Oct; 227 (10): 3363-72. doi: 10.1002 / jcp.24035.

The present invention is to provide a novel natural medicine and health functional food for the prevention and treatment of female cancer and menopausal symptoms. In particular, the present invention provides estrogen (estrogen) fruit that represents the active Taunus Serra Soi Death (Prunus cerasoides) extracts, active fractions and to identify separate the single components separate from this, women new natural medicines for the prevention and treatment of cancer and menopausal symptoms And a health functional food.

The development of a therapeutic agent for postmenopausal cancer or a therapeutic agent for cancer in which the estrogen receptor (ER), such as breast cancer or endometrial cancer, is involved in the carcinogenesis mechanism, is a substance that can have a tissue- The development of receptor modulators (SERMs) 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 while balancing estrogen efficacy in the body.

In the present invention, the efficacy, pharmacological metabolism and safety of the active ingredient of the Prunus cerasoids extract, its active fraction isolated therefrom, and its pharmacological metabolism and safety are evaluated, and it is possible to provide a pharmaceutical composition useful for the prevention and treatment of female cancer such as endometrial cancer, breast cancer, Thereby providing a novel pharmaceutical composition or food composition.

Specifically, in the present invention,

A pharmaceutical composition for the treatment and / or prophylaxis of female cancer and / or pancreatic cancer, which comprises, as an active ingredient, at least one selected from the group consisting of a Prunus cerassoide sprueus extract, an active fraction thereof, a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof: A pharmaceutical composition for the prevention and treatment of menopausal symptoms is provided.

Further, in the present invention,

A pharmaceutical composition for the treatment and / or prophylaxis of female cancer and / or pancreatic cancer, which comprises, as an active ingredient, at least one selected from the group consisting of a Prunus cerassoide sprueus extract, an active fraction thereof, a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof: A food composition for the prevention and improvement of menopausal symptoms is provided.

(I)

Prunus It is the first found in the present invention has a single component of the above general formula (Ⅰ) derived from cerasoides) to extract shows the female sex hormones.

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

The menopausal symptoms preferably include any one of facial flushing, hyperlipidemia, brain mental dysfunction, osteoporosis, venous thrombosis and atrophic vaginitis.

The food is preferably a health functional food having a form of tablet, capsule, powder, granule, liquid, or ring.

The present invention relates to a method for producing The present invention provides a new plant-derived single-component medicine and a health functional food useful for the prevention and treatment of female cancer and menopausal symptoms by isolating and identifying an active fraction showing a female hormone from the plant and a single component. In particular, the present invention is the first to confirm that prunus cerasoids and a single component derived therefrom exhibit female hormone resistance, and the pharmaceutical composition of the present invention containing such components is most frequently encountered among females And can be used for endometrial cancer, which has an increased incidence of breast cancer and similar pathogenesis.

Figure 1 shows the effect of estrogen ([< ³ > H] estrogen) labeled with ^triple dehydrogen on the pure recombinant human estrogen receptor alpha (hER & ) Extracts.
2 is a cross- Prunus (PCE T) and 21 fraction (PCE - 21) fractions of cerasoides . Prunus The ERβ / ERα ratios of the four fractions (PCE 6, 15, 17, and 19) in cerasoides extract (PCE T) and 21 fractions (PCE - 21)
3 is haejun after treatment with vehicle, estrogen (estrogen), fruit Taunus Sera the Soy extract des ^{10 -6 g / ml ~ 5x10 -5} g / ml concentrations to evaluate the estrogen receptor reactive with MCF-7 cell proliferation action MCF7 cell proliferation. Prunus cerasoides extracts induced cell proliferation at concentrations of 10 ^-6 g / ml, 5 × 10 ^-6 g / ml, and 10 ^-5 g / ml.
Figure 4 shows the transcriptional effects of vehicle, estrogen, and Prunus seraidesu extracts on MCF-7 cells transfected with an estrogen responsive element (ERE).
Figure 5 shows the transcriptional effects of vehicle, estrogen, and prunus cerasoidase fractions in MCF-7 cells transfected with an estrogen responsive element (ERE).
FIG. 6 shows the results of analysis of the ERE gene activity of a single substance isolated from prunus cerasoids in MCF-7 cells transfected with an estrogen responsive element (ERE) sequence.
Figure 7 shows the expression of the ERE gene-inducible pS2 gene expression of the extract of Prunus seraidesides in MCF-7 cells transfected with an estrogen responsive element (ERE) sequence.
Fig. 8 shows changes in pS2 gene activity in the uterine tissues of rats when administering prunus cerasoids to rats.
FIG. 9 shows changes in protein expression of estrogen receptor alpha (ERa), progesterone receptor A (PRA) and progesterone receptor B (PRB) in uterine tissues of rats when administering prunus cerasoids extract to rats.
FIG. 10 is a graph showing the effect of the Prunus cerasoids extract on the neuroglobin (Ngb) promoter activity of the human neuron SKNSH. The activity of the Ngb promoter was increased by the positive control and by the Prunus seraidesis extract when treated with vehicle, estrogen, genistein, and prunus cerasides as positive control.
11 is an evaluation of the effect of the Prunus cerasoids extract on Ngb mRNA expression of human nerve cell SKNSH. The expression of Ngb gene was increased by positive control and prunus cerasoids extract when treated with vehicle, estrogen, genistein and prunus cerasides extract as positive control.
12 is an evaluation of the effect of the Prunus cerasoids extract on the Ngb promoter activity of mouse neuron N2a. The activity of the Ngb promoter was increased by the positive control and by the Prunus seraidesis extract when treated with vehicle, estrogen, genistein, and prunus cerasides as positive control.
13 analyzes Ngb protein changes in the brains of ovariectomized female rats by the presence of Prunus seraidesis extract. (OVX + PC 250, or PC 500) after ovariectomized group (OVX + E2) and ovariectomized group (OVX + PC 250 or PC 500) The expression of Ngb protein in the rat brain of soybean extract-treated group was increased in a concentration-dependent manner.
14 shows the results of experiments on the effects of Prunus cerasoids extract on the levels of ALP, AST and ALP, which are representative enzymes of liver function, in female rats with ovariectomized rats. Compared with the vehicle group, the administration group showed improvement in liver water level.
Fig. 15 shows the results of an experiment on the effect of prunus cerasoids on changes in blood lipid profile of ovariectomized female rats. Compared with the Vehicle group, prunus cerasoids showed a decrease in triglycerides.

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

In the present invention, " menopausal symptoms " is meant to include facial flushing, hyperlipidemia, brain mental function depression, osteoporosis, venous thrombosis, and atrophic vaginitis in menopausal women.

In the present invention, " brain mental function deterioration " includes depression, memory loss, and cognitive decline in menopausal women.

In the present invention, Prunus < RTI ID = 0.0 > cerasoides ) is represented by the following chemical formula (I).

(I)

Chemical name: Prunetinoside

Chemical Formula: C ₂₂ H ₂₂ O ₁₀ (MW 446.4041)

Prunus cerasoides is a plant of Rosaceae Prunus, and Prunus cerasoides used in the present invention was distributed from the International Biomaterials Hub Center.

In the present invention, Prunus < RTI ID = 0.0 > cerasoides) process to identify and validate the three extract fractions and women and the prevention of cancer, menopausal symptoms, and separating the components of the (Ⅰ) effective in the treatment sera from pandanus fruit Soy Death (Prunus cerasoides) are as follows:

One. Prunus Serrasoides ( Prunus cerasoides )  The efficacy of female hormone

Prunus cerasoides) Human estrogen receptor for the extract alpha (hERα) competitive inhibition of binding, MCF-7 MCF-7 cells transfer analysis with the estrogen-responsive genes, uterine proliferation, pS2 gene activity in the uterine tissue and estrogen receptor alpha (ERα ), Progesterone receptor A (PRA), and progesterone receptor B (PRB).

2. Three fractions of the extract of prunus cerasoids and Isolation of active ingredient and confirmation of pharmacological activity

Prunus cerasoides) separating fractions of the extract, the experiment was the pharmacological activity of the active ingredient by separating the three fractions of 21 kinds, OK, it was confirmed that the excellent pharmacological activity of one compound of these. The compound represented by the above formula (I) is the first to confirm the female hormone pharmacological activity in the present invention.

The pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms of the present invention contains at least one selected from the group consisting of the compounds represented by the above-mentioned formula (I) and pharmaceutically acceptable salts thereof as an active ingredient .

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, for the desired effect, it is preferable to administer the compound of the formula (I) at a dose of 0.0001 to 1000 mg / kg per day based on the compound of the formula (I). 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 contains at least one selected from the group consisting of the compound represented by the above formula (I) and acceptable salts thereof as an active ingredient.

The food composition contains 0.0001 to 20% by weight of the compound represented by the formula (I) 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 the above-mentioned component (I) as an essential ingredient, and it may further contain various ingredients such as various flavors or natural carbohydrates such as ordinary foods and beverages have. 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 assay: Estrogen receptor (ER)

Recombinant human estrogen receptor alpha (hERα) was purchased from Invitrogen. 750mol of each receptor protein was diluted to 3 nM with binding buffer. The composition of the binding buffer is 10 mM Tris / pH 7.5, 10% glycerol, 1 mM DTT and 1 mg / ml BSA. A final concentration of 100 μl was added to a microcentriguge tube containing 750 mM ER, 3 nM tritiated estrogen ([ ³ H] estrogen, [ ³ H] E2) and a certain concentration of test substance (dissolved in DMSO). After incubation at 28 ° C for 3 hours, the ER-test substance complex was filtered through a glass filter using a harvester, and the unreacted free tritium estrogen ([ ³ H] E2) was removed by washing. To the glass filter, 3 ml of Ultima Gold scintillation cocktail was added and the radioactivity remaining in the glass filter was measured using a liquid scintillation counter. To measure non-specific binding, 5 nM estrogen (estrogen, E2) was used. As a comparative control drug, 10 nM estrogen (estrogen, E2) was used. For each experiment, several concentration values were tested for the same test substance to obtain a curve for the concentration-activity count through the degree of interference with the receptor binding of radioactive estrogens (E2), and the IC ₅₀ value Respectively. IC ₅₀ values were calculated using prism 3.0 software.

2. Gene reporter assay: Estrogen (ERE) reactive gene transcription experiment

For the ERE-luciferase assay, the breast cancer cell line MCF-7, known to have a high ER content, was used. MCF-7 breast cancer cell lines were purchased from American Tissue Culture Collection (USA). Twenty-four hours prior to seeding, the cells were cultured in a charcole dextran-treated medium (CD-DMEM), and cells having 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 estrogen response element-luciferase plasmid was transfected using Lipofectamine 2000 reagent (Invitrogen, USA). In the ERE-luciferase assay, estrogen (E2, 1 nM) was used as a positive control and ICI-182,780 (1 mM) was used as an antagonist control. Various concentrations of test substances (stock solution dissolved in DMSO After culturing for 24 hours, the cell culture was terminated and water-soluble cell extract was obtained by using Passive Lysis Buffer (Promega, USA). The activity of luciferase present in the cell lysiferase (Luciferase Assay System, Promega) containing a substrate and a reaction buffer, and a luminometer. The gene activity of the test substance was determined by measuring the activity exhibited by the positive control Was determined as 100%, and the degree of relative activity was shown, and the final comparative evaluation was made.

3. In vivo in vivo ) Evaluation of female hormone: uterotrophic assay

The uterus proliferation experiment is a method of indirectly evaluating female estrogenicity by measuring the increase in uterine tissue mass induced by estrogen. The effect of the test substance on the immature uterus was investigated by comparing with the control group for 3 days after subcutaneous injection in female rats on day 21 after birth. Immature female rats were used for the experiments. The animals were selected as the average body weight during the refinement period, and grouping was carried out using the random method to 5 animals per group. Animal identification was done by tail marking and tagging by box. In the positive control group, estrogen (E2) was homogenously suspended in corn oil to a concentration of 3 μg / kg, followed by stepwise dilution with corn oil, and the test substance was also suspended in corn oil. The experimental group was divided into five groups and each group was divided into two groups: vehicle (5 ml / kg), estrogen (E2, 0.003 mg / kg), prunus cerasoid (100, 200, 300 mg / kg) The combination of desdextract (300 mg / kg) and estrogen (estrogen, E2) was administered by subcutaneous injection at 24-hour intervals for 3 days. The dose was 0.05 ml per 10 g of immature rat body weight and 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 And the uterine weight obtained in each rat was accurately measured using a microtiter scale (Mettler microbalance).

4. Evaluation of transcriptional activity of intracellular endogenous hormone-responsive target genes 21-day-old immature female rats were divided into vehicle, estrogen (E2), and prunus cerasoids group, and vehicle (corn oil 5ml / kg ), Estrogen tradiol (0.003 mg / kg), and prunus cerasoids (100, 200 mg / kg) were administered subcutaneously for 3 days at intervals of 24 hours. Trizol) to extract mRNA. iScript cDNA synthesis kit (Bio-Rad) was used to reverse-transcribe a certain amount of mRNA (1 μg) to obtain cDNA. 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 steps, the expression level of the housekeeping gene, GAPDH, was measured at the same time, and the ratio of the expression level of this gene to 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 Trefoil factor 2 (or pS2) as a target gene of estrogen was measured. For real-time RT PCR, 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 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.

5. Assessment of effects on intracellular endogenous hormone reactive protein expression

21-day-old immature female rats were divided into vehicle, estrogen, and prunus cerasoids groups, and each group received vehicle (corn oil 5 ml / kg), estrogen (estrogen, 0.003 mg / kg), prunus cerasoids 100 mg, 300 mg / kg), and estrogen (E2) were administered by intraperitoneal injection every 24 hours for 3 days. . The extracted protein was quantitated by BCA method and electrophoresed in 20 μg each of 7.5% SDS-PAGE gel at 80V for 2 hours. After electrophoresis, the cells were transferred to a PVDF membrane at 25 volts for 3 hours, blocked with BSA for 1 hour at RT, and reacted with primary and secondary antibodies to detect ECL. The primary antibodies were diluted 1: 500 with ERα (sc-7207, SANTA CRUZ BIOTECHNOLOGY, INC.) And PR (sc-538, SANTA CRUZ BIOTECHNOLOGY, INC.). Β-actin was used to correct the difference in protein amount due to experimental errors.

6. Neuronal cell base Ngb  promoter luciferase  assay and cell and brain tissue Ngb mRNA , evaluation of protein expression change

(1) Ngb promoter assay : Estrogen and SERMS inhibit neuronal cell death and induce survival. SERMs also improve memory and improve behavior after stroke. In the present invention, it was confirmed whether SERMs can exhibit a brain protection effect through activation of Ngb. Ngb is a subtype of globin found in neurons and has a function to bind and transport O ₂ and is involved in the scavenge of reactive oxygen species (ROS). It is also involved in the signal transduction of G-protein coupled receptors. It is involved in the electron transfer of cytochrome C in mitochondria and regulates apoptosis and cell survival. It also shows neuron protection effect in stroke animal models. Recently, estrogen has been shown to enhance the expression of Ngb in neurons and astrocyte and has been associated with anti-inflammatory action. The luciferase experiments were carried out using Neuro2a neuroblastoma cell line (CCL-131; ATCC, Manassas, VA, USA) and SKNSH (human neuroblastoma cell line) By inserting the neuroglobin (Ngb) -luciferase sequence into the cell gene, the Ngb promoter transcriptional activity of the plant extract can be evaluated quantitatively. Cells were cultured in 10% FBS DMEM, seeded in 3 × 10 ⁴ cells in a 96-well plate, and media was replaced with 2% FBS DMEM at 40-50% confluency after about 24 hours. Cells were continuously cultured and when the 70 ~ 80% confluency was reached, the media was changed by dissolving the drug in serum free phenol red free DMEM. Cells were treated with extracts (10 ^-4 to 10 ^-7 g / ml) for 6 hours (SKNSH) or 24 hours (DMEM) using vehicle (DMSO, 0.1%), 17β-ES (1 nM) and genistein N2a), 40 μl of lysis buffer was added and incubated at RT for 15 min. The lysates were collected and the luminescence was measured by adding 30 μl of substrate.

(2) Ngb mRNA level (quantitative PCR ) : N2a cells and SKNSH cells were cultured in 10% FBS DMEM, and the media was changed by dissolving the drug in phenol red free DMEM. Cells were treated with extracts (10 ^-4 ~ 10 ^-7 g / ml) using Vehicle (DMSO, 0.1%), 17β-ES (1 nM) and genistein (1 μM) as control. Cells were treated with trizol The cells were detached from the container surface and destroyed. Cellular mRNA is isolated using Qiagen RNeasy mini kit. cDNA was obtained by reverse transcription of a certain amount of mRNA (1 μg) using an iScript cDNA synthesis kit (Bio-Rad). quantitative PCR was performed using a pair of primers capable of recognizing 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 steps, the expression level of the housekeeping gene, GAPDH, was measured at the same time, and the ratio of the amount of expression of this gene and the expression amount of the target gene was used for the final quantitative analysis. The primers recognizing human GAPDH are forward 5'-GGCTGAGAACGGGAAGCTTGTCAT; And reverse 5'-CAGCCTTCTCCATGGTGGTGAAGA. The primers recognizing Mouse GAPDH are forward 5'-TGCCAAGTATGATGACATCAAGAA; And reverse 5'-GCCCAAGATGCCCTTCAGT. The primers recognizing human Ngb are forward 5'-TGGAAGACCTGTCCTTCACTG; And reverse 5'-GAGCAGAGACTCACCCACTG. The primers recognizing Mouse Ngb are forward 5'-TACAATGGCCGCCAGTTCT; And reverse 5'-TGGTCACTGCAGCATCA. Quantitative PCR was performed at about 40 cycles (95 ° C; 15 seconds, 60 ° C; 60 seconds). Each gene expression curve was expressed by logarithm and the threshold cycle (C _T ) value was mathematically obtained. The value obtained by substituting this value into the 2 ^-ΔΔCT formula represents the relative expression amount of a specific gene. The expression level of a specific gene was divided by the expression level of a house keeping gene, and the result was compared with that of the vehicle group and the test substance group.

(3) Ngb protein expression in OVX rat brain: Expression of Ngb prtein was confirmed in vivo in order to confirm the brain protection effect of the selected plant extracts. Experimental animals were treated with rats and ovariectomy was performed to exclude intrinsic hormone action, followed by a 2-week recovery period. For the next 6 weeks, po was administered daily with 250 mg / kg of extract and 500 mg / kg of extract. The treatment group consisted of sham operated group, OVX group, OVX + 17β-ES group, OVX + 250mg / kg group and OVX + 500mg / kg group. After 6 weeks, lysate was obtained from brain tissue and 70 μg protein was loaded on 12% SDS-PAGE gel, and run at 80V for 1.5 hours and transferred to PVDF film at 25V for 3 hours. Expression of Ngb (ab37258, abcam) ERα (sc7207, SANTA CRUZ) was confirmed using ECL solution.

(4) Ngb mRNA level in OVX rat brain : Expression of Ngb mRNA was confirmed in vivo to confirm the brain protection effect of selected plant extracts. Brain tissue was treated with trizol to destroy brain tissue with a homogenizer. Brain tissue mRNA was isolated using Qiagen RNeasy mini kit. cDNA was obtained by reverse transcription of a certain amount of mRNA (1 μg) using an iScript cDNA synthesis kit (Bio-Rad). quantitative PCR was performed using a pair of primers capable of recognizing 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 steps, the expression level of the housekeeping gene, GAPDH, was measured at the same time, and the ratio of the amount of expression of this gene and the expression amount of the target gene was used for the final quantitative analysis. The primers recognizing Rat GAPDH are forward 5'-GGCTGAGAACGGGAAGCTTGTCAT; And reverse 5'-CAGCCTTCTCCATGGTGGTGAAGA. The primers recognizing Rat Ngb are forward 5'-TGGAAGACCTGTCCTTCACTG; And reverse 5'-GAGCAGAGACTCACCCACTG. Quantitative PCR was performed at about 40 cycles (95 ° C; 15 seconds, 60 ° C; 60 seconds). Each gene expression curve was expressed by logarithm and the threshold cycle (C _T ) value was mathematically obtained. This value is 2 ^{- ΔΔCT} The value obtained by substitution into the equation indicated the relative expression level of a specific gene. The expression level of a specific gene was divided by the expression level of the house keeping gene, and the result was compared with that of the OVX group and the test substance group.

7. Study on effects on blood glucose metabolism markers and weight gain

Plant extract ( Prunus cerasoides ) were orally administered to ovariectomized rats to investigate their effects on blood metabolism markers and weight gain. An 8-week old SD rat (female)

The weekly adaptation period was established, the rats were anesthetized with isoflurane, and the ovaries were removed by incising the abdomen under anesthesia. After the ovariectomy was completed, three cages were placed in the cage, followed by two weeks of recovery and blood estrogen wash-out, and a phytoestrogen-restricted diet (Harlan 2020X Teklad Global Soy Protein-Free Extruded Rodent Diet) was administered Respectively.

Conservative rats were divided into groups of 9-10 mice per group, with Sham (no OVX), OVX control, E2, low concentration selective extract, and high concentration selective material.

In the high-concentration selective material extract group, 500 mg / kg of plant extracts were added to the sham (no OVX), OVX control group, E2 0.5 mg / kg, / kg were distributed. At this time, 20-25g of food per rat was distributed to allow a quantitative component to be provided, and body weight was measured weekly to observe changes in body weight of Rat. After 6 weeks of oral dosing, the mice were fasted for 24 hours and then anesthetized with isoflurane to obtain a blood sample of 4 to 5 ml from the heart. The collected blood sample was centrifuged at 3500 rpm (15 minutes, 4 ° C) Plasma samples were obtained, stored at 4 ° C, and analyzed by lipid profile in the blood within 1-2 days.

8. Human assay for anti-inflammatory activity hematopoietic  Assessment of inhibition of prostaglandin D synthase (HPDGs)

Prunus The efficacy of anti - inflammatory activity of 16 low - molecular single compounds isolated from plant extracts, plant extracts and fractions such as cerasoides was evaluated by the inhibitory effect of human hematopoietic prostaglandin D synthase (HPDGs). The enzyme reaction included assay buffer (0.1M potassium phosphate, pH 6.5 with 1 mM EDTA), GSH 2.5 mM, CDNB 1 mM, and human hematopoietic prostaglandin D synthase (10 mg / ml). The assay buffer, CDNB, and HPDGs were first added to 96-well plates and mixed with GSH, a single compound (10 mM) isolated from plant extracts (250 mg / ml) or plant extracts and fractions. DMSO was used as a control.

After 2 minutes of reaction, the amount of CDNB and glutathione bound substance was measured at 340 nm using a VICTOR3 luminometer (Perkin Elmer) and the relative activity of HPDGs was compared with Control (DMSO) value.

9. Acute Toxicity Experiment

 Single - dose acute toxicity studies were conducted to increase the clinical applicability of candidate plant extracts. Female mice were used for fasting until 24 h before administration. (1% CMC in saline), extracts 250 mg / kg, 500 mg / kg, 1000 mg / kg, and 1500 mg / kg were administered once orally to measure weight change, behavioral observation and mortality. The total volume was 5 μl / g.

10. 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 C18-RP-medium pressure liquid 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 fractions were obtained by MPLC, HPLC and LC-MS analysis on the selected active fractions, and fractions containing plant metabolites having activity were classified and purified Respectively.

(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, COSY, HSQC, HMBC, NOESY, and ROESY). Using existing DB of medicinal plants, it was confirmed whether the finally obtained substance was new substance or known substance.

result

1. Effectiveness of female hormone pharmacological efficacy of prunus cerasoids extract and three fractions

end. Competitive Inhibition and ERβ Selectivity of Prunus seraidesu Extracts on ER Binding

The competitive binding capacity of the purified recombinant human estrogen receptor alpha (hERα) of prunus cerasoids extract and tritium estrogen ([ ³ H] E2) was measured in the in vitro system. The results are shown in FIG. 1 and Table 1. In the competitive binding assay, the IC ₅₀ of estrogen (E2) (●) was 3.83 × 10 ^-10 g / ml (hERα), respectively. The IC ₅₀ of a kind of phytoestrogen, prunus cerasoides (*), was 1.41 × 10 ^-5 g / ml (hERα). These results indicate that the prunus cerasoids extract acts in a concentration-dependent manner on the tritium estrogen ([ ³ H] E2) cleavage bond, and the relative bioavailability (RBA) of the estrogen- (0.0027% for hER [alpha]) of about 50000 times lower than that of the control (Fig. 1, Table 1).

Prunus The ER subtype selective gene transcriptional activity was measured for the 10 mg / ml sample after dissolving cerasoides extract (PCE T) and 21 fractions (PCE - 21) at appropriate concentration in DMSO or water. The relative comparison of the luciferase activity of each sample when the luciferase activity was compared with the DMSO relative to the relative value was shown in FIG. Prunus The ERβ / ERα ratio was found to be 2 or more in four fractions (PCE 6, 15, 17, 19) among the extracts of cerasoides (PCE T) and 21 fractions (PCE - 21 ) .

[Table 1] IC ₅₀ and RBA values for hER [alpha ] of prunus cerasoids extract

RBA = [Ki _(E2) / Ki _{( Prunus cerasoides )} ] × 100.

I. Prunus Serrasoides MCF -7 Through estrogen response genes MCF -7 cell proliferation and intracellular transcription analysis

To evaluate MCF-7 cell proliferation, Prunus The cerasoides extract was treated with 10 ^-6 g / ml to 5 × 10 ^-5 g / ml and confirmed MCF7 cell proliferation. 17β-ES, which was used as a positive control, induced the proliferation of MCF7 cells more than twice as much as that of the vehicle group. Genistein, one of the representative phytoestrogens, also increased the proliferation of MCF7 cells by about 1.8 times. Prunus and cerasoides extracts induced cell proliferation at 10 ^-6 g / ml, 5 × 10 ^-6 g / ml, and 10 ^-5 g / ml concentrations (FIG. 3).

In MCF-7 cells transfected with an estrogen responsive element (ERE), prunus cerasoids Experiments were conducted to determine whether the extract changed the activity of the ERE gene. The results are shown in Fig. E2 gene activity was increased by about 5 to 30 times at a concentration of 2.5 to 20 μg / ml when the extract of Prunus seraides was administered alone. Estrogen (E2) was found at concentrations of 10 μg / ml and 20 μg / It was found that prunus cerasoids induces ERE gene activity. These results suggest that ERE gene activity is induced in a concentration - dependent manner at a concentration of 2.5 μg / ㎖ or higher in the gene transcription system. Eure gene (estrogen, E2) higher than 10 μg / ≪ / RTI > activity. In addition, the Prunus Serrasoides Three fractions were tested for ERE gene reporter. Prunus Serrasoides Four fractions (three fractions: PC-16, 17, 18, and 20) were used at the 20 μg / ml concentration of the three fractions to increase the transcriptional activity by ERE up to 1.9 times compared to the estrogen activity (Fig. 5) . In addition, the effect of transcriptional activity of ERE on the concentration-dependent single component isolated from prunus cerasoid was confirmed (Fig. 6)

All. Uterine proliferative effect of prunus cerasoids

In order to evaluate the female hormone of the extract in the system excluding the action of endogenous estrogen (E2), female rats of immature 21 day old female mice with extremely low estrogen (E2) secretion were used to study uterine proliferation. The uterus proliferation test results were compared and evaluated as shown in Table 2 below. The weight ratio of uterus was 0.7 and the estrogen (E2) (3 ㎍ / ㎏) was injected subcutaneously as a positive control. The weight ratio of uterus was 1.8 and control group The uterine ratio was increased by about 2.5 times. The uterine weight ratio was 0.61 when administered with 100 ㎎ / ㎏ subcutaneous injection of prunus cerasoids extract in immature rats and 0.53 when administered with 200 ㎎ / ㎏. When administered at 300 ㎎ / ㎏, the uterine weight ratio was 0.68, and when combined with estrogen (E2) and prunus cerasoids 300 mg / kg, it was 2.34. Compared with the vehicle group treated with corn oil alone, the weight of the uterine tissue was similar to that of the vehicle group, regardless of the administration dose, when the prunus cerasoids extract was treated alone. On the other hand, when estrogen (E2) was administered together with prunus cerasoids extract, the uterine weight was increased compared with estrogen (E2) alone. The uterus proliferation test showed that the Prunus cerasoids extract alone did not induce uterine proliferation but increased estrogen (E2) -mediated uterine proliferation when acting together with estrogen (E2) I could. No weight gain was observed in rats during the drug administration period. Significantly different values for the vehicle group were marked with *** (*** P <0.001).

Table 2 Fruit Taunus Immature rat uterine proliferation by Serra soy extracts Death

la. Induction of estrogen-responsive pS2 gene activity of prunus cerasoids

Prunus cerasoides extract was treated at a concentration of 10 ^-7 g / ml to 10 ^-5 g / ml, and the expression of pS2 gene in MCF-7 cells was confirmed. 17β-ES, which was used as a positive control, increased the expression of pS2 gene by about 2.5-fold compared to the vehicle group and genistein also increased the expression of pS2 gene by about 2.5-fold. In the case of Prunus cerasoides extract, the expression of pS2 gene was slightly increased at a concentration of 10 ^-5 g / ml (FIG. 7) .

A 21-day-old immature female rat was treated with subcutaneous injection of vehicle (corn oil 5 ml / kg), estrogen (estrogen, E2, 0.003 mg / kg) and prunus cerasoids (100 mg / kg, 200 mg / kg) PS2 gene expression was observed in the uterine tissues of the rats obtained after 24 hour intervals. Experimental results showed that estrogen (E2) increased the expression of pS2 gene by about 1.9 times compared to vehicle group. It was confirmed that the administration of prunus cerasoids extract increased the expression level of pS2 gene by about 1.4 times at 100 mg / kg. On the other hand, the dose of 200 mg / kg was about 0.9 times that of the vehicle group. These results suggest that estrogenic activity of estrogen (estrogen, E2) in the uterine tissues can be confirmed by administration of Prunus cerasoids extract at a dose of 100 mg / kg. The expression level of the pS2 gene (Fig. 8).

hemp. Suppression of ERα protein expression in uterine tissues of prunus cerasoids

21-day old female rats were treated with vehicle (corn oil 5 ml / kg), estrogen (E2, 0.003 mg / kg), prunus cerasoids (100 mg / kg, 300 mg / kg), prunus cerasoids Progesterone receptor A (PRA), and progesterone receptor B (PRB) in the uterine tissues of rats obtained after subcutaneous injections of estrogen (300 mg / kg) Expression changes were confirmed. Experimental results showed that administration of estrogen compared to vehicle resulted in decreased protein expression of ERα, When 100 mg / kg of extract was administered, protein expression of ERa was expressed at the vehicle level (Fig. 9). Prunus Serrasoides When the extract was administered at a dose of 300 mg / kg, ERα protein expression was slightly reduced compared with the vehicle group, and Prunus serarosides The combination of 300 mg / kg of extract and E2 showed that protein expression of ERα was reduced to a similar level as estrogen - treated group. On the other hand, PRA and PRB increased when estrogen was administered, and Prunus seraides Both PRA and PRB increased with 100 mg / kg of extract. However, estrogen receptors and PRA / B protein expression in the uterine tissues of Prunus cerasoids extract were not dose-dependent (FIG. 9).

bar. Of human and mouse neurons neuroglobin  ( Ngb ) promoter activity and mRNA  Increased gene expression and increased Ngb protein in rat brain tissue

To elucidate the effect of Prunus cerasoides extracts on the Ngb promoter activity of human neuron SKNSH, SKNSH cells were treated with ⁵ × 10 ^-6 to 5 × 10 ^-5 g / ml of Ngb promoter activity by luciferase assay. And evaluated quantitatively. When the positive control genistein was treated, the activity of the Ngb promoter was increased about 1.4 times as compared with that of the vehicle group, and 17β-ES also increased the Ngb promoter activity by about 1.3 times. Treatment of Prunus cerasoides extract increased Ngb promoter activity by about 1.4 to 1.6 times (*** p <0.001) statistically at all concentrations (Fig. 10). Prunus cerasoides extracts were treated at a concentration of ⁵ × 10 ^-6 g / ml to 10 ^-5 g / ml, and the expression of Ngb mRNA gene in SKNSH cells was confirmed. Genistein, a positive control, increased the expression of Ngb gene by about 1.8-fold and 17β-ES also increased the expression of Ngb gene by about 1.2-fold compared to the vehicle group. The Prunus cerasoides extract showed a 1.5-fold increase in Ngb gene expression at a concentration of 5 × 10 ^-6 g / ml (FIG. 11).

In order to evaluate the effect of N2a cells on mouse neuron N2a cells, N2a cells were treated with Prunus cerasoides extract at a concentration of ⁵ × 10 ^-6 g / ml to 5 × 10 ^-5 g / ml, followed by luciferase assay. promoter activity was quantitatively evaluated. When the positive control genistein was treated, the activity of the Ngb promoter was increased by about 1.7 times as compared with that of the vehicle group, and 17β-ES did not increase the Ngb promoter activity. Treatment of the Prunus cerasoides extract increased the Ngb promoter activity by about 1.4-fold (** p <0.01) statistically at a concentration of ⁵ × 10 ^-5 g / ml (Figure 12).

To determine whether the extracts of Prunus cerasoides showed the protective effect of the brain, the ovaries of female rats were actomized to make a hormone environment similar to that of postmenopausal women. Prunus cerasoides extracts were used for 6 weeks. The changes in Ngb and ERa expression in the brain were determined at the protein level. Compared with the sham operated group, ERα and Ngb decreased in the OVX group, but 17α-ES (E2) decreased ERα and Ngb. In the group treated with Prunus cerasoides extract, the expression of Ngb and ERa was decreased and the increase of Ngb was concentration-dependent (FIG. 13).

four. A Study on the Effect of Metabolic Markers and Weight Gain on Biological Blood

Prunus The effects of cerasoides plant extracts on blood metabolism markers and weight gain were investigated by oral administration to ovariectomized rats. 8-week old SD rats (ovariectomized) received ovariectomized post-operative convalescent and blood estrogen wash-outs. Sham (no OVX) for 6 weeks in rats undergoing convalescence, preparatory feed in OVX control group, E2 0.5 mg / kg, supplemented with 250 mg / kg of plant extract in the low concentration selective extract group and 500 mg / kg of the plant extract in the high concentration selective extract group. The values of ALP, AST and ALP as the liver function representative enzymes were expressed as Prunus s cerasoides Extract when compared to the 500mg / kg group and the control group was administered, Prunu s cerasoides extract could see that the group treated with 500mg / kg Sham, reduced to the same level as the group E2 ALP, AST, ALP levels. Therefore, Prunus cerasoides The extracts were found to have some effect on improving liver water (Fig. 14). After 6 weeks of oral administration, the mice were fasted for 24 hours and blood samples were collected at 4 to 5 ml to obtain plasma samples and analyzed for lipid profile. When the lipid profile and glucose changes were measured, in case of Triglyceride, Prunus cerasoides When the extracts were compared with the Ovx group treated with 250 mg / kg and 500 mg / kg, the amount of Triglyceride was decreased to a level similar to that of Sham and E2 group. Therefore, Prunus cerasoides extract is effective in improving the triglyceride level in the body. However, for the remaining figures, there was no significant effect (Fig. 15).

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

Prunus separation evaluate anti-inflammatory effect for low-molecular active single compound of the identified one kinds of a single material (PCE20-10-po)) via the inhibitory effect of human hematopoietic prostaglandin D synthase (HPDGs) from the extract (PCE) and extract the fraction cerasoides when, Prunus cerasoides The inhibitory effect of extract (PCE T) on HPGDs was 70.92% and HPGDs inhibition effect was very high. The HPGDs inhibitory effect of one kind of single component (PCE20-10-po) was 1.01%, which showed no HPGDs inhibitory effect.

character. Safety evaluation through acute toxicity test

Prunus To evaluate the biotoxicity of cerasoides extracts, single dose acute toxicity studies were performed. When 2-week mortality, weight change and behavior were observed in female mice (10 weeks old) with po (250 mg / kg, 500 mg / kg, and 1000 mg / kg po) of vehicle (1% CMC saline) and Prunus cerasoides extract, Normal behavior was observed and feed and water intake were normal. During the 2 weeks of the study period, the mortality rate was 0% in all groups including the vehicle group, and the weight change between the groups was similar. The maximum dose of 1000 mg / kg was not considered to be acute toxicity in the oral dose, so it was evaluated as a highly safe candidate plant (Table 3).

2. Purunus Serrasoides  Analysis of bioassay-guided analysis and its physiological activity evaluation

end. Fractionation and separation of the extract

Pandanus fruit Serra Soi Death (Prunus The methanol extract (20g) of cerasoides) of chloroform and methanol as the mobile phase (CHCl ₃ : MeOH, 100: 0 to 0: 100), and 21 fractions (PC1 to PC21) were obtained. As a result of evaluating the binding capacity of ER and PR of these fractions, the ERE gene transcription activity was superior to that of estrogen in fractions PC15, 16, 17, 18 and 20, and based on the activity results, five fractions were targeted To find a single active substance.

PC20 was divided into 21 small fractions (PC20-1 to PC20-21) by performing RP-MPLC using methanol and water as mobile phase (MeOH: H ₂ O 10:90 to 100: 0) Powder was produced, and the powder was compound 1 (SK-PC1) (Table 4).

Table 4 pandanus fruit Structure and estrogenic potency of a single active ingredient present in the sera Soy Death

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

Structural analysis of isolated single substances was carried out by spectroscopic methods such as NMR and HRESIMS for chemical structure identification, and the concentration - dependent ERE activity of these monomers was evaluated. The single component, SK-PC1 (I), which shows the pharmacological activity finally, was isolated and identified. The single component of one kind is shown in Table 3. The single component SK-PC1 isolated from prunus cerasoids (Fig. 6). It is the first invention of the present invention to find Compound I as a single component showing female hormone resistance from Furunus cerassoides plants (Table 4).

(1) Compound (I)

Prunetinoside

Chemical Formula: C ₂₂ H ₂₂ O ₁₀ (MW 446.4041)

^{1 H NMR (400 MHz, CD} 3 OD): δ 8.05 (1H, s, H-2), 7.32 (2H, d, J = 8.4 Hz, H-2 ', 6'), 6.87 (2H, d, J = 8.4 Hz, H-3 ', 5'), 6.92 (1H, d, J = 2.3 Hz, H-6), 6.77 (1H, d, J = 2.3 Hz, H-8), 4.87 (1H, m, glc-1, 3.48 ( 2H, t, J = 12.5, 9.2 Hz, glc-5, 3), 3.58 (1H, t, J = 17.0, 9.3 Hz, glc-2), 3.40 (1H, t, J = 18.3, 9.2 Hz, gCl-4, 3.92 (1H, m, gCl-6), 3.72 (1H, dd, J = 12.1, 5.9 Hz, .

¹³ C (100 MHz, CD ₃ OD):? 153.3 (C-2), 126.9 (C-3), 177.6 (C-4), 160.6 C-7), 105.2 (C-8), 160.3 (C-9), 111.3 (C-10), 124.0 ), 158.7 (C-4 '), 103,4 (glc-1), 77.2 (glc-5), 75.9 (glc- , 61.2 (gLC-6), 55.2 (-OMe) ppm.

[ Example ]

Formulation example  One. Sanje  Produce

Compound (I) 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 (I) 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 (I) 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 (I) 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 (I) 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 then purified water was added thereto to adjust the total volume to 100 ml. The resulting solution was filled in a brown bottle and sterilized to prepare a liquid preparation do.

The Prunus cerasoidus extract of the present invention derived from natural products, its active fractions and female hormone substances 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 (8)

A pharmaceutical composition for the treatment and / or prophylaxis of female cancer and / or pancreatic cancer, which comprises, as an active ingredient, at least one selected from the group consisting of a Prunus cerassoide sprueus extract, an active fraction thereof, a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof: A pharmaceutical composition for the prevention and treatment of menopausal symptoms.
(I)

The pharmaceutical composition according to claim 1, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer. The pharmaceutical composition according to claim 1, wherein the menopausal symptom includes any one of facial flushing, hyperlipidemia, brain mental dysfunction, osteoporosis, venous thrombosis and atrophic vaginitis. A pharmaceutical composition for the treatment and / or prophylaxis of female cancer and / or pancreatic cancer, which comprises, as an active ingredient, at least one selected from the group consisting of a Prunus cerassoide sprueus extract, an active fraction thereof, a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof: Food compositions for the prevention and improvement of menopausal symptoms.
(I)

5. The food composition according to claim 4, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer. The food composition according to claim 4, wherein the menopausal symptom includes any one of facial flushing, hyperlipidemia, brain mental dysfunction, osteoporosis, venous thrombosis and atrophic vaginitis. The food composition according to claim 4, wherein the food is a health functional food having any one of tablet, capsule, powder, granule, liquid, and ring. The food composition according to claim 4, 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.

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