WO2017065516A1 - Composition for preventing or treating gynecological cancers and menopausal symptoms containing cajanus cajan 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 Cajanus cajan 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 Cajanus cajan 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 Kazanus Kazan extract or a compound isolated therefrom as an active ingredient

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

Women's cancers such as endometrial and breast cancers and diseases such as menopausal syndrome are associated with female hormones such as excessive and reduced estrogens and imbalances of estrogens 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 receptor (ER) or progesterone receptor (PR) 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 the high fat and high calorie westernized diet and obesity, late marriage and low fertility rate, avoiding lactation, and overexposure to estrogen. The western region is more than three times more likely to develop breast cancer than Korean women, and the demand for breast cancer drugs is very high worldwide. Breast cancer treatment is primarily performed after surgery, radiation therapy, chemotherapy, hormone therapy, etc. to prevent recurrence. Hormonal therapy is effective in lowering recurrence rates, but there is a problem that resistance is expressed after long-term administration over many years. In the case of tamoxifen, the first-choice drug, endometrial cancer induction and 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, the treatment of estrogen alone or a combination of estrogen / progesterone is almost the only treatment. This is called hormone replacement therapy (HRT). However, long-term use of HRT is known to be directly involved in increasing the incidence of cancer in tissues containing ER, such as breast, uterus and ovary. Women's Health Initiative (WHI) study found that taking Premarin and Prempro, one of the most prescribed HRT products in North America, is associated with increased breast cancer and the development of cardiovascular disease. In addition, various small-scale clinical trials suggest that the use of HRT is closely related to hypercellular growth and carcinogenesis, such as increased uterine proliferation in postmenopausal women who are taking HRT.

As a carcinogenic mechanism related to HRT, excessive cell proliferation due to E2 / ER interaction and accumulation of DNA mutations have been suggested, and genotoxicity by reactive metabolites occurring during in vivo metabolism of estrogens is also carcinogenic. It is presented as a mechanism. The pharmacological effects of these tissue-specific estrogens have been known to be due to a variety of molecular mechanisms that interact with estrogen and ER and transcription factors involved in specific tissues / cells (Jordan, 2007). For example, tamoxifen, which is used as a breast cancer drug, acts as an ER antagonist in the breast, but acts as an ER agonist in the uterus and bone, so long-term use of tamoxifen may cause side effects of endometrial cancer. Therefore, the development of a therapeutic agent or a postmenopausal symptom treatment agent for cancers in which ER is involved in carcinogenic mechanisms, such as breast cancer or endometrial cancer, has been described as a substance capable of having 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 kinds of chemical ingredients, and many of the dietary supplements have not been subjected to strict toxicity tests. none. The side effects of these products are actually impossible to monitor in government. In addition, there is a lack of scientific basis for pharmacological efficacy in its efficacy, and it is difficult to judge whether it complies with strict and precise quality control regulations. Therefore, there is a need for a safer and more potent natural material derived from the existing plant extracts.

Cajanus cajan (L.) Huth ) is a perennial plant of the Fabaceae Cajanus, which has been cultivated for more than 3,500 years ago in southern Asia and has spread to East Africa and the Americas (Van der Maeson et al. 1995, Fuller, DQ et al) 2006). The leaves of Kazanus C.cajan have been used for liver disease as a folk remedy in India (Kundu et al., 2008) and for chemotherapy in southwestern Nigeria (Ashidi et al., 2010). Roots and leaves have been used to treat diabetes in India (Grover et al., 2002) and to treat anemia in Ivory Coast (Kone et al., 2012). In Brazil, C.cajan was also used to induce various infection therapies, immune responses (Braga et al., 2007) and abortion (Lemonica and Alvarenga, 1994). In the extracts of the roots and leaves of Kazanus C.cajan, there were studies that isolated physiologically active substances, mainly flavonoids and stilbenes, betulinic acid and zenithine Genistein) (Duker-Eshun et al., 2004). Pharmacological activities include hepatocellular protective action (Ghosh and Sil, 2006, Sarkar and Sil, 2006, Manna et al., 2007b, Sinha et al., 2007), renal cell protective action (Manna et al., 2007a, Zhang et al. ., 2012), neuroprotective activity (Ruan et al., 2009, Jiang et al., 2012), cholesterol lowering activity (Luo et al., 2008, Dai et al., 2013) and anti-inflammatory (Datta S et al. 1999), antioxidant (Lai YS et al 2012), antidiabetic (Ezike AC et al. 2010, Amalraj T et al. 1998), antimicrobial (Zu YG et al. 2010), antiviral (Nwodo UU et al. 2011, Zu Y et al. 2010), antifungal (Brito SA et al. 2012), antigenic insects (Braga FG et al. 2007), anticancer activity (Ashidi JS et al. 2010) has been reported. Kazanus Kazan has been reported for its anti-osteoporosis action. Kazanus Kazan (C.cajan L). Stilbene in the extract showed anti-osteoporosis function in bone loss mice induced by ovarian resection. Kazanus Kazan (C.cajan L). Extracts reduced follicle stimulating hormone (FSH) and progesterone (LH) without affecting serum estrogen (E2) concentration and uterine weight (Zheng YY et al. 2007). Meanwhile Zheng YY et al. According to a 2007 study, Kazanus Kazan (C.cajan L). The extract confirmed the anti-osteoporosis efficacy in bone marrow-derived osteoclasts. These studies suggest that Kazanus C. cajan extract may function in postmenopausal women. However, no pharmacological activity associated with estrogen activity of C. cajan has been reported.

[Preceding technical literature]

[Non-Patent Documents]

Amalraj T1, Ignacimuthu S. (1998) Hypoglycemic activity of Cajanus cajan (seeds) in mice. Indian J Exp Biol. 36: 1032-1033.

AshidiJS, Houghton PJ, Hylands PJ, Efferth T (2010) Ethnobotanical survey and cytotoxicity testing of plants of South-western Nigeria used to treat cancer, with isolation of cytotoxic constituents from Cajanus cajan Mills pleaves. Journal of ethnopharmacology 128: 501-512.

Braga FG, Bouzada MLM, Fabri RL, Matos MD, Moreira FO, Scio E, Coimbra ES (2007) Antileishmanial and antifungal activity of plants used in traditional medicine in Brazil. Journal of thnopharmacology 111: 396-402.

Brito SA1, Rodrigues FF, Campos AR, da Costa JG (2012) Evaluation of the antifungal activity and modulation between Cajanus cajan (L.) Millsp. leaves and roots ethanolic extracts and conventional antifungals. Pharmacogn Mag. 30: 103-106.

Datta S1, Sinha S, Bhattacharyya P (1999) Effect of a herbal protein, CI-1, isolated from Cajanus indicus on immune response of control and stressed mice. J Ethnopharmacol. 67: 259-267.

Dai FJ, Hsu WH, Huang JJ, Wu SC (2013) Effect of pigeon pea (Cajanus cajan L.) on high-fat diet-induced hypercholesterolemia in hamsters. Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 53: 384-391.

Duker-Eshun G, Jaroszewski JW, Asomaning WA, Oppong-Boachie F, Christensen SB (2004) Antiplasmodial constituents of Cajanus cajan. Phytotherapy Research 18: 128-130.

Ezike AC, Akah PA, Okoli CC, Okpala CB (2010) Experimental evidence for the antidiabetic activity of cajanus cajan leaves in rats. J Basic Clin Pharm. 1: 81-84.

Fuller, D. Q .; Harvey, E. L. (2006). The archaeobotany of Indian pulses: Identification, processing and evidence for cultivation. Environmental Archeology 11: 219.

Ghosh A, Sil PC (2006) A 43-kDa protein from the leaves of the herb Cajanus indicus L. modulates chloroform induced hepatotoxicity in vitro. Drug Chem Toxicol 29: 397-413.

Grover JK, Yadav S, Vats V (2002) Medicinal plants of India with anti-diabetic potential. Journal of ethnopharmacology 81: 81-100.

Jiang BP, Yang RW, Liu XM, Liu YM, Chang Q, Si JY, Pan RL (2012) [Neuroprotective effect of longistyline A against corticosterone-induced neurotoxicity in PC12 cells]. Acta pharmaceutica Sinica 47: 600-603.

Kone WM, Koffi AG, Bomisso EL, Bi FHT (2012) Ethnomedical Study and Iron Content of Some Medicinal Herbs Used in Traditional Medicine in Cote D'ivoire for the Treatment of Anaemia. Afr J Tradit Complem 9: 81-87.

Kundu R, Dasgupta S, Biswas A, Bhattacharya A, Pal BC, Bandyopadhyay D, Bhattacharya S, Bhattacharya S (2008) Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function. Journal of ethnopharmacology 118: 440-447.

Lemonica IP, Alvarenga CM (1994) Abortive and teratogenic effect of Acanthospermum hispidum DC. and Cajanus cajan (L.) Millps. in pregnant rats. Journal of ethnopharmacology 43: 39-44.

Lai YS, Hsu WH, Huang JJ, Wu SC (2012) Antioxidant and anti-inflammatory effects of pigeon pea (Cajanus cajan L.) extracts on hydrogen peroxide- and lipopolysaccharide-treated RAW264.7 macrophages. Food Funct. 23: 1294-1301.

Luo QF, Sun L, Si JY, Chen DH, Du GH (2008) Hypocholesterolemic effect of stilbene extract from Cajanus cajan L. on serum and hepatic lipid in diet-induced hyperlipidemic mice. Acta pharmaceutica Sinica 43: 145-149.

Manna P, Sinha M, Sil PC (2007a) A 43 kD protein isolated from the herb Cajanus indicus L attenuates sodium fluoride-induced hepatic and renal disorders in vivo. J Biochem Mol Biol 40: 382-395.

Manna P, Sinha M, Sil PC (2007b) Galactosamine-induced hepatotoxic effect and hepatoprotective role of a protein isolated from the herb Cajanus indicus L in vivo. J Biochem Mol Toxic 21: 13-23.

Nwodo UU1, Ngene AA, Iroegbu CU, Onyedikachi OA, Chigor VN, Okoh AI (2011) In vivo evaluation of the antiviral activity of Cajanus cajan on measles virus. Arch Virol. 156: 1551-1557.

Ruan CJ, Si JY, Zhang L, Chen DH, Du GH, Su L (2009) Protective effect of stilbenes containing extract-fraction from Cajanus cajan L. on Abeta (25-35) -induced cognitive deficits in mice. Neuroscience letters 467: 159-163.

Sarkar K, Sil PC (2006) A 43 kDa protein from the herb Cajanus indicus L. protects thioacetamide induced cytotoxicity in hepatocytes. Toxicol in Vitro 20: 634-640.

Sinha M, Manna P, Sil PC (2007) Attenuation of cadmium chloride induced cytotoxicity in murine hepatocytes by a protein isolated from the leaves of the herb Cajanus indicus L. Archives of toxicology 81: 397-406.

Van der Maeson, L. J. G. (1995). Pigeonpea Cajanus cajan, by Smartt, J. and Simmonds, N. W. (2nd eds.), Evolution of Crop Plants. Essex: Longman pp. 251255.

Zhang DM, Li Y, Cheang WS, Lau CW, Lin SM, Zhang QL, Yao N, Wang Y, Wu X, Huang Y, Ye WC (2012) Cajaninstilbene Acid Relaxes Rat Renal Arteries: Roles

of Ca2 + Antagonism and Protein Kinase C-Dependent Mechanism. PloS one 7: e47030.

Zheng YY, Yang J, Chen DH, Sun L. (2007) Effects of the stilbene extracts from Cajanus cajan L. on ovariectomy-induced bone loss in rats. Acta pharmaceutica Sinica 42: 562-565.

Zheng YY, Yang J, Chen DH, Sun L. Yao Xue Xue Bao (2007) Effects of the extracts of Cajanus cajan L. on cell functions in human osteoblast-like TE85 cells and the derivation of osteoclast-like cells. Acta pharmaceutica Sinica 42: 386-391.

Zu YG1, Liu XL, Fu YJ, Wu N, Kong Y, Wink M. (2010) Chemical composition of the SFE-CO extracts from Cajanus cajan (L.) Huth and their antimicrobial activity in vitro and in vivo. Phytomedicine. 2010 17: 1095-1101.

Zu Y1, Fu Y, Wang W, Wu N, Liu W, Kong Y, Schiebel HM, Schwarz G, Schnitzler P, Reichling J. Forsch Komplementmed (2010). Comparative study on the antiherpetic activity of aqueous and ethanolic extracts derived from Cajanus cajan (L.) Millsp. J. Forsch Komplementmed (2010) 17: 15-20.

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 is to isolate and identify the Cajanus cajan (L.) Huth extract, active fraction and a single component separated therefrom exhibiting estrogen activity, for the prevention and treatment of female cancer and menopausal symptoms It aims to provide new natural medicines and health functional foods.

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,

Cajanus new pharmaceutical composition or functional food effective for the prevention and treatment of women's cancer and menopausal symptoms such as endometrial cancer, breast cancer, etc. by identifying the cajan ) extract, its active fraction, its efficacy, pharmacological metabolism and safety To provide a composition.

Specifically, in the present invention,

Cajanus cajan ) extract, its active fraction, the compounds represented by the formula (I) to (VI) below and any one or more selected from the group consisting of pharmaceutically acceptable salts thereof, female cancer and menopause Provided are pharmaceutical compositions for the prevention and treatment of symptoms.

In the present invention,

Cajanus cajan ) extract, its active fraction, the compounds represented by the formula (I) to (VI) below and any one or more selected from the group consisting of pharmaceutically acceptable salts thereof, female cancer and menopause Functional food compositions for the prevention and amelioration of symptoms are provided.

(Ⅰ)

(Ⅱ)

(Ⅲ)

(Ⅳ)

(Ⅴ)

(Ⅵ)

The present invention is the first confirmation that the Kazanus Kazan plant extract and its active fractions show female hormones, and the active ingredient is isolated and identified for the first time.

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, Cajanus ( Cajanus) cajan ) Obtain new plant extracts and their active fractions, identify the effects of female hormones, and identify and identify active ingredients to provide new natural medicines and health foods useful for the prevention and treatment of female cancers and menopausal symptoms. In particular, the present invention was confirmed for the first time that Kazanus Kazan plant extract and its active fraction exhibits female hormones, the pharmaceutical composition of the present invention containing the same occurs with the highest frequency among female cancers and the incidence is increased It can be usefully used for breast cancer and endometrial cancer having similar pathogenesis.

1 shows the extraction of estrogen (●) and kazanus kazan (◆) extracts against pure recombinant human estrogen receptor alpha (hERα) binding of deuterium-labeled estrogen ([ ³ H] estrogen). The effect is shown.

Figure 2 shows the results of measuring the competitive binding capacity of three fractions of Kazanus Kazan and tritium-labeled estrogen ([ ³ H] estrogen) to pure recombinant human estrogen receptor alpha (hERα).

3 is Cajanus The gene transcriptional activity of ER subtype was measured for 23 extracts (CCT) and 23 fractions ( CC1-23 ). Cajanus Nine fractions ( CC 10, 14, 15, 16, 17, 18, 19, 20, 22) of cajan extract (CCT) and 23 fractions ( CC1-23 ) showed an ERβ / ERα ratio of 2 or more.

Figure 4 MCF7 cell after treatment of vehicle, estrogen, Kazanus Kazan extract 5x10 ^-6 g / ml ~ 10 ^-6 g / ml concentration to evaluate the estrogen receptor-reactive MCF-7 cell cell proliferation effect Proliferation is confirmed. Cajanus cajan extract induced cell proliferation at concentrations of 5x10 ^-6 g / ml and 10 ^-5 g / ml.

 5 is a test result of Kazanus Kazan extract changes the activity of ERE-gene in MCF-7 cells transfected with Estrogen Responsive Element (ERE).

FIG. 6 shows the results of experiments showing that Kazanus Kazan fractions change the activity of ERE genes in MCF-7 cells transfected with Estrogen Responsive Element (ERE).

Figure 7 shows the results of experiments to increase the expression of ERE gene-induced pS2 gene of Kazanus Kazan extract in MCF-7 cells transfected with the estrogen responsive element (ERE).

Figure 8 shows the change of pS2 gene activity in the uterine tissue of rats when Kazanus Kazan is administered 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 Kazanus Kazan extract is administered to rats.

Figure 10 evaluates the effect of Kazanus Kazan extract on the Ngb promoter activity of human neurons SKNSH. When estrogen, genistein, and kazanus kazan extract were treated as vehicle, positive control group, Ngb gene expression was increased by positive control group and kazanus kazan extract.

Figure 11 evaluates the effect of Kazanus Kazan extract on the Ngb promoter activity of mouse neurons N2a. When estrogen, genistein, and kazanus kazan extract were treated as vehicle and positive control, the activity of Ngb promoter was increased by positive control and kazanus kazan extract.

In the present invention, by identifying the therapeutic efficacy, pharmacological metabolism and safety of female hormone and female cancer of Kazanus Kazan extract, a new pharmaceutical composition effective for the prevention and treatment of female cancer and menopausal symptoms such as endometrial cancer, breast cancer or Provides a functional food composition.

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.

In the present invention, a single component exhibiting feminine hormones separated from the active fraction of Cajanus cajan is represented by the following formulas (I) to (VI).

(Ⅰ)

longistylin A

Chemical Name: ( E ) -3-methoxy-2- (3-methylbut-2-en-1-yl) -5-styrylphenol

Chemical Formula: C ₂₀ H ₂₂ O ₂

MW: 294.39

(Ⅱ)

longistylin C

Chemical Name: ( E ) -3-methoxy-4- (3-methylbut-2-en-1-yl) -5-styrylphenol

Chemical Formula: C ₂₀ H ₂₂ O ₂

MW: 294.39

(Ⅲ)

genistein

Chemical Name: 5,7-dihydroxy-3- (4-hydroxyphenyl) -4 H -chromen-4-one

Chemical Formula: C ₁₅ H ₁₀ O ₅

MW: 270.24

(Ⅳ)

cajanin

Chemical Name: 5,2 ', 4'-Trihydroxy-7-methoxyisoflavone

Chemical Formula: C ₁₆ H ₁₂ O ₆

MW: 300.26

(Ⅴ)

pinosylvin monomethyl ether

Chemical Name: ( E ) -3-methoxy-5-styrylphenol

Chemical Formula: C ₁₅ H ₁₄ O ₂

MW: 226.27

(Ⅵ)

cajanol

Chemical Name: 4 ', 5-dihydroxy-2', 7-dimethoxyisoflavanone

Chemical Formula: C ₁₇ H ₁₆ O ₆

MW: 316.31

Cajanus cajan (L.) Huth ) is a perennial plant of the family Cabañas of the Fabaceae. Kazanus Kazan used in the experiments in the present invention will be sold from overseas biological material hub center.

In the present invention, Kajanus ( Cajanus) cajan ) extracts and subfractions are identified and the components of (I) to (VI), which are effective for the prevention and treatment of female cancer and menopausal symptoms , are isolated and identified from Cajanus cajan as follows. .

One. Kazanus Kazan  Extract and Subdivision  Female hormone pharmacological efficacy test

Kazanus Kazan Competitive inhibition of human estrogen receptor alpha (hERα) binding to extracts and subfractions, ER subtype selective gene transcriptional activity analysis, MCF-7 cell proliferation, Estrogen responsive element (ERE) activity, MCF- Induction of ERE gene-induced pS2 gene expression in rats, effect of uterine proliferation in rats, and expression of pS2 gene in uterine tissue and protein expression of estrogen receptor alpha (ERα) and progesterone receptors A and B (PRA, PRB) It was.

2. Kazanus Kazan  Extract and Subdivision  Pharmacological Validation Experiment for Brain Cell Protection

Kazanus Kazan The effect of the extract on the neuroglobin (Ngb) promoter activity of human neurons SKNSH and on the Ngb promoter activity of mouse neuron N2a was confirmed. In addition, the anti-inflammatory activity of the extract of Kazanus Kazan was evaluated by the inhibitory effect of human hematopoietic prostaglandin D synthase (HPDGs), and a single dose acute toxicity test was performed to evaluate the in vivo toxicity of Kazanus Kazan extract.

3. Kazanus Kazan  Separation and Pharmacological Activity of Active Ingredients

Kazaa Taunus Kazan (Cajanus The 23 fractions were isolated and identified by experimenting with the isolated fraction of cajan ) extract and pharmacological activity of the active ingredient, and 6 compounds were isolated and identified. The compounds represented by the above formulas (I) to (VI) are the first to identify and separate the burner by Kajanus cajan in the present invention.

The pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms of the present invention is effective at least one selected from the group consisting of the compounds represented by the formula (I) to (VI) and pharmaceutically acceptable salts thereof Contains as an ingredient. The pharmaceutical composition for the prevention and treatment of female cancer and menopausal symptoms of the present invention comprises Kazanus Kazan extract and fractions 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 extract or the active fraction. 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 a Kazanus Kazan extract and fractions and salts acceptable as food as an active ingredient.

The functional food composition comprises 0.0001 to 20% by weight of the extract or active fraction 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 particularly limited to other ingredients except extracts or active fractions as essential ingredients, and may further contain various ingredients such as various flavors or natural carbohydrates, such as ordinary foods or drinks. 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 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: human  Estrogen receptor alpha ( hERα )

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

2. Gene reporter evaluation test: Estrogen (ERE) reactive gene transcription test

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 (ERE) -luciferase and plasmid were transfected with Lipofectamine 2000 reagent (Invitrogen, USA). In the case of the ERE-luciferase assay, estrogen (estrogen, E2, 1 nM) as a positive control, ICI-182,780 (1mM) was treated as an antagonist control. Other test substances dissolved in various concentrations of DMSO were diluted in the medium and treated with cultured cells. After incubation for 24 hours, cell culture was terminated, and water-soluble cell extracts were obtained using Passive Lysis Buffer (Promega, USA). Luciferase activity present herein was quantitatively measured using a Luciferase Assay System (Promega) and a Luminometer including a substrate of Luciferase and a reaction buffer. The gene activity represented by the test substance was set to 100% of the activity indicated by the positive control, and the relative activity was shown.

3. Kazanus Kazan  Extracts In fractions  ER subtype selectivity evaluation

HeLa cell line, which does not express ER, is seeded in 5x10 ⁵ cells / well, 24-well plate and contains charcoal-treated fetal bovine serum (FBS), and phenol-red-free DMEM medium (hereafter estrogen-free media) is used. And incubated. Plasmids expressing ERa and ERb (0.25 mg / well) and ERE3-luciferase plasmids (0.25 mg / well) were co-transfected using Lipofectamine 2000 (Invitrogen). After 1 day of incubation, the appropriate test material was treated with the cells, and after 24 hours, water-soluble cell extracts were extracted using Passive lysis buffer (Promega). Luciferase activity in this extract was evaluated by reading the value using a VICTOR3 luminometer (Perkin Elmer) instrument using a luciferase assay system (Promega) reagent.

4. Kazanus Kazan  Of extract MCF -7 cell proliferation effect evaluation

The cell proliferation of Kazanus Kazan extract was confirmed using MCF-7 cell line. MCF7 cell line is a cell line derived from human breast cancner and expresses abundant estrogen receptor, which is useful for evaluating estrogenicity of Kazanus Kazan extract. Phenol-red free media (CDFBS-DMEM) containing charcoal dextran treated serum was used to thoroughly exclude external estrogen effects in cell culture and test material treatment. MCF-7 cells were seeded in a 96-well plate to 10 ⁴ cells / well, and after 24 hours, the medium was removed and replaced with a medium containing a certain concentration of test substance. CD-DMEM medium containing the test substance was prepared by diluting a concentration of a test substance stock solution dissolved in DMSO 500-1000 times in the medium immediately before the experiment. DMSO (0.5 ~ 0.1%) was added to CDFBS-DMEM in the vehicle control group and positive control group was treated with 1 nM 17β-ES and 1μM genistein. The final concentration of solvent in all experimental systems was 0.1% (v / v) or less. After 48 hours incubation in 37 ° C, 5% CO ₂ incubator MTT assay was performed to determine the number of live cells. After completely removing the culture solution, MTT [3- (4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide, Sigma] solution dissolved in DPBS was treated with 100 μl volume at a concentration of 1 mg / ml, followed by 37 ° C. Incubated for 1 hour in 5% CO ₂ incubator. After incubation, the solution was completely removed, and isopropanol was added to lyse the cells. At the same time, purple MTT metabolite (crystalline) produced by living cells was dissolved. The amount of MTT metabolite dissolved in isopropanol was measured at 540 nm by ELISA Plate Reader (UVmax, USA). As absorbance is known to be proportional to the number of living cells, it is possible to quantitatively assess the degree of cell proliferation by the test substance.

5. Kazanus Kazan  By extract MCF Of the target genes of the -7 cells on the transcriptional activity

In order to confirm the estrogenicity of Kazanus Kazan extract at the mRNA level, experiments were carried out on pS2 (TFF1) gene, a well-known estrogen-reactive gene. Phenolic-red free media containing charcoal dextran treated serum (CDFBS-DMEM) when 90% of the MCF7 cells were seeded at 5x10 ⁵ in 60 mm ² petri dish to measure the change of pS2 gene expression by test substance. The vehicle control group was treated with DMSO (0.1%), positive control group 17β-ES (1 nM), genistein (1μM), and a certain concentration of the test substance. Typically, after 24 hours of drug treatment, the medium was removed, washed with DPBS to completely remove the cell culture, and then treated with trizol to separate from the surface of the culture vessel and destroy the cells. Intracellular mRNA 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 GAPDH are forward 5'-CTCTCTGCTCCTCCTGTTCGAC; And reverse 5'-TGAGCGATGTGGCTCGGCT. Primers that recognize pS2 are forward 5'-CGTGAAAGAC AGAATTGTGGTTTT; And reverse 5'-CGTCGAAACAGCAGCCCTTA. Quantitative PCR was performed at about 40 cycles (95 ° C; 30 seconds, 60 ° C; 30 seconds, 72 ° C; 30 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 vehicle group, the 17β-ES treatment group, and the test substance treatment group.

6. In vivo  ( in vivo ) Female hormone rating: uterotrophic  assay

The uterine growth test is a method of indirectly evaluating female hormone sex by measuring the increase in uterine tissue mass induced by estrogen. The effect of uterine proliferation was measured by examining the effect of the test substance on the immature uterus when subcutaneous injection was administered to female rats at 21 days of age for 3 days. Immature female rats were used for the experiment. During the acclimatization period, the subjects were selected to be close to the average body weight, and grouped by random method. Individual identification of animals was done by tail marking and tag by breeding box. In the positive control group, estrogen (E2) was homogeneously suspended in corn oil to 0.003 mg / kg and diluted in corn oil stepwise, and the test substance was also suspended in corn oil. The experimental group was divided into three groups, and each group was administered with subcutaneous injection of vehicle (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), and kazanus kazan extract (300 mg / kg) at 24 hour intervals for 3 days. It was. 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).

7. intracellular hormone inherent reactivity target transcription activating effect evaluation method 21 days immature female command vehicle, estrogen mice for the gene (estrogen, E2, 0.003 mg / kg), Kazaa Taunus Kazan MRNA was extracted from the rat uterine tissues obtained by subcutaneous injection of (300 mg / kg) extract at 24 hours intervals for 3 days using Trizol. cDNA was obtained by reverse transcription of a predetermined amount of mRNA (1 μg) using an iScript cDNA synthesis kit (Bio-Rad). The expression levels of the genes were quantitatively measured by real-time PCR using cDNA, a pair of primers capable of recognizing the cDNA of the target gene, and PCR SYBR green kit (Qiagen) reagent. In order to make up for the technical mistakes in the various stages of the reaction, the expression level of GAPDH, a housekeeping gene, was measured at the same time. Primers that recognize GAPDH include forward 5'-CTCTCTGCTCCTCCTGTTCGAC; And reverse 5'-TGAGCGATGTGGCTCGGCT. The expression level of Trefoil factor 2 (pS2) was measured as a target gene of estrogen. Primers that recognize pS2 for real-time RT PCR reactions are forward 5'-CGTGAAAGAC AGAATTGTGGTTTT; And reverse 5'-CGTCGAAACAGCAGCCCTTA. Real-time PCR was performed at about 40 cycles (95 ° C; 30 seconds, 60 ° C; 30 seconds, 72 ° C; 30 seconds). Each gene expression curve is expressed in logarithm, and then the threshold cycle (C _T ) is mathematically obtained. The value obtained by substituting this value with 2-DDC _T represents the relative expression level of a specific gene. The expression level of the specific gene divided by the expression level of the house keeping gene was selected as the final value for the gene expression level, and the ER antagonist or agent treatment value was compared with that of the test substance.

8. Intracellular  Assessing the Effect of Altered Endogenous Hormone Reactive Protein Expression

21-day-old immature female rats were divided into vehicle, estrogen, and kazanus kazan groups, and vehicle (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), and kazanus kazan Changes in protein expression of estrogen receptor alpha (ERα), progesterone receptor A (PRA), and progesterone receptor B (PRB) in uterine tissues of rats obtained after subcutaneous injection of (300 mg / kg) extract at 24 hour intervals for 3 days. It was confirmed. Proteins were isolated by adding lysis buffer containing protease inhibitor and phosphatase inhibitor to rat uterine tissues, and the extracted proteins were quantified by BCA method at 20 μg, 7.5% SDS-PAGE gel at 80V for 2 hours. It moved. After electrophoresis, transfer to PVDF membrane at 25V for 3 hours, blocking with RT for 1 hour with BSA, and reacted with primary and secondary antibodies were detected as ECL solution. At this time, the primary antibody was used by diluting ERα (sc-7207, SANTA CRUZ BIOTECHNOLOGY, INC.) And PR (sc-538, SANTA CRUZ BIOTECHNOLOGY, INC.) In a 1: 500 ratio. Β-actin was used to correct the difference in protein amount due to experimental error.

9. Neuron based Ngb  promoter luciferase  assay and in cells Ngb mRNA  Expression change assessment

(1) Ngb promoter assay: Estrogen and was known to suppress the SERMS neuron cell death and the induction of 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). It is also involved in signal transduction of G-protein coupled receptors and is known to regulate apoptosis and cell survival by inducing the transport of cytochrome C in mitochondria and also has neuron protective effects in stroke animal models. Recently, neuroprotective activity was studied based on previous studies showing that estrogen increases Ngb expression in neurons and astrocytes and is associated with 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 Kazanus Kazan extract shows brain protective effect. The neuroglobin (Ngb) -luciferase sequence was inserted into the cell gene to quantitatively evaluate the Ngb promoter transcriptional activity of Kazanus kazan extract. 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 were replaced by dissolving the test substance in serum free phenol red free DMEM when the cells 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 incubated in 10% FBS DMEM, and then the media were replaced by dissolving the test material 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. Intracellular mRNA 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 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.

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

The anti-inflammatory activity of Kazanus Kazan extract was evaluated by the inhibitory effect of human hematopoietic prostaglandin D synthase (HPDGs). Enzyme reactions included assay buffer (0.1M potassium phosphate with 1mM EDTA, pH 6.5), GSH 2.5mM, CDNB 1mM, human hematopoietic prostaglandin D synthase (10mg / ml). The assay buffer, CDNB, HPDGs were mixed first in a 96-well plate, and GSH and Kazanus Kazan extract (250mg / ml) were added thereto. DMSO was used for vehicle 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.

11. Acute Toxicity

Single dose acute toxicity experiments were performed to increase the clinical applicability of Kazanus Kazan extract. Animals were used as female mice and fasted 24 h before administration. Body weight (1% CMC in saline), extract 500mg / kg, 1000mg / kg, 1500mg / kg, 2000mg / 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.

12. Analysis of Active Ingredients on Plant Extract Fractions

The major subfractions with pharmacological effects were found by separating the active fractions from crude extracts. The specific experimental method is as follows.

(1) Extraction and separation: Kazanus Kazan extract was dissolved in MeOH, centrifuged and the supernatant was subjected to high performance liquid chromatography to obtain 23 fractions. Instruments used for the analysis include UV detectors at 254 nm and 280 nm, equipped with a Waters Fraction Collector II, and Agilent Eclipse XDB-C18 column (250 mm × 21.2 mm, 7 μm particle size).

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

(3) Single component separation and purification of the active fraction: The single component was purified by HPLC analysis for the active fractions CC14 and CC23 selected by UPLC / TOF-ESIMS, TLC and HPLC analysis.

(4) Chemical structure analysis and characterization of single component: HPLC, ESIMS, NMR, etc. separation of a single component on chromatogram, after obtaining a single component NMR analysis ( ¹ H, ¹³ C, DEPT, COSY, HSQC, HMBC , NOESY, ROESY) and various structural decision analysis methods were used. Existing medicinal plant DB was used to confirm whether the final substance was new or known.

result

Kazanus Kazan Pharmacological Effects of Female Extracts and Fractions on Female Hormones

end. For ER binding Kazanus Kazan  extract, Subdivision  Competitive inhibition and ERβ selectivity

In vitro system Kajanus extract Competitive binding of cajan extract (CCE) and tritium estrogen ([ ³ H] E2) to pure recombinant human estrogen receptor alpha (hERα) was measured. The results are shown in FIG. 1 and Table 1. In a competitive binding assay, the IC ₅₀ of estrogen (E2) (•) was 1.81 × 10 ⁻¹⁰ g / mL (hERα). IC ₅₀ of Kazanus Kazan, a kind of phytoestrogen, was 8.18 × 10 ⁻⁶ g / mL (hERα). These results confirm that Kazanus Kazan extract acts concentration-dependently on tritium estrogen ([ ³ H] E2) separation and binding, and RBA is about 45,000 times lower than that of endogenous estrogen (0.0022%). for hERα). (Fig. 1, Table 1).

As a result of analysis of the ER binding force of the three fractions, the hERα binding strength of the CC, CC20, CC21, CC22, and CC23 fractions was excellent (FIG. 2, Table 2).

The ER subtype selective gene transcriptional activity was measured for 10 mg / ml samples after dissolving in Kazanus Kazan extract (CCT) and 23 fractions (CC1-23) at an appropriate concentration in DMSO or water. The relative comparison results of luciferase activity of each sample when extracted and compared relative to luciferase activity compared to DMSO are shown in FIG. 3. Nine fractions ( CC 10, 14, 15, 16, 17, 18, 19, 20, 22) of the extracts (CCT) and 23 fractions (CCT) of Kazanus Kazan have an ERβ / ERα ratio of 2 or more. (FIG. 3) .

Table 1 IC ₅₀ and RBA Values for hERα of Kazanus Kazan Extract

(Table 2) Comparison of ERα binding capacity of 23 kinds of extracts subdivided from Kazanus Kazan extract and extract

RBA = [Ki _(E2 / Ki _{( Cajanus cajan} ] × 100.

I. Kazanus Kazan  Extract and Subdivision MCF Through -7 estrogen-responsive genes MCF -7 cell proliferation and Intracellular  Transcription analysis

In order to evaluate the MCF-7 cell proliferation effect, after treatment with Kazanus Kazan extract at a concentration of 10 ^-6 g / ml ~ 10 ^-5 g / ml and confirmed the MCF7 cell proliferation. The 17β-ES used as a positive control induced about 2.3-fold proliferation of MCF7 cells compared with vehicle group, and genistein also increased 1.8-fold proliferation of MCF7 cells. Kazanus kazan extract was found to induce about 1.5-fold cell proliferation at a concentration of ⁵ × 10 ⁻⁶ g / ml and 10 ⁻⁵ g / ml (FIG. 4) .

In the MCF-7 cells transfected with the Estrogen Responsive Element (ERE), it was tested whether Kazanus Kazan altered the activity of the ERE-gene. When treated with Kazanus Kazan alone, the ERE gene activity was increased by about 10- to 40-fold compared to the vehicle at a concentration of 1-20 μg / ml, and concentration-dependently induced ERE activity. When the ERE activity of estrogen (estrogen, E2) was 100%, the ERE activity of Kazanus Kazan extract was 33% ~ 118%. In particular, at 15 μg / ml and 20 μg / ml, the gene activity was higher than that of estrogen (E2), suggesting the potent ERE gene activity of Kazanus Kazan. These results confirm that Kazanus Kazan extract induces ERE gene activity in a concentration-dependent manner at a concentration of 1 μg / mL or higher during gene transcription, and shows higher ERE gene activity than estrogen above 15μg / mL. It became. In the case of subfractions, it was confirmed that subfractions 14, 16, 17, 18, 19, 20, 21, and 22 showed higher ERE gene activity than estrogen (FIG. 6).

All. Kazanus Kazan  Estrogen reactivity of pS2  Gene activity induction

Kazaa Taunus after haejun handle Kazan extract to ^{10 -7 g / ml ~ 10 -5} g / ml concentration were confirmed pS2 gene expression changes in the MCF7 cells. In the case of 17β-ES used as a positive control, the expression of pS2 gene was increased by 2.7 times compared with vehicle group, and genistein also increased the expression of pS2 gene by more than 2.2 times. In the case of Kazanus Kazan extract, the expression of pS2 gene was increased 2.7-fold at a concentration of 10 ^-5 g / ml, but the expression of pS2 gene was not induced at low concentrations. (* p <0.05) (FIG. 7) .

la. Kazanus Kazan  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 using immature 21-day-old female rats with very low estrogen secretion. Uterine growth results were compared and evaluated as shown in Table 3 below. Uterine weight (mg) ratio was 0.67 compared to that of corn oil in the vehicle group, and uterine weight as a positive control was administered by subcutaneous injection of estrogen (Etrogen, E2, 3 ㎍ / ㎏). The ratio was 1.88, which was about 2.82 fold higher than the control group. When the Kazanus Kazan extract was injected subcutaneously in immature rats at a dose of 300 mg / kg, the uterine weight ratio was 0.88 and increased by about 1.32 times compared to the vehicle group. Through this uterine growth experiment, it was evaluated that Kazanus Kazan extract induces uterine tissue growth. Weight gain in rats was not observed during drug administration, and the vehicle group was markedly marked with *** or ** (*** P <0.001, ** P <0.05).

Table 3 Immature Rat Uterine Proliferation by Kazanus Kazan Extract

hemp. Kazanus Kazan  In uterine tissue pS2  Gene activity induction

After 21 days of age in immature female rats, vehicle (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), and kazanus kazan (300 mg / kg) extracts were administered subcutaneously in a 24-hour interval for 3 days. PS2 gene expression was confirmed in the obtained uterine tissue. As a result of the experiment, when the estrogen (estrogen, E2) was administered compared with the vehicle, the expression of the pS2 gene was confirmed to be increased by about 1.7 times, and when the Kazanus Kazan extract was administered, it was confirmed that the increase was about 1.44 times at 300 mg / kg (FIG. 5). . These results confirm that Kazanus Kazan extract promotes estrogen activity in the uterine tissue at 300 mg / kg, and induces pS2 gene expression at about 80% of estrogen. It could be confirmed that (Fig. 8).

bar. Kazanus Kazan  In uterine tissue ERα  Inhibit protein expression

Vehicles (corn oil 5ml / kg), estrogen (estrogen, E2, 0.003 mg / kg), kazanus kazan in 21-day-old immature female rats Changes in protein expression of estrogen receptor alpha (ERα), progesterone receptor A (PRA) and progesterone receptor B (PRB) in rat uterine tissues obtained after subcutaneous injection of (300 mg / kg) extract at 24 hour intervals for 3 days. It was confirmed. Experimental results show that ERα protein expression is decreased when estrogen is administered compared to vehicle and Kazanus Kazan Administration of the extract also reduced the expression of ERα protein. Protein expression of PRA and PRB, on the other hand, compared to the vehicle group. When the extract was administered it was confirmed that the expression increased. Through these experimental results, it was confirmed that Kazanus Kazan extract inhibited estrogen receptor alpha (ERα) protein expression in uterine tissues and increased progesterone receptor A and B protein expression (FIG. 9).

four. Of human and mouse neurons neuroglobin  ( Ngb promoter activity mRNA  Increased gene expression

In order to have an effect on the Ngb promoter activity of SKNSH in human neurons, Kazanus Kazan extract was treated with SKNSH cells at a concentration of 5x10 ^-6 g / ml to 5x10 ^-5 g / ml, followed by luciferase assay, and then Ngb promoter activity by the extract. 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 Kazanus Kazan extract significantly increased Ngb promoter activity by about 1.9 to 2 fold at all concentrations (** p <0.001) (FIG. 10) .

The effect of Kazanus Kazan extract on the Ngb promoter activity of mouse neuron N2a was evaluated. Kazanus Kazan extract was treated with N2a cells at a concentration of 5x10 ^-6 g / ml to 5x10 ^-5 g / ml and then subjected to luciferase assay to quantitatively evaluate Ngb promoter activity by the extract. Treatment with the positive control genistein increased the activity of the Ngb promoter by 1.7 times compared to the vehicle group, and 17β-ES did not increase the Ngb promoter activity. Treatment with Kazanus Kazan extract significantly increased Ngb promoter activity by about 1.8-2.2 times at all concentrations (** p <0.01) (FIG. 11) .

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

When the anti-inflammatory activity of the extract of Kazanus Kazan (CCT) was evaluated through the inhibitory effect of human hematopoietic prostaglandin D synthase (HPDGs), the HPGDs inhibitory effect of Kazanus Kazan extract (CCT) was 17.16%. They had low levels (Table 4).

Table 4 HPDGs Inhibitory Effect by Kazanus Kazan Extract

character. Through acute toxicity test Safety evaluation

Single dose acute toxicity experiments were performed to evaluate the biotoxicity of Kazanus Kazan extract. Female mice (10 weeks old) were treated with vehicle (1% CMC saline), Kazanus Kazan extract at doses of 500 mg / kg, 1000 mg / kg, 1500 mg / kg and 2000 mg / kg for two weeks of mortality, body weight change and When the behavior was observed, normal behavior was observed, and feed and water intake were also observed normally. The mortality rate was 0% in all groups, including the vehicle group, during the two-week experiment. The maximum dose of 2000 mg / kg oral dose did not show acute toxicity and was evaluated as having high safety (Table 5) .

Table 5 Acute Toxicity Assessment of Kazanus Kazan Extract

2. Kazanus Kazan  Bioassay-guided analysis

end. Fraction and Purification of Extracts

Kazaa Taunus Kazan (Cajanus cajan ) methanol extract (0.9884 g) was dissolved in MeOH and centrifuged to obtain only supernatant. The supernatant was analyzed using a Waters 2545 HPLC (UV detector at 254 nm and 280 nm, equipped with a Waters Fraction Collector II) equipment. The column used for analysis was an Agilent Eclipse XDB-C18 column (250 mm × 21.2 mm, 7 μm particle size) with a flow rate of 8 mL / min. The mobile phase was mixed with 0.1% aqueous formic acid (A) and methanol (B). Fractions were collected over time (4 min / fraction) and a total of 23 fractions were obtained. As a result of evaluating the ERα binding capacity of these fractions, high ERα binding capacity and ERβ selective ERE activity were observed in fractions CC14 and CC23. Based on the activity results, the experiment was to find a single substance that targets two fractions. ).

I. Isolation and Structure Identification of Single Components

To identify chemical structures, six single components (compounds (I) to (VI)) were separated and identified by structural analysis using spectroscopic methods such as HPLC, ESIMS chromatogram, and NMR. Six single components are shown in Table 6.

Table 6- Kazanus Single active ingredient present in Kazan

(Ⅰ)

longistylin A

Chemical Name: ( E ) -3-methoxy-2- (3-methylbut-2-en-1-yl) -5-styrylphenol

Chemical Formula: C ₂₀ H ₂₂ O ₂

MW: 294.39

¹ H NMR (CD ₃ OD, 400 MHz) δ _H : 7.52 (2H, d, J = 7.6 Hz, H-2 ', 6'), 7.33 (2H, t, J = 7.6 Hz, H-3 ', 5 '), 7.22 (1H, t, J = 7.6 Hz, H-4'), 7.05 (2H, br s, H-7, 8), 6.64 (1H, br s, H-2), 6.63 (1H, br s, H-6), 5.19 (1H, t, J = 8.0 Hz, H-2``), 3.84 (3H, s, -OCH <sub>3</sub> ), 3.33 (2H, d, J = 8.0 Hz, H- 1 ''), 1.76 (3H, s, H-4``), 1.65 (3H, s, H-5 ''); ESI-MS m / z : negative: 293.33 [M−H] ⁻ ; positive: 318.44 [M + H + Na] ^{2 +} , 274.41 [MC ₃ H ₇ + Na] ⁺ .

(Ⅱ)

longistylin C

Chemical Name: ( E ) -3-methoxy-4- (3-methylbut-2-en-1-yl) -5-styrylphenol

Chemical Formula: C ₂₀ H ₂₂ O ₂

MW: 294.39

¹ H NMR (CD ₃ OD, 400 MHz) δ _H : 8.53 (1H, brs, -OH), 7.48 (2H, d, J = 7.6 Hz, H-2 ′, 6 ′), 7.34 (2H, t, J = 7.6 Hz, H-3 ', 5'), 7.33 (1H, d, J = 16.0 Hz, H-7), 7.23 (1H, t, J = 7.6 Hz, H-4 '), 6.93 (1H, J = 16.0 Hz, H-8), 6.66 (1H, d, J = 2.4 Hz, H-4), 6.37 (1H, J = 2.4 Hz, H-6), 5.06 (1H, t, J = 7.6 Hz , H-2``), 3.78 (3H, s, -OCH <sub>3</sub> ), 3.39 (2H, d, J = 6.8 Hz, H-1 ''), 1.79 (3H, s, H-4 ''), 1.66 (3H, s, H-5``); ESI-MS m / z : negative: 293.22 [M−H] ⁻ ; positive: 318.47 [M + H + Na] ²⁺ , 274.44 [MC ₃ H ₇ + Na] ⁺ .

(Ⅲ)

genistein

Chemical Name: 5,7-dihydroxy-3- (4-hydroxyphenyl) -4 H -chromen-4-one

Chemical Formula: C ₁₅ H ₁₀ O ₅

MW: 270.24

¹ HNMR (CD ₃ OD, 400MHz) δ _H : 8.07

1H, s, H-2), 7.37 (2H, d, J = 8.4Hz, H-2 ', 6'), 6.85 (2H, d, J = 8.4Hz, H-3 ', 5'), 6.35 (1H, s, H-8), 6.23 (1H, s, H-6); (-)-ESI-MS m / z : 269.16 [MH] ^- .

(Ⅳ)

cajanin

Chemical Name: 5,2 ', 4'-Trihydroxy-7-methoxyisoflavone

Chemical Formula: C ₁₆ H ₁₂ O ₆

MW: 300.26

¹ H NMR (CD ₃ OD, 400MHz) δ _H : 8.07 (1H, s, H-2), 7.05 (1H, d, J = 8.4Hz, H-6 '), 6.57 (1H, d, J = 2.0 Hz, H-3 '), 6.40 (1H, d, J = 2.0 Hz, H-6), 6.38 (1H, d, J = 2.0 Hz, H-8), 6.37 (1H, dd, J = 8.4, 2.0 Hz, H-5 '), 3.89 (3H, s, H-OCH ₃ ); (+)-ESI-MS m / z : 301.16 [M + H] ⁺ , (-)-ESI-MS m / z : 299.14 [MH] ^- .

(Ⅴ)

pinosylvin monomethyl ether

Chemical Name: ( E ) -3-methoxy-5-styrylphenol

Chemical Formula: C ₁₅ H ₁₄ O ₂

MW: 226.27

¹ HNMR (CD ₃ OD, 400MHz) δ _H : 8.55 (1H, s, -OH), 7.53 (2H, d, J = 8.4Hz, H-2 ', 6')), 7.34 (2H, dd, J = 8.4,8.0Hz, H-3 ', 5'), 7.23 (1H, t, J = 8.0Hz, H-4 '), 7.08 (1H, s, H-7), 7.06 (1H, s, H -8), 6.61 (1H, s, H-2), 6.60 (1H, s, H-6), 6.29 (1H, s, H-4), 3.79 (3H, s, H-OCH ₃ ); ¹³ CNMR (CD ₃ OD, 150 MHz) δ _C : 128.4,128.3,128.2,127.1,126.1,54.2.

(Ⅵ)

cajanol

Chemical Name: 4 ', 5-dihydroxy-2', 7-dimethoxyisoflavanone

Chemical Formula: C ₁₇ H ₁₆ O ₆

MW: 316.31

¹ H NMR (CD ₃ OD, 400MHz) δ _H : 6.92 (1H, d, J = 8.4Hz, H-6 '), 6.47 (1H, s, H-3'), 6.36 (1H, d, J = 8.4Hz, H-3 '), 6.06 (1H, s, H-6), 6.04 (1H, s, H-8), 4.52 (1H, t, J = 6.8Hz, H-3), 4.38 ( 1H, dd, J = 10.8,5.2 Hz, H-2a), 4.23 (1H, dd, J = 10.8,5.2 Hz, H-2b), 3.83 (3H, s, 2'-OCH ₃ ), 3.75 (3H , s, 7-OCH ₃ ); ¹³ CNMR (CD ₃ OD, 150MHz) δ _C : 199.6,169.3,165.6,165.0,159.9,159.8,132.2,131.4,116.3,115.1,108.4,104.4,100.5,99.2,95.7,94.7,71.6,57.5,56.3, 55.9.

[ 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.

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

Claims (9)

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

   (Ⅰ)

   

   (Ⅱ)

   

   (Ⅲ)

   

   (Ⅳ)

   

   (Ⅴ)

   

   (Ⅵ)

   
2. The pharmaceutical composition of claim 1, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer.
3. 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.
4. Cajanus cajan ) extract, its active fraction, the compounds represented by the formula (I) to (VI) below and any one or more selected from the group consisting of pharmaceutically acceptable salts thereof, female cancer and menopause Functional food composition for the prevention and improvement of symptoms.

   (Ⅰ)

   

   (Ⅱ)

   

   (Ⅲ)

   

   (Ⅳ)

   

   (Ⅴ)

   

   (Ⅵ)

   
5. The functional food composition of claim 4, wherein the female cancer is any one of endometrial cancer, breast cancer, and ovarian cancer.
6. 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.
7. The functional food composition of claim 4, wherein the food is a health functional food having any one of tablets, capsules, powders, granules, liquids and pills.
8. The functional 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.
9. The functional food composition of claim 7 or 8, wherein the food is a health functional food.

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