WO2014185601A1 - Pharmaceutical composition for preventing or treating menopausal symptoms containing flavonoid compound derived from toxicodendron vernicifluum extract as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating menopausal symptoms, the composition containing Toxicodendron vernicifluum extract, or a flavonoid compound derived therefrom or a pharmaceutically acceptable salt thereof, as an active ingredient. The Toxicodendron vernicifluum extract or flavonoid compound derived therefrom of the present invention exhibits activity which is more selective for oestrogen receptor beta (ER-β) than for oestrogen receptor alpha (ER-α) and thus, being a phytoestrogen with fewer side effects, can be used to advantage in preventing and treating menopausal symptoms.

Description

Pharmaceutical composition for preventing and treating menopausal syndrome, using flavonoid compounds derived from sumac extract

The present invention relates to a pharmaceutical composition for preventing and treating menopausal syndrome, which contains a flavonoid compound derived from Rhus verniciflua extract or a pharmaceutically acceptable salt thereof as an active ingredient.

Estrogens have a variety of biological actions, including cell number growth and differentiation. The biological actions of estrogens are exerted through two estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β) belonging to the nuclear receptor superfamily (Nilsson et al., 2001, Physiol. Rev., 81 (4): 1535-1565.). The mechanism of action of the estrogen receptor (ER) is a combination of estrogen and intracellular estrogen receptors to form dimers of the receptors, which are located in specific estrogen receptor elements located in the promoters of target genes. elements, EREs) to regulate the expression of related genes. Since the two estrogen receptors have different tissue distribution patterns, ligand binding forces and sites of metastatic activity, these differences allow different selective roles of agonists and antagonists of estrogen receptors in different tissues (Kuiper et et al. al., 1997, Endocrinology, 138 (3): 863-870.). Compounds acting on estrogen receptors have been used as targets for hormone replacement therapy in postmenopausal women and for the development of chemotherapeutic agents in genital cancer.

Among the compounds that can bind to estrogen receptors, compounds that can act as tissue selectors and selectively act as inhibitors or inhibitors of two estrogen receptors are called selective ER modulators (SERMs) (Paech et al. , 1997, Science, 277: 1508-1510.). Representative SERMs are raloxifene and tamoxifen.

Commonly known hormonal agents can be supplemented with estrogen, but they also carry a risk of developing heart disease, dementia and breast cancer (Chlebowski et al., 2003, JAMA, 289, (24): 3243-3253.). ER-α is known to induce the proliferation of breast cancer cells, while ER-β is known to be a tumor suppressor, it is histoselective and antagonizes ER-α while acting as an ER-β agonist. Various attempts have been made to derive compounds and develop them as hormone replacement therapy.

Phytoestrogens are compounds that bind to estrogen receptors and mimic or regulate the roles of estrogens produced in the body (Setchell, 1998, Am. J. Clin. Nutr., 68: 1333-1346.). These compounds are structurally diverse and include flavonoids, stilbenes and lignans. Their structural features can exert estrogen or antiestrogenic activity through binding to estrogen receptors.

Most of the phytoestrogens activate genes of both types of estrogen receptors. If phytoestrogens that selectively activate ER-α or ER-β can be identified, they can be developed as hormone replacements to minimize the cancer-causing side effects of estrogens. Can be. Phytoestrogens that selectively act on two estrogen receptors, ER-α or ER-β, can be usefully used for the treatment and prevention of various menopausal syndromes.

Accordingly, the present inventors, while studying substances having selective activity on the estrogen receptor alpha (ER-α) and the estrogen receptor beta (ER-β), purely known flavonoid compounds and novel flavonoid compounds from the sumac extract After separation and purification, the chemical structure was determined, and the present invention was completed by confirming that the compound exhibits selective activity on the estrogen receptor and thus can be usefully used for the prevention and treatment of menopausal syndrome.

It is an object of the present invention to provide a pharmaceutical composition for preventing and treating menopausal syndrome, which contains a lacquer extract-derived flavonoid compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a health food composition for preventing and improving menopausal syndrome, which contains the lacquer extract-derived flavonoid compound as an active ingredient.

In order to solve the above problems, the present invention has estrogen activity, Sulfurintin (Sulfuretin), Gabanzol (Garbanzol), Resorcinol (Resorcinol), Fustin (Fisetin), Butene (Butein) And it provides a pharmaceutical composition for preventing or treating menopausal syndrome as an active ingredient of any one or more Flavonoid compound or a pharmaceutically acceptable salt thereof selected from Butin (Butin).

Sulfurin and Garbanzol do not affect estradiol by simultaneous treatment with estradiol, have excellent estrogen activity, and have selective activity on estrogen receptor beta (ER-β). Preferred as an active ingredient in the pharmaceutical composition for preventing or treating menopausal syndrome.

In addition, the resorcinol does not affect estradiol by concurrent treatment with estradiol, has excellent estrogen activity at low concentrations, and has a selective activity on estrogen receptor beta (ER-β). It is preferable as an active ingredient of a pharmaceutical composition for preventing or treating a syndrome.

In addition, Fustin and Fisetin have lower estrogen activity than Sulfurin, Garbanzol and Resorcinol, but dose-dependently increases estrogen activity. , Does not affect the estradiol action, has selective activity on the estrogen receptor beta (ER-β), and also contains more than 10 g, 1 g or more for each 100 g of Rhus verniciflua extract solids It is preferable as an active ingredient of the pharmaceutical composition for preventing or treating menopausal syndrome in that it can be obtained.

The present invention provides a having estrogenic activity, sumac (Rhus verniciflua) extract the menopausal syndrome preventive or therapeutic pharmaceutical composition of which, as an active ingredient.

In the present invention, sulfuretin, Garbanzol, Resorcinol, Fustine, Fistine, Butein and Butin have estrogen activity. Provided is a nutraceutical composition for preventing or improving menopausal syndrome, which comprises at least one flavonoid compound selected as an active ingredient.

The present invention provides a health functional food composition for preventing or improving menopausal syndrome having an estrogen activity, Rhus verniciflua extract as an active ingredient.

Flavonoid compounds and lacquer extracts of the present invention have selective activity on estrogen receptor beta (ER-β), and thus are phytoestrogens with reduced side effects, compositions for the prevention and treatment of menopausal syndrome, or health functions for preventing or improving menopausal syndrome. It can be usefully used as a food composition.

1 is a schematic view showing a process of separating the flavonoid compound of the present invention from the sumac extract in Example 2.

Figure 2 shows the structural formula of the compound isolated in Example 2.

Figure 3a is a graph showing the cytotoxicity according to the concentration of the lacquer extract (RV10) and the cytotoxicity according to the mixed administration of the lacquer extract and estradiol (E2) in MCF7 / ERE cells in Experimental Example 1, Figure 3b It is a graph showing the cytotoxicity according to the concentration of seven species of flavonoid compounds isolated from the lacquer extract against MCF7 / ERE cells.

Figure 4a is a graph showing the in vitro estrogen activity according to the concentration of the lacquer extract (RV10), Figure 4b is an estrogen when treated with ICI 182.770 (ICI), an estrogen receptor inhibitor with the lacquer extract (RV10) It is a graph showing the activity, Figure 4c is a graph showing the estrogen activity according to the concentration of the seven flavonoid compounds isolated from the lacquer extract.

5A and 5B show estrogen-dependent genes, namely estrogen receptor alpha (ER-α), of seven species of the flavonoid compounds isolated in Example 2 and the lacquer extract powder of Example 1 in the estrogen dependent gene expression confirming experiment of Experimental Example 3; Northern blot images of estrogen receptor beta (ER-β), progesterone receptor (PR) and pS2 genes are shown.

6A and 6B show the expression rate of DMSO using the result of Experiment 3 as a negative control group as 1, and estrogen receptor alpha (ER-α), estrogen receptor beta (ER-β) and pS2 according to the type and concentration of the sample, respectively. It is a graph showing the change in the expression rate of the gene.

7A to 7F are results of confirming the selective expression of the estrogen receptor alpha or beta genes of Experimental Example 4, respectively, according to the concentrations of the lacquer extract (RV10), sulfuretin, gabansol, resorcinol, phycetin and fustin, respectively. It is a graph showing the estrogen activity of the estrogen receptor alpha (ER-α) and beta (ER-β).

8A to 8C show the body weight, the weight of the uterus (wet) and the weight of the uterus (dry) on the fourth day after the sample intraperitoneal injection in the Uterotrophic assay of Experimental Example 5.

In the present invention, sulfuretin, Garbanzol, Resorcinol, Fustine, Fistine, Butein and Butin have estrogen activity. The present invention relates to a pharmaceutical composition for preventing or treating menopausal syndrome, which comprises at least one selected flavonoid compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, sulfuretin, Garbanzol, Resorcinol, Fustine, Fistine, Butein and Butin have estrogen activity. The present invention relates to a pharmaceutical composition for preventing or treating menopausal syndrome, which comprises at least one selected flavonoid compound or a pharmaceutically acceptable salt thereof as an active ingredient.

One or more flavonoids selected from Sulfurintin (Sulfuretin), Garbanzol, Resorcinol, Resorcinol, Fustin, Fisetin, Butein and Butin Flavonoid) compounds can be isolated from Rhus verniciflua extract.

In order to obtain the lacquer extract, the wood portion of the lacquer is cut and dried in the shade for at least one week, preferably at least one month, more preferably at least three months, and then crushed or used as is. The upper limit of the drying period does not need to be set separately, and one that is dried for one year or several years may be used. In addition, it can be used to produce wood chips or sawdust without drying the wood part of the lacquer.

The lacquer tree processed as described above may be prepared by using water, an organic solvent or a mixture thereof as an extraction solvent. The lacquer extract may be extracted. The kind of organic solvent used at this time and the mixing ratio of water and an organic solvent are not specifically limited.

For example, the organic solvent may be one or more solvents selected from the group consisting of lower alcohols, hexane, acetone, ethyl acetate, chloroform, and diethyl ether. The lower alcohol may be an alcohol having 1 to 6 carbon atoms. For example, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol may be used as the lower alcohol. Organic solvents include polar solvents such as acetic acid, dimethyl-formamide (DMFO) and dimethyl sulfoxide (DMSO), acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, 2,2,4-trimethylpentane, and decane. , Cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene And nonpolar solvents such as benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride, and THF (Tetrahydrofuran).

In one embodiment of the present invention, the lacquer extract may be an extract of water, a lower alcohol or a mixture thereof, preferably, a lacquer hydrothermal extract or a lacquer ethanol aqueous solution, for example, lacquer alcohol extract.

As used herein, the term 'extract' also includes fractions that additionally fractionate the extract. That is, the sumac extract includes not only those obtained by using the aforementioned extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, the lacquer extract may be an ethyl acetate fraction obtained by fractionation with hexane to remove the nonpolar portion and the remaining aqueous layer fractionated with ethyl acetate.

In addition, the term 'extract' as used herein referring to the lacquer extract includes not only the crude extract obtained by treating the extracting solvent on the lacquer, but also the processed product of the lacquer extract. For example, the sumac extract may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, fractions obtained by passing the extract or fraction through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Selected from Sulfurintin, Garbanzol, Resorcinol, Fustin, Fisetin, Butein and Butin Any one or more flavonoid compounds may be isolated.

In one embodiment of this separation process, first extract the lacquer extract with hexane to separate the non-polar part, and the remaining aqueous layer is fractionated with ethyl acetate and concentrated under reduced pressure, the hexane layer, ethyl acetate layer, water divided by polarity Three fractions of the layer can be separated by silica gel and then separated using a variety of chromatographic methods such as sephadex LH-20, ODS-A, diol, preparative MPLC, or preparative HPLC. Figure 1 shows a schematic process of obtaining each compound through this process.

The present invention includes not only the flavonoid compounds but also all pharmaceutically acceptable salts thereof, and possible solvates, hydrates, racemates, or stereoisomers which may be prepared therefrom.

The flavonoid compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and the salt is an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, Xylene Sulfonate, Phenyl Acetate, Phenylpropionate, Phenyl Butyrate, Citrate, Lactate, Hydroxybutyrate, Glycolate, Maleate, Tartrate, Methanesulfonate, Propanesulfonate, Naphthalene-1-sulfonate , Naphthalene-2-sulfonate or mandelate.

The acid addition salts according to the invention can be prepared by conventional methods, for example, by dissolving the flavonoid compound in an excess of aqueous acid solution, and using the water miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation.

An equivalent amount of the flavonoid compound and an aqueous acid solution or alcohol may be heated, and then the mixture is evaporated to dryness, or the precipitated salt may be produced by suction filtration.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-dissolving compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).

Sulfurin, Garbanzol, Resorcinol, Fustine, Fistine, Butene, Butin and Butin of the present invention Flavonoid compounds or lacquer extract showed MCF7 / ERE cell viability of at least 80% up to 100 μg / ml, showing no cytotoxicity.

In addition, when the flavonoid compound or lacquer extract of the present invention was treated with an estrogen receptor inhibitor ICI 182.770, the activity was lowered, the flavonoid compound or lacquer extract showed a concentration dependence and was dependent on the estrogen receptor.

In addition, it was confirmed that, among the flavonoid compounds of the present invention, phycetin and fustin, which are the main components of the lacquer extract, did not exhibit aromatase inhibitory activity.

In addition, as a result of confirming the estrogen activity through the Uterotrophic assay of the lacquer extract of the present invention was not able to confirm the concentration dependence, the uterine weight was significantly increased at a dose of 20 mg / kg or more in vivo ( in In vivo ) estrogen activity was confirmed.

On the other hand, the term 'comprising as an active ingredient' in the present specification is a sulfurintin (Sulfuretin), Gabanzol (Garbanzol), Resorcinol (Resorcinol), Fustin (Fisetin), Butane It is meant to include an amount sufficient to achieve the efficacy or activity of any one of the flavonoid compounds selected from (Butein) and Butin (Butin) or a pharmaceutically acceptable salt thereof, or lacquer extract. The pharmaceutical composition for preventing or treating menopausal syndrome of the present invention preferably comprises 0.1 to 50% by weight of the flavonoid compound or lacquer extract based on the total weight of the composition, but is not limited thereto.

The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.

The composition according to the present invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. have. Carriers, excipients and diluents that may be included in the compositions of the present invention 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 preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the composition of the present invention, for example, starch, calcium carbonate, sucrose (sucrose), 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, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The composition of the present invention can be administered orally or parenterally, any parenteral administration method can be used, systemic or topical administration is possible, but systemic administration is more preferred, and intravenous administration is most preferred.

Preferred dosages of the compositions of the present invention vary depending 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, the composition of the present invention is preferably administered at 0.0001 to 0.03 g / kg, preferably at 0.001 to 8 mg / kg. 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.

In addition, the present invention is any one selected from Sulfurintin (Sulfuretin), Garbanzol, Resorcinol (Resorcinol), Fustine (Fustin), Pisetin (Fisetin), Butein (Butein) and Butin (Butin) It provides a health functional food composition for preventing and improving menopausal syndrome containing one flavonoid compound or lacquer extract as an active ingredient.

In the health functional food composition, there is no particular limitation on the type of food. Examples of foods to which the above-mentioned substances may be added include dairy products, various soups, drinks, meat, sausages, breads, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and the like includes all the health functional foods in the conventional sense.

The flavonoid compound or lacquer extract of the present invention may be added to food as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). Generally, the amount of the compound in the health food can be added at 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

When preparing a beverage, there are no special limitations to the other ingredients except for containing the flavonoid compound or lacquer extract as essential ingredients in the ratios indicated, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . 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 natural carbohydrates is generally from about 1 to 20 g, preferably from about 5 to 12 g per 100 compositions of the present invention.

In addition to the flavonoid compounds or lacquer extract of the present invention, various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, coloring and neutralizing agents (such as cheese, 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 flavonoid compound or lacquer extract of the present invention may contain a pulp for the production of 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 generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the flavonoid compounds or lacquer extract of the present invention.

As used herein, 'menopausal syndrome' refers to a symptom caused by a decrease in hormone production in postmenopausal women, and the menopausal or menopausal symptoms generally include hot flashes, sweating, sweating during sleep, dry skin, vaginal dryness, vaginal atrophy, Lower urethral atrophy, intercourse pain, vaginitis, cystitis, urination pain, urination, concentration disorders, short-term memory disorders, anxiety, nervousness, decreased memory, decreased libido, myalgia or joint pain.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Preparation Examples. However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Preparation Examples.

Example 1 Preparation of Sumac Extract

The wood parts of the sumac collected from Wonju-si, Gangwon-do were naturally dried for more than 3 months, and then crushed to about 11 × 1 × 0.2cm in the wood chip maker.

10 times of water was added to the lacquer wood chips, followed by extraction of hot water at 90 to 110 ° C. for 3 hours, followed by filtration to remove impurities, and concentration under reduced pressure to prepare 15 brix lacquer extract concentrates.

With respect to 100 parts by weight of the concentrate, 10 parts by weight of ethanol was added, and then left for 3 days, and concentrated again to remove ethanol. The same amount of dextrin was added as an excipient based on the solids of the concentrate from which the alcohol was removed, followed by spray drying to prepare a lacquer extract powder (hereinafter also referred to as 'RV10').

Example 2: Isolation of Flavonoid Compounds

The lacquer extract powder (490 g) of Example 1 was separated with hexane to separate the non-polar part, and the remaining aqueous layer was partitioned with ethyl acetate, and concentrated under reduced pressure. The hexane layer, the ethyl acetate layer, and the water layer divided by polarity were separated. In the ethyl acetate layer of the fractions, a single compound was isolated and purified by applying various chromatographic techniques.

200 g of the concentrated ethyl acetate layer was fixed as silica gel (silica gel, 230-400mesh, Merck, Germany), mobile phase chloroform (Chroloform, Dae-jung chemical, Korea), methanol (Methanol, Dae-jung chemical, Korea) Open column chromatography was performed using a mixed solvent composition and primary fraction fractionation was performed.

3 out of 10 fractions (SB3-6-3,3g) consisted of hexane (Hexane, Dae-jung chemical, Korea), and ethyl acetate (Ethyl acetate, Dae-jung chemical, Korea) And garbanzol (2) was isolated by silica gel open column, and resorcinol (4) and new material (10) were purified using prep-HPLC (High Performance Liquid Chromatography, Varian system Prostar 210, USA). It was. Fraction No. 4 (SB3-6-4, 3g) was eluted with 4-chlorobenzoic acid (11) by eluting with chloroform and methanol in a silica open column, and using 2-plybenzne- prep-HPLC in the remaining fractions. 1,4-diol (17) was purified. Fraction 5 (SB3-6-5, 3g) was subjected to a silica open column under chloroform and methanol solvent conditions to obtain 5-deoxykaempferol (7), and the precipitate produced when the remaining fraction was dissolved in ethyl acetate was filtered. Butin (8) and butein (5) were purified through a reverse phase chromatography (ODS-A, 12nm S-75㎛, TMC GEL) column process. Fraction 6 (SB3-6-6, 8g) was subjected to a silica open column using chloroform and methanol to obtain Sulfuretin (9) and urs-12-en-18αH-3β-O-β-D-glucopyranoside (18). Was isolated, and the remaining fractions were purified by 4-hydroxybenzoic acid (11) and guibourtacacidin (19) through prep-HPLC, respectively. Fraction No. 7 (SB3-6-7, 60 g), which had the highest amount, eluted the silica open column with chloroform and methanol solvent composition and separated the fraction by quipcetin (6), 2,3 through prep-HPLC. trans taxifolin (12) was isolated, and the remaining fractions were prepared by reverse phase chromatography open column and prep-HPLC purification, respectively, 2.3 cis fustin (14), 2.3 cis taxifolin (15), 2- (3 ', 4'-). Dihydroxyl) -2,6 dihydroxubenzofuran-3- (2H) -one (13) was obtained. In addition, the precipitate obtained by dissolving ethyl acetate was filtered to obtain fustin (3). Filtin (1) was obtained by filtering the precipitate formed after dissolving fraction No. 9 (SB3-6-9, 24 g) in chloroform (see FIG. 1).

A total of 19 single compounds isolated from the lacquer extract were analyzed by TLC (Thin Layer Chromatography, Silica gel 60 F _254, Silica gel 60 RP-18 F ₂₅₄ S, Merck, Germany) and analytical HPLC (Varian 920-LC, USA). Purity was confirmed using NMR (Nuclear Magnetic Resonance, Varian systems 500MHz, USA) and MS (LC-MS; UPLC / ESI Q-TOF MS, waters, USA, GC-MS; 7790/5973 MS system, Agilent, USA) spectroscopy equipment was used to confirm the structural identification, molecular weight, and molecular formula.

The names and molecular formulas, molecular weights and separation amounts of the separated substances are summarized in Table 1, and the structural formulas of the separated substances are shown in FIG. 2.

Table 1

division	Substance	Molecular weight	Separation
(One)	Fisetin	C 15 H 10 O 6 MW = 286	2 g
(2)	Garbanzol	C 15 H 12 O 5 MW = 272	750 mg
(3)	Fustin	C 15 H 12 O 6 MW = 288	13 g
(4)	Resorcinol	C 6 H 6 O 2 MW = 110	38 mg
(5)	Butein	C 15 H 12 O 5 MW = 272	60 mg
(6)	Quercetin	C 15 H 10 O 7 MW = 302	7 mg
(7)	5-deoxykaempferol	C 15 H 10 O 5 MW = 270	25 mg
(8)	Butin	C 15 H 12 O 5 MW = 272	400 mg
(9)	Sulfuretin	C 15 H 10 O 7 MW = 270	125 mg
10	New	C 15 H 10 O 5 MW = 288	220 mg
(11)	4-hydroxybenzoic acid	C 7 H 6 O 3 MW = 270	3 mg
(12)	2,3 trans Taxifolin	C 15 H 12 O 7 MW = 304	140 mg
(13)	2- (3 ', 4'-dihydroxyl) -2,6 dihydroxubenzofuran-3- (2H) -one	C 15 H 12 O 6 MW = 288	280 mg
(14)	2.3 cis Fustin	C 15 H 12 O 6 MW = 288	50 mg
(15)	2.3 cis Taxifolin	C 15 H 12 O 7 MW = 304	5 mg
16)	2.4 dihydroxybenzaldehyde	C 7 H 6 O 3 MW = 138	2 mg
(17)	2.methoxybenzne-1,4-diol	C 7 H 8 O 6 MW = 140	3 mg
(18)	urs-12-en-18αH-3β-O-β-D-glucopyranoside	C 36 H 60 O 6 MW = 588	100 mg
(19)	Guibourtacacidin	C 15 H 14 O4 MW = 274	4 mg

Experimental Example 1: Cytotoxicity

Seven kinds of flavonoid compounds isolated from Example 2 for MCF7 / ERE cells in which MCF7 cells, which are breast cancer cell lines, contain 3x estrogen response element (ERE) in a promoter region and were permanently transfected with plasmid using luciferase as a reporter gene. The cytotoxicity of the sumac extract powder of Example 1 was confirmed by the following method.

MCF7 / ERE cells were plated at a concentration of 1.2 X 10 ⁴ in 96 well plates and the samples were treated at various concentrations after 24 hours. After 24 hours of incubation at 37 ° C. in a 5% CO ₂ incubator, cytotoxicity was performed using a CellTiter 96 AQ _ueous Non-Radioactive Cell Proliferation Assay kit (Promega, USA). After removing the cell culture medium, the medium was changed to a medium containing MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfopheny) -2H-tetrazolium) solution. After standing at 1 ° C. for 1 hour, the absorbance values were compared with the control group by reading the absorbance at 490 nm using a microplate reader. The negative control group was treated with 0.1% DMSO instead of the sample, and the positive control group was treated with 100 pM of ethynyl estradiol (E2).

Cytotoxicity according to the concentration of the lacquer extract (RV10) and the cytotoxicity when 100 pM simultaneous treatment with the lacquer extract and ethynyl estradiol (E2) was shown in Figure 3a. Sumac extract (RV10) showed MCF7 / ERE cell viability of at least 90% up to 100 μg / ml, showing no cytotoxicity. In addition, even when the sumac extract and ethynyl estradiol (E2) 100 pM co-treatment did not show cytotoxicity.

In addition, the concentrations of Sulfurin, Garbanzol, Resorcinol, Fustin, Fisetin, Butein and Butin were isolated from Sumac extract. Check the cytotoxicity according to the shown in Figure 3b. Seven flavonoid compounds isolated from the lacquer extract also exhibited MCF7 / ERE cell viability of at least 80% to 100 μg / ml, and did not show cytotoxicity.

Experimental Example 2: In Vitro in vitro Estrogen activity

In order to confirm the in vitro estrogen activity of seven species of the flavonoid compounds isolated in Example 2 and the lacquer extract powder of Example 1, luciferase reporter gene analysis was performed by the following method. MCF7 / ERE cells incubated with 5% charcoal stripped FBS phenol-red free DMEM medium for 24 hours were plated with 1.2 X 10 ⁴ cells per well in 96 well plates and treated with various concentrations of lacquer extract or isolates after 24 hours. . After 24 hours of incubation at 37 ° C. in a 5% CO ₂ incubator, luciferase activity in lysates obtained by lysis of cells was measured using a mono luciferase assay kit (Promega, USA). The total protein mass in lysate was quantified using a Bio-Rad protein assay kit (Bio-Rad, USA) to correct luciferase values.

When the sumac extract (RV10) was treated to a concentration of 100 mg / ml, it showed a concentration-dependent estrogen action (see FIG. 4A), which was completely inhibited by the estrogen receptor inhibitor ICI 182.770 (ICI) (see FIG. 4B). Ethyl estradiol (E2) and RV10 treatment at the same time did not show an anti-estrogen action, but rather showed an increase in estrogen activity.

Among the seven flavonoid compounds, sulfuretin, gabansol, butin and butane showed stronger estrogen action than other substances, and high concentrations of antiestrogenic action appeared (see FIG. 4C). And even when co-administered with ethynyl estradiol (E2), estrogen action was not inhibited by the lacquer isolates.

Experimental Example 3: Confirm estrogen dependent gene expression

RT-PCR (Reverse transcription-polymerase chain reaction) was carried out in order to confirm the estrogen-dependent gene expression level of seven species of the flavonoid compounds isolated in Example 2 and the lacquer extract powder of Example 1 in the following manner.

The expression of estrogen receptor alpha (ER-α) is reduced by estrogen, but the expression of progesterone receptor (PR) and pS2 genes is increased, and the expression of these genes was observed to observe the estrogen action of the isolates. However, estrogen receptor beta (ER-β) was observed to determine whether the expression of the ER-β-mediated reporter gene assay is increased by the expression of flavonoid compounds isolated.

Among the flavonoid compounds isolated in Example 2, the estrogen-dependent genes of the lacquer extract powders of the seven species and Example 1, namely the estrogen receptor alpha (ER-α), the estrogen receptor beta (ER-β), the progesterone receptor (PR) and pS2 Northern blot images of genes are shown in FIGS. 5A and 5B. Beta-actin (β-actin) was used as a control to confirm gene expression. In addition, the expression rate of DMSO used as a negative control group was reported as 1, and changes in the expression rates of the estrogen receptor alpha (ER-α), the estrogen receptor beta (ER-β), and the pS2 gene according to the type and concentration of the sample were shown in FIGS. Shown in

The extract of Rhus oleracea (RV10) and Resosinol showed estrogen action at low concentrations, and the expression of pS2 gene was decreased at low concentrations, especially the expression of estrogen receptor beta (ER-β). In addition, sulfuretin, butein, and butin have higher expression of estrogen-dependent genes than lacquer extract (RV10). Gene expression was also significantly increased, which may contribute to the estrogen action of R. chinensis extract (RV10).

Experimental Example 4: Confirmation of Selective Expression of Estrogen Receptor Alpha or Beta Gene

HEK293T cells were seeded in a 48-well plate at a concentration of 4.0 x 10 ⁴ cells / well. After 24 hours, the medium was removed and ER-α-plasmid, ER-β-plasmid, pERE reporter gene plasmid, and pRL-TK lipofectamine. 2000 (invitrogen, USA) was mixed with medium mixed with 5% charcoal stripped FBS phenol-red free DMEM and temporarily transfected. After 24 hours the samples were treated at various concentrations. After 24 hours of incubation at 37 ° C. in a 5% CO ₂ incubator, luciferase activity in lysates obtained by lysis of cells was measured using a dual luciferase assay kit (Promega, USA). The results were determined by the ratio of firefly luciferase activity and RL-TK derived renilla luciferase activity.

According to FIG. 7A, the lacquer extract (RV10) was able to confirm that estrogen receptor beta (ER-β) mediated estrogen action was much greater, and estrogen receptor beta (ER-β) mediated in sulfuretin and gaborazole of FIGS. 7B and 7C. It was confirmed that the estrogen action is remarkably large, the estrogen receptor beta (ER-β) mediated estrogen action in the case of the resorcinol, phycetin and fustin of Figure 7d to 7f compared to the estrogen receptor alpha (ER-α) It was somewhat larger, but not large compared to sulfuretin and gabansol.

Experimental Example 5: Uterotrophic assay

Seventy seven-week-old female Sprague-Dawley rats were purchased and adapted for one week, and 60 uterus were extracted under ether anesthesia. After 2 weeks recovery, 1) sham control, 2) control, 3) ethynylestradiol (EE) alone, 4) sumac extract (RV10) low concentration (20mg / kg) , 5) medium concentration group of lacquer extract (100mg / kg), 6) high concentration of lacquer extract and 7) simultaneous administration of ethynyl estradiol (EE) and lacquer extract (500mg / kg). The sumac extract was suspended orally by 1% CMC, and ethynyl estradiol (EE) was administered intraperitoneally for 3 consecutive days at a dose of 0.2 μg / kg / day. On day 4, rats were sacrificed under ether anesthesia, and uterus was extracted, and wet and dry weights were measured. When uterine weight was significantly increased compared to uterine extraction control group, estrogen action was determined.

Figure 8a shows the weight, Figure 8c shows the weight of the uterus, Figure 8d shows the weight of the dried uterus. Uterine weight of the ethynyl estradiol (EE) treated group was similar to that of the Sham group. Sumac extract (RV10) treated group significantly increased uterine weight but showed no concentration dependence. In the case of co-administration with ethynyl estradiol (EE), wet uterine weight was not significantly higher than that of ethynyl estradiol (EE) treatment group, but increased. Body weight did not change significantly by administration of the sumac extract.

Formulation Example 1 Preparation of Pharmaceutical Formulation

1-1. Manufacture of powder

500 mg of the RV10 of Example 1 or the flavonoid compound of Example 2

Lactose 100 mg

Talc 10 mg

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

1-2. Manufacture of tablets

500 mg of the RV10 of Example 1 or the flavonoid compound of Example 2

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.

1-3. Manufacture of capsule

500 mg of the RV10 of Example 1 or the flavonoid compound of Example 2

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

1-4. Preparation of Injectables

500 mg of the RV10 of Example 1 or the flavonoid compound of Example 2

Appropriate sterile distilled water for injection

pH adjuster

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

1-5. Preparation of liquid

100 mg of RV10 of Example 1 or the flavonoid compound of Example 2

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, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled into a brown bottle. The liquid is prepared by sterilization.

Preparation Example 2 Preparation of Health Functional Food (Powder Type)

1000 mg of RV10 of Example 1 or the flavonoid compound of Example 2

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folic acid 50 mg

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Preparation Example 3 Preparation of Health Functional Food (Drink Type)

1000 mg of RV10 of Example 1 or the flavonoid compound of Example 2

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional method for preparing healthy beverages, and stirring and heating at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2l container, sealed sterilization and refrigerated and then stored in a healthy beverage Used to prepare the composition.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Preparation Example 4 Preparation of Health Functional Food (Chewing Gum)

Gum base 20%

Sugar 76.36-76.76%

0.24 to 0.64% of RV10 of Example 1 or the flavonoid compound of Example 2

1% fruit flavor

Water 2%

Chewing gum was prepared using conventional methods using the above composition and content.

Preparation Example 5 Preparation of Health Functional Food (Flour Food)

0.5 to 5 parts by weight of the RV10 of Example 1 or the flavonoid compound of Example 2 was added to 100 parts by weight of wheat flour, and the mixture was used to prepare bread, cake, cookies, crackers, and noodles to prepare health promoting food.

Preparation Example 6 Preparation of Health Functional Food (Dairy Product)

5-10 parts by weight of the RV10 of Example 1 or the flavonoid compound of Example 2 was added to 100 parts by weight of milk, and various dairy products such as butter and ice cream were prepared using the milk.

Flavonoid compounds and lacquer extract of the present invention can be usefully used as a composition for the prevention and treatment of menopausal syndrome, or a health functional food composition for the prevention or improvement of menopausal syndrome.

Claims (5)

1. Any one selected from Sulfurintin, Garbanzol, Resorcinol, Fustin, Fisetin, Butin and Butin with estrogen activity A pharmaceutical composition for preventing or treating menopausal syndrome, comprising at least one flavonoid compound or a pharmaceutically acceptable salt thereof as an active ingredient.
2. The method of claim 1, wherein the flavonoid compound is Sulfurintin (Sulfuretin), Garbanzol, Resorcinol (Resorcinol), Fustin (Fustin) or Pisetin (Fisetin), or the prevention of menopausal syndrome Therapeutic pharmaceutical composition.
3. A pharmaceutical composition for preventing or treating menopausal syndrome having an estrogen activity as an active ingredient of Rhus verniciflua extract.
4. Any one selected from Sulfurintin, Garbanzol, Resorcinol, Fustin, Fisetin, Butin and Butin with estrogen activity A health functional food composition for preventing or improving menopausal syndrome comprising at least one flavonoid compound as an active ingredient.
5. A health functional food composition for preventing or improving menopausal syndrome having an estrogen activity as an active ingredient of Rhus verniciflua extract.

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