WO2017065514A1 - Composition for preventing or treating gynecological cancers and menopausal symptoms containing prunus cerasoides extract or compound isolated therefrom as active ingredient - Google Patents

Abstract

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

Description

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

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

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

Endometrial cancer

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

Breast cancer

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

Menopause syndrome

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

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

On the other hand, pomegranate extract, soybean extract and isoflavone formulations, evening primrose oil, etc. are used as health foods for improving women's menopausal symptoms and preventing / treating osteoporosis. These products, made from vegetable raw materials, are rapidly expanding in the market, giving consumers a sense of safety. However, there is no scientific basis for the recognition that it is safe because it is vegetable, and these products are composed of complex extracts composed of various chemical components, and many of the dietary supplements have not been subjected to rigorous toxicological tests, so their safety cannot be proved. . The side effects of these products are actually impossible to monitor in government. In addition, there is a lack of scientific basis for pharmacological efficacy in its efficacy, and it is difficult to judge whether it complies with strict and precise quality control regulations. Therefore, there is a need for a safer and more potent natural material derived from the existing plant extracts.

Prunus cerasoides cerasoides ) is a plant of the Rosaceae Prunus and is called wild Himalayan cherry or sour cherry. It is known to inhabit the Himalayan Mountains, Southwestern China, Burma Province, Thailand from the Himachal Pradesh province of northern India. Aqueous extracts of the branches of the Prunus cerasoides plant have been reported to be used in India to prevent abortion (Kirtkar, KR et al. , 1975). Leaves and bark of the Prunus cerasoides plant have been used for coughing, asthma, indigestion and diarrhea, and 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 agent for digestive and gastric ulcers, and heartwood is known to be useful for pitta , burning sensations, sprains, and skin bleaching (Vaidyaratnam, PS et al ., 1995). To date, it has been found that Prunus cerasoides contains flavonoids, steroids, and terpene. It contains glucogenkwanin as a flavone family and naringenin as a flavanone. Isoflavonoids contain genistein and prunetin. As a steroid and a terpene, it contains beta-sitosterol and ursoric acid. As a study on the components identified from Prunus cerasoides , studies on naringenin, prunetin, beta-sitosterol, and ursoric acid have. Naringenin has been reported to act as an estrogen agonist in the absence of estrogen in breast cancer cell lines and as an antagonist in high estrogen levels (Kim S et al ., 2013). Prunetin (prunetin) has been reported to show a concentration-dependent action in the Syrian hamster embryo (SHE) cell model (Tsutsui T et al ., 2003). Beta sitosterol (β-sitosterol) It has been reported to reduce the risk of ER-negative tumors and to reduce the risk of ER-positive breast cancer in postmenopausal women (Touillaud MS et al ., 2005). Ursoric acid has been shown to increase p53 gene expression, inhibit the growth of breast cancer cells and inhibit the growth of MCF-7 breast cancer cells that exhibit tamoxifen resistance (Gu G et al ., 2012). .

[Preceding technical literature]

[Non-Patent Documents]

Kirtkar, K. R .; Basu, B.D. Indian Medicinal Plants, M / s Periodicals, New Delhi, 1975, 2, 959.

Chopra, RN; Handa, KL; Kapoor, LD; 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. 2013 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, Ando S .; 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 new natural medicine and health functional food for the prevention and treatment of female cancer and menopausal symptoms. In particular, the present invention Prunus cerasoides ( Prunus ) showing the estrogen activity (estrogen) cerasoides) identified by separating the extract, fractions and single active ingredients separated therefrom, to provide a new women's natural health medicines and functional foods for the prevention and treatment of cancer and menopausal symptoms for the purpose.

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

In the present invention, by identifying the efficacy, pharmacological metabolism and safety of Prunus cerasoides extract, its active fraction, the active ingredient isolated therefrom, it is effective in the prevention and treatment of female cancers such as endometrial cancer, breast cancer and menopausal symptoms New pharmaceutical compositions or functional food compositions are provided.

Specifically, in the present invention,

Female cancer, containing any one or more selected from the group consisting of Prunus cerasoides prunus extract, its active fraction, a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof Provided are pharmaceutical compositions for the prevention and treatment of menopausal symptoms.

In the present invention,

Female cancer and menopause, containing any one or more selected from the group consisting of Prunus cerasoides prunus extract, its active fraction, the compound represented by the following formula (I), and a food acceptable salt thereof Functional food compositions for the prevention and amelioration of symptoms are provided.

(I)

Prunus cerasoides It was first discovered in the present invention that a single component of formula (I) derived from cerasoides ) extract exhibits female hormones.

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

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

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

The present invention, Prunus cerasoides By separating and identifying active fractions and single components showing female hormones from plants, the present invention provides new plant metabolism-derived single-component drugs and health functional foods useful for the prevention and treatment of female cancer and menopausal symptoms. In particular, the present invention is the first confirmation that Prunus cerasoides and a single component derived therefrom exhibit female hormones, and the pharmaceutical composition of the present invention containing such components occurs most frequently among female cancers. It can be useful for breast cancer, which has an increased incidence, and endometrial cancer, which has a similar pathogenesis.

1 shows estrogen (●) and Prunus cerasides (◆) for pure recombinant human estrogen receptor alpha (hERα) binding of tritium-labeled estrogen ([ ³ H] estrogen). ) Shows the effect of the extract.

2 is Prunus ER subtype selective gene transcriptional activity of extracts of cerasoides (PCE T) and 21 fractions (PCE-21) were measured. Prunus Among the extracts of cerasoides (PCE T) and 21 fractions (PCE-21), 4 fractions (PCE 6, 15, 17, 19) showed an ERβ / ERα ratio of 2 or more.

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 was confirmed. Prunus cerasoides extract induced cell proliferation at concentrations of 10 ^-6 g / ml, 5x10 ^-6 g / ml, and 10 ^-5 g / ml.

Figure 4 shows the transcriptional effect of the vehicle, estrogen, prunus cerosaides extract in MCF-7 cells transfected with the estrogen responsive element (ERE).

Figure 5 shows the transcriptional effects of vehicles, estrogens, prunus cerasoides fractions in MCF-7 cells transfected with the estrogen responsive element (ERE).

FIG. 6 shows the results of analyzing the activity of ERE gene of a single substance isolated from Prunus cerosaides in MCF-7 cells transfected with Estrogen Responsive Element (ERE).

Figure 7 shows the increase in ERE gene-induced pS2 gene expression of prunus cerosaides extract in MCF-7 cells transfected with the estrogen responsive element (ERE).

Figure 8 shows the change in pS2 gene activity in the uterine tissue of rats when Prunus cerasoides administration to rats.

Figure 9 shows the changes in protein expression of estrogen receptor alpha (ERα), progesterone receptor A (PRA), progesterone receptor B (PRB) in rat uterine tissues when Prunus cerosaides extract is administered to rats.

Figure 10 evaluates the effect of Prunus cerasoides extract on neuroglobin (Ngb) promoter activity of human neurons SKNSH. When estrogen, genistein and prunus cerosaides extracts were treated as vehicle and positive controls, the activity of Ngb promoter was increased by positive control and prunus cerosaides extract.

Figure 11 evaluates the effect of Prunus cerosaides extract on Ngb mRNA expression of human neurons SKNSH. When estrogen, genistein, and prunus cerosaides extracts were treated as vehicle and positive control, Ngb gene expression was increased by positive control and prunus cerosaides extract.

Figure 12 evaluates the effect of Prunus cerosaides extract on the Ngb promoter activity of mouse neurons N2a. When estrogen, genistein and prunus cerosaides extracts were treated as vehicle and positive controls, the activity of Ngb promoter was increased by positive control and prunus cerosaides extract.

FIG. 13 analyzed Ngb protein changes by Prunus cerosaides extract in the brains of female rats whose ovaries were excised. Prunus serra when divided into Sham group, ovarian ablation group (OVX), E2 administration group after ovarian resection (OVX + E2), Prunus cerasoides extract administration group (OVX + PC 250, or PC 500) after ovarian resection Rat brain Ngb protein expression in the group treated with Soides extract was found to increase concentration-dependently.

FIG. 14 shows the results of experiments of the effects of Prunus cerasoides extract on the levels of ALP, AST and ALP, which are representative enzymes of liver function, in female rats from which ovaries were removed. Compared with the vehicle group, the administration group showed improvement in liver count.

FIG. 15 shows the results of experiments of the effects of Prunus cerasoides on blood lipid profile changes in female rats from which ovaries were removed. Prunus cerosaides has been shown to reduce triglycerides compared to the Vehicle group.

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

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

In the present invention, "decrease in brain mental function" is meant to include all the depression, memory loss and cognitive decline that appear in menopausal women.

Prunus cerasoides in the present invention ( Prunus) A single component exhibiting feminine hormones isolated from cerasoides ) is represented by the formula (I) below.

(Ⅰ)

Chemical Name: Prunetinoside

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

Prunus cerasoides is a plant of the Rosaceae Apricot (Prunus), Prunus cerasoides used in the experiments in the present invention is sold from the Center for Overseas Biomaterials .

Prunus cerasoides in the present invention ( Prunus) cerasoides ) extracts and subfraction fractions are identified and the process of separating and identifying the components of (I ) from Prunus cerasoides effective for the prevention and treatment of female cancer and menopausal symptoms is as follows.

One. Prunus Cerasoides ( Prunus cerasoides )  Female Hormonal Pharmacological Effectiveness Test of Extracts

Prunus cerasoides cerasoides ) competitive inhibition of human estrogen receptor alpha (hERα) binding, MCF-7 intracellular transcription analysis through MCF-7 estrogen-responsive genes, uterine proliferation effect, pS2 gene activity in uterine tissues and estrogen receptor alpha (ERα) ), Protein expression confirmation experiments of progesterone receptor A (PRA) and progesterone receptor B (PRB) were performed.

2. Prunus Cerasoides  Of extract Subdivision  And Separation of Active Ingredients and Confirmation of Pharmacological Activity

Prunus cerasoides The pharmacological activity of the cerasoides ) extract and the active ingredient were examined and identified 21 subfractions. Among them, one compound showed excellent pharmacological activity. The compound represented by the above formula (I) is the first confirmed 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 comprises any one or more selected from the group consisting of a compound represented by the 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 commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical composition of the present invention may further include other pharmaceutically active ingredients or active mixtures.

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

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

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

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

Functional food composition for the prevention and improvement of female cancer and menopausal symptoms of the present invention comprises any one or more selected from the group consisting of compounds represented by the formula (I) and their food acceptable salts as an active ingredient.

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

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

The functional food composition of the present invention is not essential to any other ingredients except the above-mentioned (I) component as essential ingredients, and may further contain various ingredients such as various flavors or natural carbohydrates, such as ordinary foods and beverages. Can be. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

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

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

[ Experimental Example ]

Experiment method

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

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

2. Gene reporter evaluation test: Estrogen (ERE) Reactivity  Gene transcription experiment

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

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

The uterine growth test is a method of indirectly evaluating female hormone (estrogenicity) by measuring the increase in uterine tissue mass (induced by estrogen). The effect of uterine proliferation was measured by examining the effect of the test substance on the immature uterus when subcutaneous injection was administered to female rats at 21 days of age for 3 days. Immature female rats were used for the experiment. During the acclimatization period, the subjects were selected to be close to the average body weight, and grouped by random method. Individual identification of animals was done by tail marking and tag by breeding box. In the positive control group, estrogen (E2) was uniformly suspended in corn oil to 3 μg / kg, and then diluted in corn oil stepwise, and the test substance was also suspended in corn oil. The experimental group was divided into five groups, each group containing vehicle (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), prunus cerasoides (100, 200, 300 mg / kg) extract, prunus cerasoy Co-administration of death extract (300 mg / kg) and estrogen (estrogen, E2) was administered by subcutaneous injection at 24 hour intervals for 3 days. The dosage was 0.05 ml per 10 g body weight of immature rats, and solution preparation of the substance was performed on the day of administration. The test items to be tested in this test are weight and uterine weight. Body weight is measured for all animals immediately before administration and just before autopsy, and uterine weight is sacrificed by cervical dislocation 25 days after birth about 24 hours after the last administration, and the uterus is carefully removed to remove fat and fibrous tissue. After thorough drying of the water on the room, the weight of the uterus obtained from each rat was accurately measured using a micro balance (Mettler microbalance).

4. The vehicle effects evaluated 21 days command immature female rats for intracellular hormone intrinsic transcriptional activity of target genes responsive, estrogen (estrogen, E2), fruit Taunus sera Soy gave the des vehicle group for each group (corn oil 5ml / kg ), Estrogen tradiol (0.003 mg / kg), Prunus cerasoides (100, 200 mg / kg) extract subcutaneously injected for 3 days at a 24-hour interval, trisol ( MRNA was extracted using Trizol). cDNA was obtained by reverse transcription of a predetermined amount of mRNA (1 μg) using an iScript cDNA synthesis kit (Bio-Rad). The expression levels of the genes were quantitatively measured by real-time PCR using cDNA, a pair of primers capable of recognizing the cDNA of the target gene, and PCR SYBR green kit (Qiagen) reagent. In order to make up for the technical mistakes in the various stages of the reaction, the expression level of GAPDH, a housekeeping gene, was simultaneously measured, and the ratio between the expression level of the gene and the expression level of the target gene was used for the final quantitative analysis. Primers that recognize GAPDH include forward 5'-CTCTCTGCTCCTCCTGTTCGAC; And reverse 5'-TGAGCGATGTGGCTCGGCT.

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

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

The 21-day-old immature female rats were divided into vehicle, estrogen, and prunus cerosaides groups, and each group was assigned to vehicle (corn oil 5ml / kg), estrogen (estrogen, 0.003 mg / kg), and prunus cerasoides ( 100, 300 mg / kg) extract, Prunus cerasoides extract and estrogen (estrogen, E2) were administered by subcutaneous injection at 3 hours and 24 hours intervals. Extracted. The extracted protein was quantified by BCA method, and electrophoresed at 7.5% SDS-PAGE gel at 20 μg for 2 hours at 80V. After electrophoresis, transfer was made to PVDF membrane at 25 Volt for 3 hours, then blocked with RT for 1 hour at RT and reacted with primary and secondary antibodies to detect ECL solution. At this time, the primary antibody was diluted 1: 500 ERα (sc-7207, SANTA CRUZ BIOTECHNOLOGY, INC.), PR (sc-538, SANTA CRUZ BIOTECHNOLOGY, INC.) In a ratio of 1.500. Β-actin was used to correct the difference in protein amount due to experimental error.

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

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

(2) Ngb mRNA level (quantitative PCR ) : N2a cells and SKNSH cells were cultured in 10% FBS DMEM, and the media were dissolved by dissolving the drug in phenol red free DMEM. Treated with cells (DMSO, 0.1%), 17β-ES (1nM), genistein (1μM) as a control, the extract (10 ^-4 ~ 10 ^-7 g / ml) to the cells and incubated with trizol after 24 hours Isolate from the vessel surface and destroy the cells. Isolate mRNA in cells using Qiagen RNeasy mini kit. cDNA was obtained by reverse transcription of a predetermined amount of mRNA (1 μg) using an iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using a pair of primers capable of recognizing cDNA and cDNA of a target gene and a PCR SYBR green kit (Qiagen) reagent to quantitatively measure the expression level of the gene. In order to make up for the technical mistakes in the various stages of response, the expression level of GAPDH, which is a housekeeping gene, was simultaneously measured, and the ratio between the expression level of the gene and the expression level of the target gene was used for the final quantitative analysis. Primers that recognize human GAPDH include forward 5'-GGCTGAGAACGGGAAGCTTGTCAT; And reverse 5'-CAGCCTTCTCCATGGTGGTGAAGA. Primers that recognize Mouse GAPDH include forward 5'-TGCCAAGTATGATGACATCAAGAA; And reverse 5'-GCCCAAGATGCCCTTCAGT. Primers that recognize human Ngb are forward 5'-TGGAAGACCTGTCCTTCACTG; And reverse 5'-GAGCAGAGACTCACCCACTG. Primers that recognize Mouse Ngb are forward 5'-TACAATGGCCGCCAGTTCT; And reverse 5'-TGGTCACTGCAGCATCA. Quantitative PCR was performed in about 40 cycles (95˚C; 15 seconds, 60˚C; 60 seconds). Each gene expression curve was expressed in logarithm, and then the threshold cycle (C _T ) was mathematically obtained. The value obtained by substituting this value into the 2 ^-ΔΔCT expression represents the relative expression level of the specific gene. The expression level of the specific gene divided by the expression level of the house keeping gene was selected as the final value for the gene expression level, and compared with that of the vehicel group and the test substance administration group.

(3) Ngb protein expression in OVX rat brain: In order to confirm the brain protective effect of the selected plant extracts, the expression of Ngb prtein was confirmed in vivo . Rats were used as rats and two weeks of recovery were performed after ovariectomy to rule out endogenous hormonal action. After that, for 6 weeks, the extract was administered at a dose of 250 mg / kg and 500 mg / kg daily. Treatment groups consisted of sham operate group, OVX group, OVX + 17β-ES group, OVX + 250mg / kg group, OVX + 500mg / kg group. After 6 weeks, lysate was obtained from brain tissue and loaded with 70μg protein on 12% SDS-PAGE gel, which was run at 80V for 1.5 hours and transferred to PVDF film at 25V for 3 hours. The expression of Ngb (ab37258, abcam) ERα (sc7207, SANTA CRUZ) was confirmed using ECL solution.

(4) Ngb mRNA level in OVX rat brain : In order to confirm the protective effect of the selected plant extracts, the expression of Ngb mRNA was confirmed in vivo . The brain tissue was trizol treated to destroy the brain tissue with a homogenizor. MRNA in brain tissue was isolated using Qiagen RNeasy mini kit. cDNA was obtained by reverse transcription of a predetermined amount of mRNA (1 μg) using an iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using a pair of primers capable of recognizing cDNA and cDNA of a target gene and a PCR SYBR green kit (Qiagen) reagent to quantitatively measure the expression level of the gene. In order to make up for the technical mistakes in the various stages of response, the expression level of GAPDH, which is a housekeeping gene, was simultaneously measured. Primers that recognize Rat GAPDH include forward 5'-GGCTGAGAACGGGAAGCTTGTCAT; And reverse 5'-CAGCCTTCTCCATGGTGGTGAAGA. Primers that recognize Rat Ngb include forward 5'-TGGAAGACCTGTCCTTCACTG; And reverse 5'-GAGCAGAGACTCACCCACTG. Quantitative PCR was performed in about 40 cycles (95˚C; 15 seconds, 60˚C; 60 seconds). Each gene expression curve was expressed in logarithm, and then the threshold cycle (C _T ) was mathematically obtained. This value is 2 ^{- ΔΔCT} The value obtained by substituting the equation indicated the relative expression level of the specific gene. The expression level of the specific gene divided by the expression level of the house keeping gene was selected as the final value for the gene expression level, and compared with that of the OVX group and the test substance administration group.

7. In vivo blood metabolism Marker  Of the effects on body weight gain

Plant extract ( Prunus cerasoides ) were orally administered to ovarian isolated rats to investigate their effects on blood metabolic markers and weight gain. Eight-week-old SD rats (females) were purchased and allowed to acclimate for one week. Rats were anesthetized with isoflurane, and anesthesia was excised to remove the ovaries. At the end of ovarian extraction, three animals were placed in cages for two weeks of recovery and blood estrogen wash-out and administered with a phytoestrogen-limited diet (Harlan 2020X Teklad Global Soy Protein-Free Extruded Rodent Diet) to avoid affecting experimental results. It was.

Rats undergoing recovery were divided into Sham (no OVX), OVX control, E2 administration, low concentration extract selection group, and high concentration selection material administration group.

For 6 weeks, Sham (no OVX) and OVX control group prepared feed, E2 administered group E2 0.5 mg / kg, low concentration selective extract group, 250 mg / kg plant extract, high concentration selective extract group, 500 mg plant extract. A formulated feed containing / kg was distributed. At this time, the distribution of 20-25g of food per rat so that the quantitative components can be provided, and the weight of the rat was measured every week to observe the weight change of the rat. After 6 weeks of oral administration, fasted for 24 hours, anesthetized with isoflurane, and collected blood samples from 4 to 5 ml from the heart.The blood samples were centrifuged at 3500 rpm (15 minutes, 4 ° C). Plasma samples were obtained, stored at 4 ° C. and requested to analytical institutions within 1-2 days to analyze lipid profiles in blood.

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

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

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

9. Acute Toxicity Test

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

10. Analysis of active ingredients for plant extract fractions

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

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

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

(3) Detailed separation and purification of single-component fractions of active fractions: The fractions containing purified plant metabolites can be subdivided and purified by obtaining multiple fractions through MPLC, HPLC, and LC-MS analysis on selected active fractions. Was carried out.

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

result

One. Prunus Cerasoides  Extract and Subdivision  Female hormone pharmacological efficacy

end. For ER binding Prunus Cerasoides  Competitive inhibition of extracts ERβ selectivity

Competitive binding of Prunus cerasoides extract and tritium estrogen ([ ³ H] E2) to pure recombinant human estrogen receptor alpha (hERα) was measured in an in vitro system. The results are shown in FIG. 1 and Table 1. In a competitive binding assay, the IC ₅₀ of estrogen (E2) (•) was 3.83 × 10 ⁻¹⁰ g / mL (hERα), respectively. The IC ₅₀ of Prunus cerasoides (◆), a kind of phytoestrogen, was 1.41 × 10 ⁻⁵ g / mL (hERα). These results confirm that Prunus cerosaides extract acts concentration-dependently on tritium estrogen ([ ³ H] E2) separation and binding. Relative bioavailability (RBA) compared to endogenous estrogen. Was found to exhibit about 50000 times lower potency (0.0027% for hERα) (FIG. 1, Table 1).

Prunus The ER subtype selective gene transcriptional activity was measured for 10 mg / ml samples after dissolving cerasoides (PCE T) and 21 fractions (PCE-21) in DMSO or water. When the relative comparison of the luciferase activity compared to the DMSO by the relative value, the relative comparison results of the luciferase activity of each sample is shown in FIG. Prunus Among the extracts of cerasoides (PCE T) and 21 fractions (PCE-21), four fractions (PCE 6, 15, 17, 19) showed an ERβ / ERα ratio of 2 or more (FIG. 2) .

(Table 1) IC ₅₀ and RBA values for hERα of Prunus cerosaides extract

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

I. Prunus Cerasoides MCF Through -7 estrogen-responsive genes MCF -7 cell proliferation and Intracellular  Transcription analysis

Prunus to evaluate MCF-7 cell cell proliferation After treating cerasoides extract at a concentration of 10 ^-6 g / ml to 5x10 ^-5 g / ml, MCF7 cell proliferation was confirmed. In the case of 17β-ES used as a positive control, MCF7 cells proliferated more than two times compared to the vehicle group, and genistein, one of the representative phytoestrogens, increased the proliferation of MCF7 cells by about 1.8 times. Prunus The cerasoides extract was found to induce cell proliferation at concentrations of 10 ⁻⁶ g / ml, 5 × 10 ⁻⁶ g / ml, and 10 ⁻⁵ g / ml (FIG. 3).

Prunus cerasoides in MCF-7 cells transfected with Estrogen responsive element (ERE) Whether the extract changes the activity of the ERE-gene and the results are shown in FIG. When treated with Prunus cerasoides extract alone, ERE gene activity was increased by 5 ~ 30 times compared to vehicle at the concentration of 2.5 ~ 20μg / mL, and estrogen (estrogen, E2) at 10μg / mL and 20μg / mL It showed higher gene activity, indicating that Prunus cerosaides induces ERE gene activity. Based on the results of this experiment, Prunus cerasoides extract induces ERE gene activity in a concentration-dependent manner at a concentration of 2.5 μg / ml or higher and ERE gene higher than estrogen (E2) at a concentration of 10 μg / ml or higher. It was confirmed to exhibit activity. Prunus cerasoides also Three fractions were performed with the ERE gene reporter experiment. Prunus cerasoides Four fractions were tested at a concentration of 20 μg / ml, resulting in up to 1.9-fold increase in transcriptional activity through ERE compared to the activity of estrogen (fraction number: PC-16, 17, 18, 20). Could be confirmed (FIG. 5) . In addition, the concentration-dependent ERE transcriptional activity of a single component isolated from Prunus cerasoides was confirmed (FIG. 6).

All. Prunus Cerasoides  Uterine growth effect

In order to evaluate the female hormone properties of the extract in the system excluding the action of endogenous estrogen (E2), uterine proliferation was tested in 21-day-old female rats with very low estrogen (estrogen, E2) secretion. Uterine growth results were compared and evaluated as shown in Table 2 below. Uterine weight ratio (relative to body weight) was 0.7 when corn oil was administered to the vehicle group, and the uterine weight ratio was 1.8 as a positive control after subcutaneous injection of estrogen (E2) (3 μg / kg). Compared to that, the uterine rate was increased about 2.5 times. The uterine weight ratio was 0.61 when subcutaneous injection of Prunus cerosaides extract was 100 mg / kg in immature rats and 0.53 when 200 mg / kg was administered. The uterine weight ratio was 0.68 when the drug was administered at 300 mg / kg and 2.34 when the estrogen (estrogen, E2) was used in combination with Prunus cerosaides at 300 mg / kg. When treated with Prunus cerosaides extract alone, the weight of the uterine tissues was similar to that of the vehicle group compared to the vehicle group administered with corn oil alone. On the other hand, when the Prunus cerasoides extract and estrogen (estrogen, E2) were administered together, the weight of the uterus was increased compared to the administration of estrogen (estrogen, E2) alone. This uterine proliferation experiment confirms that Prunus cerasoides extract alone does not induce uterine tissue proliferation, but when it works together with estrogen (Etrogen), it further increases uterine proliferation by estrogen (Etrogen). Could. Weight gain in rats was not observed during drug administration. Significantly different values for vehicle groups were marked with *** (*** P <0.001).

Table 2 Prunus Immature rat uterine proliferation by Serra soy extracts Death

la. Prunus Cerasoides  Estrogen Reactivity pS2  Gene activity induction

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. In the case of 17β-ES used as a positive control, the expression of pS2 gene was increased by 2.5 times compared with the vehicle group, and genistein also increased the expression of pS2 gene by more than 2.5 times. In the case of Prunus cerasoides extract, the expression of pS2 gene was slightly increased at the concentration of 10 ^-5 g / ml (FIG. 7) .

21-day-old immature female rats were subcutaneously injected with vehicle (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg) and Prunus cerosaides (100mg / kg, 200mg / kg) for 3 days. PS2 gene expression was confirmed in uterine tissues of rats obtained after administration at 24 hour intervals. As a result of the experiment, when the estrogen (estrogen, E2) was administered compared with the vehicle group, the expression of the pS2 gene was confirmed to be increased by 1.9 times, and when the Prunus cerosaides extract was administered, it was confirmed that the 1.4-fold was increased by 100 mg / kg. On the other hand, when administered at 200mg / kg was confirmed that there is almost no difference by about 0.9 times compared to the vehicle group. These results confirmed the estrogen activity in the uterine tissues when Prunus cerosaides extract was administered at a dose of 100 mg / kg, and the expression level of pS2 gene rather than the positive control estrogen (Etrogen). Was lower (FIG. 8).

hemp. Prunus Cerasoides  In uterine tissue ERα  Inhibit protein expression

In 21-day-old immature female rats, vehicle (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), prunus cerosaides (100 mg / kg, 300 mg / kg) extract, prunus cerosaides (300 mg / kg) and estrogen receptor alpha (ERα), progesterone receptor A (PRA) and progesterone receptor B (PRB) proteins in rat uterine tissue Expression change was confirmed. Experimental results show that ERα protein expression decreases when estrogen is administered as compared to vehicle and Prunus cerosaides When 100 mg / kg of the extract was administered, the protein expression of ERα was expressed at the vehicle level (FIG. 9). Prunus cerasoides Administration of 300 mg / kg of extract showed a slight decrease compared to the protein expression vehicle group of ERα and Prunus cerosaides When 300 mg / kg of the extract was administered in combination with E2, the protein expression of ERα was confirmed to decrease to a similar level as the estrogen-administered group. PRA and PRB, on the other hand, were increased with estrogen and Prunus cerosaides Administration of 100 mg / kg of extract increased both PRA and PRB. However, the estrogen receptor and PRA / B protein expression in uterine tissues of Prunus cerasoides extract was not dose dependent (FIG. 9).

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

Prunus cerasoides extracts were treated with SKNSH cells at concentrations of 5x10 ^-6 g / ml ~ 5x10 ^-5 g / ml and then subjected to luciferase assay to determine Ngb promoter activity by extracts. It was evaluated quantitatively. In treatment with positive control genistein, Ngb promoter activity was increased by 1.4-fold compared to vehicle group, and 17β-ES also increased Ngb promoter activity by 1.3-fold. Treatment with Prunus cerasoides extract significantly increased Ngb promoter activity by 1.4-1.6 fold at all concentrations (*** p <0.001) (FIG. 10). After treatment with Prunus cerasoides extract at a concentration of 5x10 ^-6 g / ml ~ 10 ^-5 g / ml, changes in Ngb mRNA gene expression of SKNSH cells were confirmed. Genistein used as a positive control increased the expression of Ngb gene by 1.8 times compared with vehicle group, and 17β-ES also increased the expression of Ngb gene by 1.2 times. In the case of Prunus cerasoides extract, the expression of Ngb gene was increased about 1.5-fold at a concentration of 5 × 10 ⁻⁶ g / ml (FIG. 11).

Prunus cerasoides extract was treated with N2a cells at concentrations of 5x10 ^-6 g / ml ~ 5x10 ^-5 g / ml and then luciferase assay was performed to evaluate the effect on Ngb promoter activity of mouse neuron N2a cells. Quantitative evaluation of promoter activity was made. Treatment with the positive control genistein increased the activity of the Ngb promoter by 1.7 times compared to the vehicle group, and 17β-ES did not increase the Ngb promoter activity. If haejun handle Prunus cerasoides extract 5x10 ^{- 5} g / ml a statistically significant at a concentration Ngb the promoter activity increased about 1.4-fold (** p <0.01) (Fig. 12).

Ngb protein expression in OVX rat brain: To determine whether Prunus cerasoides extracts show brain protection in postmenopausal women, actomizes the ovary of female rats to create a hormone environment similar to postmenopausal women. Administration of Ngb and ERα expression in the brain was confirmed at the protein level. Compared to the Sham operate group, ERα and Ngb were increased in OVX group, but ERα and Ngb were increased by 17β-ES (E2). In the group treated with Prunus cerasoides extract, the expression of Ngb and ERα was decreased and Ngb increase was concentration-dependent (FIG. 13).

four. Biological blood metabolism Marker  Of the effects on body weight gain

Prunus Plant extracts of cerasoides were orally administered to ovarian-extracted rats to investigate their effects on blood metabolic markers and weight gain. 8-week-old SD rats (females) undergoing postoperative recovery and blood estrogen wash-out after 6 weeks of ovarian extraction were treated with Sham (no OVX) and OVX control groups for 6 weeks. 0.5 mg / kg, low concentration selective material extract administration group was administered 250 mg / kg of plant extract, high concentration selective material extract administration group was formulated feed formulation containing 500 mg / kg. As a representative liver enzyme, Prunu s calculates the levels of ALP, AST and ALP. cerasoides Prunu s compared with control group and 500 mg / kg extract cerasoides The ALP, AST, and ALP levels were reduced in the group administered with 500 mg / kg of the extract, similar to the Sham and E2 groups. Thus Prunus cerasoides The extract was found to be effective in improving liver count to some extent (FIG. 14). After 6 weeks of oral administration, fasted for 24 hours, blood samples were collected from 4 to 5 ml, plasma samples were obtained, and lipid profiles were analyzed. Prunus for Triglycerides when Lipid Profile and Glucose Changes were Measured cerasoides When the extracts were compared with the Ovx group and the 250 mg / kg, 500 mg / kg group, the triglycerides were reduced to the same level as the Sham and E2 groups. Thus Prunus cerasoides Extract is effective in improving triglyceride levels in the body. However, for the remaining values, nothing showed a significant effect (FIG. 15).

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

Prunus The anti-inflammatory activity of the cerasoides extracts (PCE) and one single substance (PCE20-10-po) isolated from the fractions of the extracts was evaluated through the inhibitory effect of human hematopoietic prostaglandin D synthase (HPDGs). Prunus cerasoides The HPGDs inhibitory effect of the extract (PCE T) was 70.92%, which was very high. The HPGDs inhibitory effect of one single component (PCE20-10-po) was 1.01%, indicating no HPGDs inhibition effect.

character. Through acute toxicity test Safety evaluation

Prunus To evaluate the biotoxicity of cerasoides extract, a single dose acute toxicity test was performed. When female mice (10 weeks old) were dosed with vehicle (1% CMC saline), Prunus cerasoides extract 250 mg / kg, 500 mg / kg, 1000 mg / kg, at 2 weeks of mortality, weight change and behavior, Normal behavior was observed and feed and water intake were normal. The mortality rate was 0% in all groups, including the vehicle group, during the two-week experiment. The maximum dose of 1000 mg / kg oral dose did not show acute toxicity, so it was evaluated as a high safety candidate plant (Table 3).

2. Purunus Cerasoides  Bioassay-guided analysis and evaluation of its bioactivity

end. Fraction and Purification of Extracts

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) A total of 21 fractions (PC1 to PC21) were obtained by silica gel open column chromatography. As a result of evaluating the binding force of ER and PR of these fractions, ERE gene transcription activity was superior to that of estrogen in fractions PC15, 16, 17, 18 and 20. An attempt was made to find a single substance with activity.

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

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

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

For the chemical structure of the isolated single substances, structural analysis was performed by spectroscopic methods such as NMR and HRESIMS, and the concentration-dependent ERE activity of the single components was evaluated. Finally, one single component (Compound SK-PC1 (I)) showing effective pharmacological activity was isolated and identified. One single component is shown in Table 3. Concentration of single component SK-PC1 isolated from Prunus cerosaides It was confirmed that it exhibits ERE transcriptional activity in dependence (Fig. 6) The present invention is the first time that Compound I was discovered from a Prunus cerosaides plant as a single component exhibiting female hormone activity (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, glc-4, 3.92 (1H, m, glc-6), 3.72 (1H, dd, J = 12.1, 5.9 Hz, glc-6), 3.90 (3H, s, -OMe) ppm .

¹³ C NMR (100 MHz, CD ₃ OD): δ 153.3 (C-2), 126.9 (C-3), 177.6 (C-4), 160.6 (C-5), 97.2 (C-6), 165.6 ( C-7), 105.2 (C-8), 160.3 (C-9), 111.3 (C-10), 124.0 (C-1 '), 131.8 (C-2', 6 '), 116.2 (C-3 ', 5'), 158.7 (C-4 '), 103,4 (glc-1), 77.2 (glc-5), 75.9 (glc-3), 73.3 (glc-2), 69.9 (glc-4) , 61.2 (glc-6), 55.2 (-OMe) ppm.

[ Example ]

Formulation example  One. Powder  Produce

Compound (I) 1 mg

Lactose 100 mg

Talc 10 mg

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

Formulation example  2. Preparation of Tablets

Compound (I) 1 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  3. Manufacture of capsule

Compound (I) 1 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation example  4. Preparation of Injectables

Compound (I) 1 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

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

Formulation example  5. Liquid  Produce

Compound (I) 1 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, and lemon flavor is added thereto. do.

Prunus cerasoides extract of the present invention derived from natural products, active fractions thereof, and female hormone substances thereof can be used in the fields of medicines and health functional foods as effective substances for the treatment and prevention of female cancer and menopausal symptoms.

Claims (9)

Female cancer, containing any one or more selected from the group consisting of Prunus cerasoides prunus extract, its active fraction, a compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof Pharmaceutical composition for the prevention and treatment of menopausal symptoms. (Ⅰ)

The pharmaceutical composition of claim 1, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer. The pharmaceutical composition of claim 1, wherein the menopausal symptoms include any one of hot flashes, hyperlipidemia, decreased brain mental function, osteoporosis, venous thrombosis, and atrophic vaginitis. Female cancer and menopause, containing any one or more selected from the group consisting of Prunus cerasoides prunus extract, its active fraction, the compound represented by the following formula (I), and a food acceptable salt thereof Functional food composition for the prevention and improvement of symptoms. (Ⅰ)

The functional food composition of claim 4, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer. The functional food composition of claim 4, wherein the menopausal symptoms include any one of hot flashes, hyperlipidemia, cerebral psychiatric function, osteoporosis, venous thrombosis, and atrophic vaginitis. The functional food composition of claim 4, wherein the food has any one of a tablet, a capsule, a powder, a granule, a liquid, and a ring. The food composition of 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. The functional food composition of claim 7 or 8, wherein the food is a health functional food.

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