KR101683344B1 - Composition comprising carnosic acid for preventing or improving menopausal symptoms - Google Patents

Abstract

The present invention relates to a composition for preventing or ameliorating symptoms of menopausal symptoms comprising a carnoic acid compound as an active ingredient and a composition (pharmaceutical composition, food composition) for preventing or ameliorating the symptoms of menopausal symptoms, And a medicament. The carnosic acid compound according to the present invention is useful for the prevention and treatment of weight gain due to decreased estrogen appearing in menopausal or menopausal period; Fat accumulation; Increased inflammatory cytokines; And the effect of reducing the blood level increase of insulin, leptin, triglyceride and free fatty acid is remarkable. Therefore, the carnosic acid of the present invention is a substance that can prevent, treat, or ameliorate symptoms appearing in menopausal or menopausal period, and can be utilized not only as a pharmaceutical such as a pharmacological composition, but also as a functional food. It is expected that the value will be very high when applied to functional food industry.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or improving menopausal symptoms comprising carnosic acid as an active ingredient,

The present invention relates to a composition for preventing or ameliorating symptoms of menopausal symptoms comprising a carnoic acid compound as an active ingredient and a composition (pharmaceutical composition, food composition) for preventing or ameliorating symptoms of menopausal symptoms, And a medicament.

In the life of modern industrialized by the development of science and civilization, the life span of human being is gradually extended, and at the same time, the immune system is degraded due to various stresses, pollution and spontaneous aging phenomenon, and aging progresses. Especially, women suffer physical and mental difficulties due to the menopausal symptoms gradually starting after their forties.

On the other hand, menopause is a kind of endocrine syndrome, which is the transition period in which physiological function and sexual function are reduced or lost due to the reduction of female hormone, estrogen, caused by the general and gradual aging of ovarian function. Symptoms of these menopausal symptoms include symptoms due to vascular changes such as facial flushing, tachycardia, sweating or headache, symptoms due to musculoskeletal changes such as myalgia, arthralgia and back pain, symptoms due to genitourinary changes such as urinary frequency or urinary incontinence And depression, depression, loss of concentration, and dizziness. In addition, symptoms such as decreased visual acuity and changes in skin and hair occur, and osteoporosis and cardiovascular diseases caused by hormonal changes Which can lead to health-related diseases.

Therefore, in order to improve the physical and mental health and quality of life of middle-aged women, it was required to develop a therapeutic agent capable of improving the symptoms of menopausal symptoms. To improve these symptoms, hormone replacement therapy and non-steroidal drugs were developed . However, most of these drugs are known to have side effects such as headache and weight gain. Especially, in the case of estrogen replacement therapy, since the hormone is artificially administered to the body, (Swaran L., et al., Obstetrics & Gynecology , 91, 678-684, 1998). It is known that the risk of stroke, breast cancer and uterine cancer may increase.

Therefore, due to these problems, there is a growing interest in replacing estrogen therapy with natural methods of ingestion in the form of food or additives, and it is required to develop a new menopausal remedy that has the effect of alleviating symptoms of menopause without side effects In fact.

On the other hand, carnosic acid is a main active ingredient that can be separated from rosemary or sage, and has a structure of 11,12-dihydroxy-8,11,13-abietatrien-20-oic acid, and a phenolic diterpene having a molecular weight of 332.4 Da (phenolic diterpenes), which are known to have excellent antioxidative effects.

Therefore, the inventors of the present invention have been developing a new composition for improving the symptoms of menopausal symptoms without side effects while improving the symptoms of menopausal symptoms. In the case of feeding a carnoic acid compound to ovariectomized menopausal model mice, weight gain ; Fat accumulation; Increased inflammatory cytokines; And an effect of preventing and ameliorating symptoms of menopausal or menopausal symptoms by effectively reducing the blood level increase of insulin, leptin, triglyceride and free fatty acid.

Korean Patent No. 10-1087608 Korean Patent No. 10-0874778 Korean Patent Publication No. 10-2010-0115840

Accordingly, an object of the present invention is to provide a composition which is stable to human body even after long-term use and can effectively prevent, treat or ameliorate symptoms of menopausal symptoms.

Another object of the present invention is to provide a health functional food which is stable to human body and can prevent or ameliorate menopausal symptoms effectively even for a long period of use.

It is still another object of the present invention to provide a medicament which is stable to human body and can prevent or treat symptoms of menopausal symptoms for a prolonged period of time.

In order to achieve the above-mentioned object of the present invention,

The present invention provides a composition for preventing, treating or ameliorating menopausal symptoms comprising, as an active ingredient, a carnosic acid compound or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the composition may be a pharmaceutical composition or a food composition.

The present invention also provides a health functional food for preventing or ameliorating menopausal symptoms comprising the composition.

In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foodstuff, confectionery, pizza, ramen, other noodles, rice cake, gum, candy, ice cream, alcoholic beverage, ≪ / RTI >

The present invention also provides a medicament for the prevention or treatment of menopausal symptoms comprising the composition.

The carnosic acid compound according to the present invention is useful for the prevention and treatment of weight gain due to decreased estrogen appearing in menopausal or menopausal period; Fat accumulation; Increased inflammatory cytokines; And the effect of reducing the blood level increase of insulin, leptin, triglyceride and free fatty acid is remarkable. Therefore, the carnosic acid of the present invention is a substance that can prevent, treat, or ameliorate symptoms appearing in menopausal or menopausal period, and can be utilized not only as a pharmaceutical such as a pharmacological composition, but also as a functional food. It is expected that the value will be very high when applied to functional food industry.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram illustrating the outline of the step-wise molecular mechanism in the relevant liver and adipose tissue following the feeding / treatment of a carnosic acid compound.
FIG. 2 is a schematic diagram schematically showing an experimental plan of an animal experimental group according to the type of dietary and ovariectomy.
FIG. 3 is a graph showing the degree of phosphorylation of AMPK (FIG. 3A) and ACC (FIG. 3B) by western blot analysis as indexes closely related to lipid metabolism in the liver tissues of animal experimental groups according to dietary type and ovariectomy to be.
Fig. 4 shows the results of SREBP1c (Fig. 4A), FAS (Fig. 4B), PPAR? (Fig. 4C) and CPT-1 (Fig. 4D) genes related to lipid biosynthesis in liver tissues according to the animal test groups of the present invention, MRNA expression levels of the cells were measured by Real-time PCR.
Fig. 5 shows the results of PPARy (Fig. 5A), aP2 (Fig. 5B) and LPL (Fig. 5C) gene mRNA The expression level was measured by real-time PCR.
FIG. 6 is a graph (FIG. 6A) showing the results of histological analysis of adipocyte size according to an animal test group of the present invention depending on dietary type and ovariectomy (FIG. 6A) .
FIG. 7 shows mRNA expression levels of the inflammatory cytokines TNF-a (FIG. 7A) and IL-6 (FIG. 7B) genes of animal experimental groups according to the type of diet and ovariectomy by real-time PCR to be.

The present invention is characterized by providing a composition for preventing, treating or ameliorating menopausal symptoms comprising a carnosic acid compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The carnosic acid compound defined in the present invention is characterized by having the following formula (1).

≪ Formula 1 >

The carnosic acid compounds according to the present invention can be isolated from nature or can be prepared by chemical synthesis methods known in the art.

Such carnosic acid compounds of the present invention may be prepared as pharmaceutically acceptable salts according to methods conventional in the art.

Pharmaceutically acceptable salts include acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be filtered by suction.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid glutamic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid may be used as the acidic compound, such as glutaric acid, glutamic acid, glutaric acid, glucuronic acid,

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving a compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of the present invention. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate (Salicylate) salts, which are known in the art and which are known in the art, such as, for example, ≪ / RTI >

The composition of the present invention may be a pharmaceutical composition or a food composition.

First, the composition of the present invention is a pharmaceutical composition comprising the carnosic acid as an active ingredient, and can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients, A disintegrant, a sweetening agent, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the carnosic acid of the present invention may be contained in the composition at a concentration of 1 to 1000 μM, and the carnosic acid of the present invention may be contained in the composition in an amount of 0.1 to 90 % ≪ / RTI > by weight.

Examples of diseases for which the pharmaceutical composition of the present invention can exhibit a therapeutic effect include diseases associated with decreased estrogen appearing in the menopausal period (or menopausal period) such as facial flushing, sweating, dry skin, vaginal dryness, vaginal atrophy, , Sexual intercourse, vaginitis, cystitis, dysuria, diarrhea, concentration disorders, short term memory impairment, anxiety, nervousness, memory loss, loss of libido, myalgia, arthralgia or osteoporosis.

The pharmaceutical composition of the present invention may further contain, as an active ingredient, a substance having an effect of treating a disease (symptom) due to estrogen depression appearing in the menopausal period (or menopause) in addition to carnosic acid.

In addition, the present invention provides the use of a composition comprising, as an active ingredient, carnosic acid for the manufacture of a medicament for the prevention or treatment of menopausal symptoms (symptoms). The composition of the present invention containing carnosic acid as an active ingredient can be used for the manufacture of medicines for the prevention or treatment of menopausal symptoms.

The present invention also provides a method for the prevention or treatment of a menopausal disease (symptom) comprising administering to a mammal carnosic acid.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like.

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

The composition according to the present invention can be used alone or in combination with methods for the prevention and treatment of menopausal symptoms (symptom) or using surgery, hormone therapy, drug therapy and biological response modifiers.

Since the carnosic acid compound itself according to the present invention has little toxicity and side effects, it can be safely used for long-term use for preventive purposes.

The present invention also provides a food composition comprising carnosic acid as an active ingredient.

Such a food composition may contain, as an additional ingredient, various flavors or natural carbohydrates such as an ordinary food composition in addition to an active ingredient carnosic acid.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition to the carnosic acid as an active ingredient, the food composition may also contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates such as cheese and chocolate, Acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

Carnosic acid, which is an active ingredient of the present invention, is a compound derived from a natural substance and has little toxicity and side effects. Therefore, it can be safely used for long-term administration for the purpose of improving menopausal symptoms.

The present invention also provides a health functional food for preventing or ameliorating symptoms of menopausal (or menopausal) symptoms comprising carnosic acid as an active ingredient.

The health functional foods of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of preventing and improving menopausal symptoms.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in tablets may be prepared by granulating a mixture of carnosic acid, which is an active ingredient of the present invention, with an excipient, a binder, a disintegrant and other additives in a usual manner, And the mixture can be directly compression-molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule type health functional food can be prepared by filling a mixture of carnosic acid, which is an active ingredient of the present invention, with an additive such as an excipient into a normal hard capsule, and the soft capsule is a carnoic acid carnosic acid) with an excipient such as an excipient into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of carnosic acid, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like by a conventionally known method, It can be applied to other skin preparations, or the surface can be coated with materials such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of carnosic acid, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like into a granule by a conventionally known method, , A mating agent, and the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example >

Statistical analysis

Statistical analysis was performed using the SAS package (9.3, SAS Institute Inc., NC, USA) and the data were expressed as mean ± standard deviation. One-way ANOVA and Duncan's multiple test were used to analyze the statistical differences between groups, and P <0.05 was considered statistically significant.

< Example  1>

Animal experiment and ovariectomy

Seventy-seven-week-old female C57BL / 6J mice were purchased from a total of 78 dogs. After a week of adaptation, animals were sacrificed at 8 weeks of age. (N = 13) in each group. The non-excision group was classified into sham-operation group and non-noxious group. And the ovariectomized group removed the ovaries. At this time, 8-week-old C57BL / 6J mice were anesthetized by intraperitoneal injection (47.5 mg / kg body weight) of anesthetics mixed with 1: 2 ratio of zoletile and xylazine, Respectively. After general surgical treatment, the surgical site and its surroundings were disinfected, and skin, muscle, and peritoneal incisions were performed under sterile conditions. Both ovaries were exposed and the ovaries were removed and the resection site was ligated and sutured. The diets fed to the animals were divided into three groups: general diet based on AIN-93G diet (fat 15%), high fat diet (45% fat), normal diet + 0.02% carnosic acid, high fat diet + 0.02% Respectively. During the experimental period, fresh diets were fed once every 2 to 3 days and body weight was measured and administered once a week (see FIG. 2). When the animals reached 20 weeks of age, all animals were sacrificed and tissues such as blood, uterus, liver, and fat were collected. Blood was centrifuged at 1,550 × g for 20 minutes, serum was separated and stored at -80 ° C., and stored at -80 ° C. until analysis of tissues such as liver, uterus, and fat. Some of the adipose tissue was stored in 10% formalin for tissue staining.

Animal test group according to diet Experimental group Ovariectomy and dietary type Abbreviation One Non-cow extract + general diet group SHAM + ND 2 Ovariectomy + general diet OVX + ND 3 Ovariectomized + normal diet + carnosic acid OVX + ND + CA 4 Declassification Extraction + Notification Method SHAM + HD 5 Ovariectomy + high fat diet OVX + HD 6 Ovariectomy + high fat diet + carnot fat OVX + HD + CA

< Example  2>

Ovariectomy Success  Confirm

In this experiment, in order to investigate whether ovariectomy was actually successfully performed in the ovariectomized mouse prepared in Example 1, the mouse uterine weight and 17β-estradiol (Calbiotech, CA, USA, # ES180S-100). It is known that 17β-estradiol is a representative ovarian hormone and plays an important role in females' menstruation. In this experiment, in order to determine whether ovariectomy was performed normally, we measured the uterine weight of each experimental group, Was used as an indicator. At this time, the serum concentration of 17β-estradiol was measured according to the manufacturer's instructions.

Results of the measurement of uterine weight and 17β-estradiol in the mouse by experimental group Experimental group The uterus (g) Estradiol (pg / ml) SHAM + ND 0.46 0.10 ^a 18.92 ± 2.37 ^a OVX + ND 0.11 + - 0.10 ^c 5.97 ± 1.93 ^b OVX + ND + CA 0.11 ± 0.12 ^c 7.01 ± 2.06 ^b SHAM + HD 0.36 ± 0.09 ^b 18.99 ± 2.21 ^a OVX + HD 0.08 0.09 ^c 6.52 ± 1.56 ^b OVX + HD + CA 0.11 + - 0.14 ^c 7.43 ± 1.78 ^b Significance <.0001 <.0001

As a result, as shown in Table 2, both the uterine weight and the level of 17β-estradiol in the blood were lower in the ovariectomized experimental groups 2, 3, 5 and 6, and it was confirmed that the ovariectomy was successfully performed I could.

< Example  3>

Of ovariectomized mice Carnoic acid  Changes in body weight and fat mass according to salary

In this study, we examined the effect of carnosic acid supplementation on the changes in body weight and tissue fat mass in the menopausal model mice treated with surgical ovariectomy. For this, in each of the experimental groups prepared in Example 1, body weight, mammary fat pad, abdominal fat, gonadal fat, perirenal fat (Perirenal fat, upper mesenteric fat, and total fat were measured and are shown in Table 3 below.

Results of measurement of body weight and fat mass of mice by experimental group Experimental group Weight (g) Wired fat pad stomach
Fat reproduction
Sam Province Around the kidney
Fat Upper mesentery
Fat Total fat content SHAM + ND 22.82 ± 1.84 ^c 0.83 ± 0.21 ^d 0.77 + - 0.44 ^d 0.60 ± 0.37 ^d 0.40 ± 0.12 ^d 0.57 ± 0.35 ^d 3.18 ± 1.24 ^d OVX + ND 26.70 ± 1.39 ^b 1.63 ± 0.49 ^bc 2.04 ± 0.59 ^bc 1.23 ± 0.56 ^bcd 1.03 + - 0.33 ^c 1.08 ± 0.34 ^bc 7.02 ± 1.92 ^bc OVX + ND + CA 24.45 + - 1.12 ^c 1.28 ± 0.42 ^cd 1.50 + - 0.76 ^cd 0.99 ± 0.50 ^cd 0.70 + - 0.27 ^c 0.78 ± 0.32 ^cd 5.25 ± 2.02 ^cd SHAM + HD 28.11 ± 3.45 ^b 1.97 + - 0.68 ^cb 2.44 ± 1.13 ^bc 1.69 + 0.66 ^b 1.17 ± 0.48 ^cb 1.34 ± 0.40 ^b 8.61 ± 3.17 ^b OVX + HD 32.32 + 4.79 ^a 2.95 ± 1.75 ^a 3.88 ± 2.08 ^a 2.52 + 1.49 ^a 2.15 ± 1.10 ^a 1.78 ± 0.86 ^a 13.27 ± 6.88 ^a OVX + HD + CA 28.06 ± 3.26 ^b 2.07 ± 0.79 ^b 2.95 ± 1.52 ^ab 1.58 ± 0.76 ^cb 1.54 ± 0.72 ^b 1.15 ± 0.55 ^cb 9.30 + 4.09 ^b Significance <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001

As shown in Table 3, weight and body weight of the mice were significantly increased in both the high fat diet and the ovariectomized group, while the surgically induced menopausal condition In the experimental groups 3 and 6, in which the carnosic acid was administered to the mice, the body weight and the amount of fat in the tissues were decreased compared to the control group (2, 4) in which the carnosic acid was not fed.

As a result of the above results, the present inventors have found that the increased body weight and fat in the ovariectomized menopausal model mice can effectively reduce the carnosic acid supplementation.

< Example  4>

Of ovariectomized mice Carnoic acid  Changes in Fat Accumulation with Benefits

In this study, we examined the effects of carnosic acid supplementation on the changes of fat accumulation in these models in the menopausal model mice through surgical ovariectomy. For this purpose, insulin (Millipore Corp., Billerica, MA, USA, # EZRMI-13K), leptin (R & D, MI, USA, # MOB00) Triglyceride (Wako, OSAKA, JAPAN, # 290-63701) and free fatty acid (Wako, OSAKA, JAPAN, # 294-63601) were measured. For reference, these blood indicators are known to be elevated in the blood of overweight or obese people, and are used as an index of fat accumulation. At this time, the blood indicators were measured according to the manufacturer's instructions.

Measurement of insulin, leptin, triglyceride and free fatty acids in the blood of mice by experimental group Experimental group insulin
(ng / ml) Leptin
(mg / ml) Triglyceride
(mg / dL) Free fatty acid
(mEq / L) SHAM + ND 1.33 ± 0.29 ^c 0.59 + - 0.38 ^c 56.97 ± 22.37 ^b 0.85 ± 0.17 ^c OVX + ND 1.82 ± 0.49 ^c 1.61 + - 0.81 ^b 133.51 + - 74.22 ^c 1.16 ± 0.18 ^b OVX + ND + CA 1.55 + - 0.34 ^c 1.05 ± 0.58 ^bc 102.61 ± 63.99 ^cd 0.90 + - 0.14 ^c SHAM + HD 3.24 ± 0.59 ^b 1.62 ± 0.72 ^b 214.89 ± 78.12 ^b 1.12 ± 0.18 ^b OVX + HD 4.54 + 1.42 ^a 2.32 ± 0.90 ^a 273.43 ± 72.97 ^a 1.39 ± 0.12 ^a OVX + HD + CA 3.40 0.43 ^b 1.40 ± 0.73 ^b 189.71 + - 82.33 ^b 1.13 ± 0.20 ^b Significance <.0001 <.0001 <.0001 <.0001

As a result, as shown in Table 4, the levels of fat accumulation indicators in the mice were significantly increased in both the high fat diet and the ovariectomized group, while the ovariectomy was performed surgically The levels of insulin, leptin, triglyceride and free fatty acids in the experimental groups 3 and 6 fed with carnosic acid were lower than those in the groups without carnoic acid supplementation (2, 4) I could.

Through the above results, the present inventors have found that the carnosic acid supplementation can effectively reduce the increased fat accumulation in ovariectomized menopausal model mice.

< Example  5>

Of ovariectomized mice Carnoic acid  In the liver tissue according to salary AMPK  And ACC Change in the expression level of

In this study, we examined the effect of carnosanic acid supplementation on the lipid metabolism in these models by using surgical ovariectomy. For this purpose, the degree of phosphorylation of AMPK (AMP-activated protein kinase) and ACC (acetyl CoA carboxylase) protein was measured by western blot analysis as an index closely related to lipid metabolism. For reference, AMPK is an enzyme that regulates glucose metabolism and lipid metabolism. It is known that this enzyme is phosphorylated to increase fatty acid oxidation and reduce lipid biosynthesis process. On the other hand, ACC is known to be involved in lipid accumulation by being influenced by phosphorylation as a fatty acid synthase, a substrate of AMPK enzyme. Therefore, in this experiment, we measured the degree of phosphorylation of two enzymes AMPK and ACC involved in lipid metabolism as described above, thereby examining the effect of carnosic acid on the lipid metabolism in the menopausal model mouse through surgical ovariectomy I would like to see.

For Western blot analysis, the mouse liver tissues of experimental groups were extracted with PRO-PREP protein extraction solution (Intron Biotechnology, Gyeonggi, Korea). The amount of extracted protein was determined by Bradford method using Bio-RAD protein assay reagent (Bio-RAD, CA, USA). All protein samples were quantitated at 50 μg, separated by 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were blocked with 2% skim milk and pAMPK (dilution 1: 1,000, cell signaling, MA, USA), AMPK (dilution 1: Cell signaling, MA, USA) and ACC (dilution 1: 1,000, Cell signaling, MA, USA). Membranes were washed with PBS / Tween 20 (PBST), and reacted bands were visualized with enhaced chemiluminescene (ECL). The visualized bands were identified using LAS 3000 (Fujifilm, Tokyo, Japan) and the bands were quantified using the Quantity One program (Bio-Rad Laboratories Ins., CA, USA).

As a result, as shown in FIG. 3, it was confirmed that phosphorylation was reduced in the experimental group (2, 5) mice in which ovariectomy was performed in AMPK and ACC, whereas in the mouse model, 3, and 6), the degree of phosphorylation of AMPK and ACC was markedly increased.

Through the above results, the inventors could prove that the carnosic acid supplementation can effectively reduce / suppress the increased fat accumulation in ovariectomized menopausal model mice.

< Example  6>

Of ovariectomized mice Carnoic acid  Changes in expression levels of genes related to lipid biosynthesis in liver tissue by feeding

In this study, we examined the effect of carnosic acid supplementation on the expression of lipid biosynthesis-related genes in the menopausal model mice through surgical ovariectomy. For this, mRNA expression levels of SREBP1c, FAS, PPARα, and CPT-1 genes were measured by real-time PCR as an indicator gene closely related to lipid biosynthesis in liver. SREBP1c is one of the genes whose expression level is decreased by estrogen. It increases the expression level of FAS (fatty acid synthase), which is involved in biosynthesis of lipid. Thus, SREBP1c (sterol regulatory element-binding protein-1c) Is one of the genes. On the other hand, PPAR alpha (peroxisome proliferator-activated receptor alpha) and CPT-1 (carnitine palmitoyltransferase-1) are known to be involved in fatty acid synthesis and beta-phosphorylation. Therefore, in this experiment, we examined the effect of carnosic acid supplementation on lipid biosynthesis in the menopausal model mice through surgical ovariectomy by measuring the expression levels of various genes involved in lipid biosynthesis.

For real-time quantitative PCR analysis, RNA extracted with Trizol in liver and abdominal adipose tissues of each experimental mouse was converted into cDNA using cDNA synthesis kit (PhileKorea Technology, Seoul, Korea). Real-time PCR analysis was performed using the synthesized cDNA and the following primer set of each gene, and the expression of the selected gene was quantitated. Real-time PCR was performed using QuantiMix SYBR Kit (PhileKorea Technology, Seoul, Korea) and 7500 Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA). Real-time PCR was carried out at 95 ° C for 15 minutes. The first cycle was performed at 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds. Respectively.

Primer set gene Primer sequence SREBP1c Forward 5'-ATCTCCTAGAGCGAGCGTTG-3 ' Reverse 5'-TATTTAGCAACTGCAGATATCCAAG-3 ' FAS Forward 5'-CCCTTGATGAAGAGGGATCA-3 ' Reverse 5'-GAACAAGGCGTTAGGGTTGA-3 ' PPARα Forward 5'-CAGTGGGGAGAGAGGACAGA-3 ' Reverse 5'-AGTTCGGGAACAAGACGTTG-3 ' CPT-1 Forward 5'-CCAGGCTACAGTGGGACATT-3 ' Reverse 5'-GAACTTGCCCATGTCCTTGT-3 '

As a result, as shown in FIGS. 4A and 4B, the expression levels of SREBP1c and FAS genes, which increase lipid biosynthesis, are increased in mice treated with high-fat diet (experimental group 4) and mice subjected to ovariectomy (2, 5) In contrast, the expression levels of SREBP1c and FAS genes in the experimental group (3, 6) fed with carnosic acid in the ovariectomized mouse model were significantly decreased. As shown in FIG. 4C and FIG. 4, the genes (PPARα and CPT-1) involved in fatty acid synthesis and beta-phosphorylation in the mouse experimental group (2, 5) 1 gene was significantly decreased, and the expression of these genes was increased by the supply of carnosic acid.

Based on the above results, the present inventors have been able to demonstrate that the expression of an increased fat biosynthesis gene in ovariectomized postmenopausal model mice can effectively reduce / inhibit the carnosic acid supplementation.

< Example  7>

Of ovariectomized mice Carnoic acid  Changes in expression levels of genes related to fat cell differentiation and fat accumulation in white adipose tissue following feeding

In this study, we examined the effect of carnosic acid supplementation on the expression of adipocyte differentiation and fat accumulation-related genes in white adipose tissue in these models by using the ovariectomized male model. For this purpose, mRNA expression levels of PPARγ, aP2 and LPL genes were measured by real-time PCR as an indicator gene closely related to lipid differentiation and fat accumulation in adipose tissue. For example, PPARγ (proliferator-activated receptor gamma) is the only regulator of adipocyte differentiation in a PPAR family. AP2 (adipocyte protein 2) is an adipocyte accumulation and adipocyte-specific gene, and LPL lipase) is an enzyme gene that acts when fat accumulates in adipocytes. Therefore, in this experiment, by measuring the expression levels of various genes involved in adipocyte differentiation and fat accumulation as described above, it is possible to observe the adipocyte differentiation and fat accumulation by the feeding of carnosic acid in the menopausal model mouse through surgical ovariectomy And to investigate the effects of these factors.

Real-time quantitative PCR analysis was performed for the analysis of gene expression levels, and this was carried out in the same manner as in Example 6 using the primer set shown in Table 6 below.

Primer set gene Primer sequence PPARγ Forward 5'-GATGGAAGACCACTCGCATT-3 ' Reverse 5'-AACCATTGGGTCAGCTCTTG-3 ' aP2 Forward 5'-GGGGCCAGGCTTCTATTCC-3 ' Reverse 5'-GGAGCTGGGTTAGGTATGGG-3 ' LPL Forward 5'-CTGCTGGCGTAGCAGGAAGT-3 ' Reverse 5'-GCTGGAAAGTGCCTCCATTG-3 '

As a result, as shown in FIGS. 5A to 5C, the expression levels of PPARγ, aP2, and LPL genes related to lipid differentiation and fat accumulation in the mouse experimental group (2, 5) receiving the high fat diet (experimental group 4) In contrast, in the experimental group (3, 6) in which carnosic acid was fed to the ovariectomized mouse model, the expression level of the genes was significantly decreased.

From the above results, the present inventors have been able to demonstrate that the expression of a gene associated with increased adipocyte differentiation and fat accumulation in ovariectomized postmenopausal model mice can effectively reduce / inhibit the carnosic acid supplementation.

< Example  8>

Of ovariectomized mice Carnoic acid  Histochemical analysis of adipocyte size by feeding

In this study, we examined the effect of carnosic acid supplementation on the size of adipocytes in the postmenopausal model mice through surgical ovariectomy. For this, the fat cell size in each experimental group prepared in Example 1 was confirmed by histological analysis.

For the morphological observation of adipose tissue cells, mouse adipose tissue was fixed in a 10% formalin solution in each of the extracted experimental groups, the tissue was dehydrated under ethanol, and then embedded through paraffin permeation process. And then stained with hematoxylin-eosin (H & E), sealed with xylene, sealed, and then observed with an optical microscope.

As a result, as shown in FIG. 6, when comparing the sizes of adipocytes according to the respective experimental groups, it was found that the adipocyte size in the experimental group (2, 5) However, in the experimental group (3, 6) fed with carnosic acid in the ovariectomized mouse model, the size of adipocytes was significantly reduced again.

Through the above results, the present inventors have been able to demonstrate that the carnosic acid supplementation can effectively reduce the increased fat cell size in ovariectomized menopausal model mice.

< Example  9>

Of ovariectomized mice Carnoic acid  Expression of Inflammatory Cytokine by Saliva side anger

In this study, we examined the effects of carnosic acid supplementation on the expression of inflammatory cytokines in these models by using surgical ovariectomy in postmenopausal model mice. For this purpose, gene expression levels of TNF-alpha (tumor necrosis factor-alpha) and IL-6 (interleukin-6) were measured as inflammatory cytokines.

Real-time quantitative PCR analysis was performed for the analysis of gene expression levels, and this was carried out in the same manner as in Example 6 using the primer set shown in Table 7 below.

Primer set gene Primer sequence TNF-a Forward 5'-ATGGGCTTTCCGAATTCAC-3 ' Reverse 5'-GAGGCAACCTGACCACTCTC-3 ' IL-6 Forward 5'-ACCAGAGGAAATTTTCAATAGG-3 ' Reverse 5'-TGATGCACTTGCAGAAAACA-3 '

As a result, as shown in FIG. 7, it was confirmed that the expression levels of TNF-α and IL-6 gene were increased in the mouse experimental group (2, 5) receiving high-fat diet (experimental group 4) and ovariectomy surgery , Whereas in the experimental group (3, 6) in which carnosic acid was fed to the ovariectomized mouse model, the expression level of the genes was significantly reduced.

Through the above-described results, the present inventors have been able to demonstrate that the expression of an inflammatory cytokine gene that is increased in ovariectomized postmenopausal model mice can effectively reduce / inhibit the carnosolate feeding.

In conclusion, hyperlipidemia and estrogen depletion promotes body fat accumulation and adipocyte differentiation, leading to lipid metabolism abnormalities in postmenopausal women. Carnosic acid activates AMPK degraded by high fat diet and ovariectomy, . This may prove that carnosic acid has the effect of preventing the related diseases such as type 2 diabetes, hypertension, cardiovascular disease and the like by controlling lipid metabolism abnormality in postmenopausal women.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

CA: Carnosic acid
OVX: ovariectomized
ND: normal diet
HD: high-fat diet
SHAM + ND: noncancer extraction + general diet group
OVX + ND: ovariectomy + general diet group
OVX + ND + CA: ovariectomized + normal diet + carnosic acid group
SHAM + HD: Declassification + Highlighting
OVX + HD: ovary extraction + high fat diet
OVX + HD + CA: ovariectomy + high fat diet +

Claims (5)

A pharmaceutical composition for the treatment or prevention of abdominal obesity in postmenopausal women, which comprises a carnosic acid compound or a pharmaceutically acceptable salt thereof as an active ingredient. delete A health functional food composition for improving or preventing abdominal obesity in postmenopausal women comprising a carnosic acid compound or a pharmaceutically acceptable salt thereof as an active ingredient. The method of claim 3,
Wherein the food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, rice cakes, gums, candy, ice cream, alcoholic beverages, alcoholic beverages, Composition.
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