KR20230159273A - Composition for preventing, improving or treating menopausal disorder containing Hippophae rhamnoides fruits extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, improving, or treating female menopausal symptoms containing sea buckthorn fruit extract as an active ingredient.
The composition according to the present invention has the effect of preventing, improving or treating various female menopausal symptoms by containing sea buckthorn fruit extract or a fraction thereof as an active ingredient, and reduces muscle mass, muscle weakness, fatty liver, abdominal obesity, etc., which mainly occur in female menopause. It is particularly effective in treating body fat gain, weight gain, blood lipid disorders, and hyperlipidemia. The active ingredient contained in the composition according to the present invention is derived from plants, and has no side effects or toxicity even when administered to the human body, ensuring safety. Therefore, the sea buckthorn fruit extract or its fraction according to the present invention can be usefully used as a material for medicine or health functional food to prevent, improve, or treat female menopausal symptoms.

Description

Composition for preventing, improving or treating menopausal disorder containing Hippophae rhamnoides fruits extract as an active ingredient}

The present invention relates to a composition for preventing, improving, or treating female menopausal symptoms containing sea buckthorn fruit extract as an active ingredient.

Female menopause is a type of endocrine syndrome and refers to a transitional period in which physiological and sexual functions are reduced or lost due to a decrease in female hormones, that is, estrogen, due to the overall and gradual aging of the ovaries.

In menopausal women, when ovarian function declines, a variety of symptoms appear along with decreased estrogen secretion in the body. Representative examples include frequent urination, dysuria, cystitis, lower urethral atrophy, urinary incontinence, sensitivity to cold, vaginal dryness, and vaginal atrophy due to changes in the genitourinary system. There are symptoms, symptoms due to changes in the cranial nervous system such as sleep disorders, depression, anxiety, decreased concentration, short-term memory impairment, and dizziness, and vascular changes such as facial flushing, tachycardia, blood lipid disorders, hyperlipidemia, arrhythmia, edema, sweating, and headaches. There are symptoms due to musculoskeletal changes such as loss of muscle mass, muscle weakness, muscle pain, joint pain, and back pain. In addition, there are symptoms of fatty liver disease, abdominal obesity, increase in body fat, weight gain, decreased vision, and changes in skin and hair. , Diseases that are fatal to women's health, such as osteoporosis or cardiovascular disease, can occur due to hormonal changes.

Therefore, in order to improve the physical and mental health and quality of life of middle-aged women, there was a need to develop treatments that can improve menopausal symptoms, and treatment methods for improving menopausal symptoms include hormone replacement therapy and non-steroidal agents. Treatment methods using drugs have been developed, and among them, hormone replacement therapy using estrogen is known to be the most effective method. However, it is known that most of the above drug treatments have side effects such as headaches and weight gain, and in particular, long-term use of the drug can cause endometrial cancer, breast cancer, high blood pressure, thrombosis, and biliary stones. Not only can menstrual bleeding occur, but there is a problem that it cannot be taken without a doctor's prescription due to side effects such as ovarian hyperstimulation. In addition, in the case of estrogen replacement therapy, since hormones are artificially administered into the body, it is known that the risk of uterine bleeding, stroke, heart attack, breast cancer, and uterine cancer may increase along with rejection reactions.

In addition, in the case of calcium preparations, if you consume more calcium than necessary, urinary stones may occur due to increased calcium excretion, so caution is required if you have a history of urinary stones. In addition, estrogen, a representative drug used as a bone resorption inhibitor, raises the risk of coronary artery disease and stroke, as well as risks to the cardiovascular system, and calcitonin requires injection or nasal spray and is resistant to long-term use. It has the disadvantage of requiring a rest period of several months. In addition, in the case of bisphosphonates, gastrointestinal symptoms such as nausea, vomiting, and indigestion may occur, and in the case of amino-bisphosphonates, they may cause esophagitis, so take a large cup with at least a glass of plain water and wait at least 30 minutes to 1 hour. It has the disadvantage of requiring caution when taking it, such as not lying down too much. In addition, in the case of Strontium, which was recently developed and widely used as a treatment, the most common side effect is diarrhea, and in some patients, creatinine kinase levels exceeding twice the upper limit of normal are observed.

Accordingly, there is a need for the development of materials derived from natural products that can relieve symptoms caused by the reduction or cessation of estrogen, which is an important factor in reducing women's quality of life, and can replace the synthetic hormones or synthetic drug therapy. There is a situation.

Meanwhile, sea buckthorn is a tree of the Rosaceae family, a true dicotyledonous plant, also called a vitamin tree. It is known to be effective in preventing aging, liver disease, blood circulation, and anti-cancer. Recent studies have shown that it is effective in treating bone metabolic diseases. , no studies have been reported on the relief or treatment efficacy of female menopausal symptoms.

As a result of efforts to develop a substance for treating female menopausal symptoms that is safe for the human body, the present inventors completed the present invention by confirming that sea buckthorn fruit extract or a fraction thereof can alleviate or treat various menopausal symptoms caused by decreased estrogen secretion.

KR 10-2022-0032504 A

The purpose of the present invention is to provide a food composition for preventing or improving female menopausal symptoms containing sea buckthorn fruit extract or a fraction thereof as an active ingredient.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating female menopausal symptoms containing sea buckthorn fruit extract or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention provides a food composition for preventing or improving female menopausal symptoms containing sea buckthorn fruit extract or a fraction thereof as an active ingredient.

According to one embodiment of the present invention, the sea buckthorn fruit extract is selected from water, alcohol with 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, hexane, benzene, chloroform, glycerin, butylene glycol, and propylene glycol. It may be an extract of one or more solvents.

According to one embodiment of the present invention, the solvent may be 20 to 80% by volume of methanol, ethanol, butanol, or propanol.

According to one embodiment of the present invention, the solvent may be hexane.

According to one embodiment of the present invention, the sea buckthorn fruit fraction may be a hexane fraction of sea buckthorn fruit.

According to one embodiment of the present invention, the sea buckthorn fruit fraction may be a fraction separated at a retention time (RT) of 61 to 81 minutes when the hexane fraction of sea buckthorn fruit is separated by HPLC.

According to one embodiment of the present invention, the female menopausal symptoms may be due to decreased estrogen secretion.

According to one embodiment of the present invention, the female menopausal symptoms include facial flushing, dry skin, arrhythmia, edema, sweating, urgent urination, pain during urination, frequent urination, urinary incontinence, cystitis, lower urethral atrophy, decreased muscle mass, muscle weakness, muscle pain, joint pain, Osteoporosis, sensitivity to cold, vaginal dryness, vaginal atrophy, fatty liver, abdominal obesity, increased body fat, weight gain, blood lipid disorder, hyperlipidemia, difficulty concentrating, short-term memory disorder, memory loss, headache, palpitations, dizziness, sweating during sleep, sleep. It may be one or more types selected from disorders, depression, anxiety, and nervousness.

According to one embodiment of the present invention, the female menopausal symptoms may be one or more selected from the group consisting of decreased muscle mass, decreased muscle strength, fatty liver, abdominal obesity, increased body fat, increased body weight, blood lipid disorder, and hyperlipidemia.

According to one embodiment of the present invention, the food composition may be a powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation, or beverage formulation.

According to one embodiment of the present invention, the food composition may be a health functional food for alleviating female menopausal symptoms.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating female menopausal symptoms, comprising sea buckthorn fruit extract or a fraction thereof as an active ingredient.

According to one embodiment of the present invention, the pharmaceutical composition may be an oral preparation.

According to one embodiment of the present invention, the oral preparation may be powder, granule, tablet, capsule, troche, suspension, emulsion, syrup or aerosol.

The composition according to the present invention has the effect of preventing, improving or treating various female menopausal symptoms by containing sea buckthorn fruit extract or a fraction thereof as an active ingredient, and has the effect of reducing muscle mass, muscle weakness, fatty liver, abdominal obesity, etc. induced by female menopause. It is particularly effective in preventing, improving, or treating body fat gain, weight gain, blood lipid disorders, and hyperlipidemia. The active ingredient contained in the composition according to the present invention is derived from plants, and has no side effects or toxicity even when administered to the human body, ensuring safety. Therefore, the sea buckthorn fruit extract or its fraction according to the present invention can be usefully used as a material for medicine or health functional food to prevent, improve, or treat female menopausal symptoms.

Figure 1a shows the degree of cell damage in normal and induced control groups after treating mouse myoblast C2C12 cells with sea buckthorn fruit extract or fractions thereof according to an embodiment of the present invention and simultaneously treating them with hydrogen peroxide to induce cell damage. This is a graph compared to . # indicates a significant difference at p<0.001 (Student's t-test) compared to the normal group (Control), and *, **, and *** compared to the induced control group (H ₂ O ₂ ), respectively. It means that there is a significant difference at p<0.05, p<0.01 and p<0.001 (Student's t-test).
Figure 1b shows mouse myoblast C2C12 cells treated with sea buckthorn fruit extract or fractions thereof according to an embodiment of the present invention and simultaneously treated with hydrogen peroxide to induce cell damage, followed by treatment with antioxidant enzymes Cu/Zn-SOD and This is a graph comparing the mRNA expression level of GPx with the normal group and induced control group. # means significant difference at p<0.01 compared to the normal group (Control) (Student's t-test), * and ** mean p<0.05 and ** compared to the induced control group (H ₂ O ₂ ), respectively. It means there is a significant difference at p<0.01 (Student's t-test).
Figure 2 is for confirming the effect of inhibiting adipocyte differentiation after treating C3H10T1/2 cells, a pluripotent stem cell line, with sea buckthorn fruit extract or fractions thereof according to an embodiment of the present invention. Figure 2a shows Oil Red O staining method. This is a photograph for confirming the degree of fat production by staining fat globules produced in adipocytes. Figure 2b shows the degree of adipogenesis by melting fat globules stained with Oil Red O and measuring their absorbance compared to the normal group. This is a conversion graph. * and ** indicate significant differences (Student's t-test) at p<0.05 and p<0.01, respectively, compared to the normal group (NC).
Figure 3 shows sea buckthorn fruit ethanol extract (Fruit-E), sea buckthorn leaf ethanol extract (Leaf-E), sea buckthorn seed ethanol extract (Seed-E), and sea buckthorn fruit hexane in C3H10T1/2 cells, a pluripotent stem cell line. This was to confirm the effect of inhibiting adipocyte differentiation after treatment with the extract (Fruit-H), sea buckthorn leaf hexane extract (Leaf-H), and sea buckthorn seed hexane extract (Seed-H), respectively. Figure 3a shows the effect of Oil Red O. This is a photograph for confirming the degree of fat production by staining fat globules produced in adipocytes through a staining method. Figure 3b shows the degree of adipogenesis by melting fat globules stained with Oil Red O and measuring their absorbance, which is normal. This is a conversion graph compared to the military. *, **, and † indicate significant differences (Student's t-test) at p<0.05, p<0.01, and p<0.1, respectively, compared to the normal group (NC).
Figure 4 is a conversion graph of the proliferation rate of MCF-7 cells treated with each extract compared to the normal group (NC) to evaluate the estrogen receptor activation effect of the sea buckthorn fruit extract and sea buckthorn leaf extract or seed extract of the present invention. am. * and † indicate significant differences (Student's t-test) at p<0.05 and p<0.1, respectively, compared to the normal group (NC).
Figure 5 is a conversion graph of the proliferation rate of MCF-7 cells treated with each sample compared to the control group to evaluate the estrogen receptor activation effect of the sea buckthorn fruit extract or fractions thereof of the present invention. *, **, and † indicate significant differences (Student's t-test) at p<0.05, p<0.01, and p<0.1, respectively, compared to the control group (Control).
Figure 6 shows changes in blood lipid components due to administration of the sea buckthorn fruit extract or fractions thereof of the present invention in an ovariectomized animal model, specifically, blood triglyceride content, blood cholesterol content, blood HDL content, and blood LDL content. and a graph showing fatty acid content in blood. * and ** indicate significant differences (Student's t-test) at p<0.05 and p<0.01, respectively, compared to the control group (OVX; oophorectomy group).
Figure 7 is a graph showing body weight change due to administration of the sea buckthorn fruit extract or fractions thereof of the present invention in an ovariectomized animal model. * and ** indicate significant differences (Student's t-test) at p<0.05 and p<0.01, respectively, compared to the control group (OVX; oophorectomy group).
Figure 8 is a graph showing changes in estrogen receptor expression due to administration of the sea buckthorn fruit extract or fractions thereof of the present invention in an ovariectomized animal model. *, **, and † indicate significant differences (Student's t-test) at p<0.05, p<0.01, and p<0.1, respectively, compared to the control group (OVX; oophorectomy group).
Figure 9 is a graph showing changes in muscle strength (grip strength) caused by administration of the sea buckthorn fruit extract or fractions thereof of the present invention in an ovariectomized animal model. * and ** indicate significant differences (Student's t-test) at p<0.05 and p<0.01, respectively, compared to the control group (OVX; oophorectomy group).
Figure 10 is a graph showing changes in the expression level of muscle-related biomarkers (MyoD and MurF1) by administration of the sea buckthorn fruit extract or fractions thereof of the present invention in an ovariectomized animal model. * and ** indicate significant differences (Student's t-test) at p<0.05 and p<0.01, respectively, compared to the control group (OVX; oophorectomy group).

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition that can prevent, improve, or treat female menopausal symptoms by containing sea buckthorn fruit extract or a fraction thereof as an active ingredient.

The sea buckthorn fruit (Seabuckthorn, Hippophae rhamnoides L.) is generally a deciduous shrub belonging to the Elaeagnaceae family and is also known as sea buckthorn tree and vitamin tree, and is used in Russia, China, and Japan respectively as Siberian pineapple and sageuk (莎). It is called by various names such as 棘, sajee, etc. Sea buckthorn grows well even in extreme climates and poor soil, and its fruits, leaves, and seeds are all edible, and are also used as raw materials for cosmetics, and are widely used in countries such as Russia, Europe, Canada, China, Mongolia, and countries surrounding the Himalayan Mountains. It is widely cultivated. In particular, sea buckthorn fruits are known to contain different physiologically active substances depending on the ripeness, size, variety, production area, climate, and extraction method of the fruit, and cryptoxanthin is found in yellow, orange, and red fruits. It is due to carotenoids such as cryptoxanthin, lycopene, γ-carotene, β-carotene, lutein, and zeaxanthin, and carotenoids are lipophilic. It exists in plant membranes as a pigment, has photosynthetic and photoprotective functions, and is reported to exhibit various activities such as antioxidant, anti-mutagenic, and anti-cancer activities.

“Female menopausal symptoms” referred to herein refers to the symptoms and diseases that appear in women before and after menopause as the secretion of estrogen decreases due to aging of the ovaries, including facial flushing, dry skin, arrhythmia, edema, sweating, Urinary urgency, painful urination, frequent urination, urinary incontinence, cystitis, lower urethral atrophy, decreased muscle mass, muscle weakness, muscle pain, joint pain, osteoporosis, sensitivity to cold, vaginal dryness, vaginal atrophy, fatty liver, abdominal obesity, increased body fat, weight gain, blood lipid disorder, hyperlipidemia , concentration disorder, short-term memory disorder, memory loss, headache, heart palpitations, dizziness, sweating during sleep, sleep disorder, depression, anxiety, or nervousness are preferred, but are not limited to these.

The present invention relates to a food composition for preventing or improving female menopausal symptoms containing sea buckthorn fruit extract or a fraction thereof as an active ingredient.

In this specification, 'including as an active ingredient' means containing a sufficient amount to achieve the efficacy or activity of the sea buckthorn fruit extract or fractions thereof. The present invention is a composition extracted from sea buckthorn fruit, which is a natural plant material. Since there are no side effects on the human body even when administered in excessive amounts, the upper quantitative limit of the sea buckthorn fruit extract or its fraction contained in the composition of the present invention is within an appropriate range by a person skilled in the art. You can select and implement it.

Sea buckthorn fruit extract

In this specification, 'extract' includes not only crude extract obtained by treating extraction raw materials with an extraction solvent, but also processed products of the crude extract. For example, it may include a concentrate obtained by additionally concentrating the extract, a dried product thereof, or a powder obtained by pulverizing the extract.

The sea buckthorn fruit extract may be an extract using one or more solvents selected from water, alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, hexane, benzene, chloroform, glycerin, butylene glycol, and propylene glycol. there is.

The alcohol having 1 to 4 carbon atoms does not need to be specifically limited, but for example, methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, or n-butanol can be used, and ethanol is preferable.

The solvent does not need to be particularly limited, but may be 20 to 80% by volume of alcohol, and preferably 60 to 80% by volume of alcohol.

Specifically, the solvent may be 20 to 80% by volume of methanol, ethanol, butanol, or propanol, and preferably 60 to 80% by volume of ethanol.

Additionally, the solvent may be hexane.

The extraction solvent is not particularly limited, but hexane-extracted sea buckthorn fruit extract or fractions thereof have the best estrogenic activity, and thus work favorably in preventing, improving, or treating female menopausal symptoms.

There is no need to specifically limit the preparation of the water extract, but it can be prepared by extracting the extraction raw material with water at 10 to 100 ° C. for 2 to 60 hours.

The preparation of the alcohol extract or the extract of a mixed solvent of water and alcohol does not need to be particularly limited, but for example, the extraction raw material is mixed with 20 to 80% by volume of ethanol, preferably 60 to 80% by volume of ethanol. It can be prepared by extraction at 60°C for 2 to 48 hours.

Sea buckthorn fruit fraction

In this specification, 'sea buckthorn fruit fraction' refers to a fraction obtained by refractionating the sea buckthorn fruit extract with a solvent, or a fraction obtained by passing the extract through an ultrafiltration membrane with a certain molecular weight cut-off value, or various chromatography methods (size It may be a fraction obtained through various additional purification methods, such as separation by charge, hydrophobicity, or affinity).

When preparing a fraction using a solvent, the solvent used may be one or more selected from water, alcohol having 1 to 4 carbon atoms, hexane, acetone, ethyl acetate, chloroform, and diethyl ether, preferably hexane and ethyl acetate. , butanol or water, more preferably hexane or ethyl acetate, and even more preferably hexane. The hexane fraction of sea buckthorn fruit showed a superior effect in preventing, improving, or treating female menopausal symptoms compared to other solvent fractions.

More preferably, the sea buckthorn fruit fraction of the present invention may be a fraction separated at a retention time (RT) of 61 to 81 minutes when the hexane fraction of sea buckthorn fruit is separated by HPLC. If the retention time (RT) is outside the above range, the effectiveness of preventing, improving, or treating female menopausal symptoms may be significantly reduced.

In addition, the sea buckthorn fruit “fraction” used in the present invention includes the above fractions, concentrates obtained by additionally concentrating these fractions, and purified products obtained by purifying or separating them, and dried products or pulverized products of the fractions, concentrates or purifications. It is used to mean that it contains one powder.

The sea buckthorn fruit extract or its fraction exhibits estrogenic activity and can be usefully used to prevent, improve, or treat female menopausal symptoms due to decreased estrogen secretion.

Although not specifically described in the following examples, the present inventors confirmed that estrogen secretion increased in a concentration-dependent manner in cells treated with sea buckthorn fruit extract or fractions thereof.

Accordingly, through these experimental results, the present inventors have found that the sea buckthorn fruit extract or fraction thereof of the present invention can be usefully used for preventing, improving, or treating female menopausal symptoms caused by decreased estrogen secretion.

Furthermore, the inventors of the present invention investigated whether the sea buckthorn fruit extract or fraction thereof of the present invention could actually alleviate, improve, or treat the major symptoms seen in menopausal women, such as muscle weakness, weight gain, body fat increase, and blood lipid content increase. To confirm, after oral administration of sea buckthorn fruit extract or fractions thereof to an ovariectomized animal model, changes in body fat mass, blood lipid composition, body weight, estrogen receptor expression level, and muscle strength were evaluated. As a result, oral administration of sea buckthorn fruit extract or fractions thereof increased body fat mass and blood lipid content (e.g., blood triglyceride content, blood cholesterol content, blood HDL content, blood LDL content, and blood fatty acid content due to ovarian extraction). ) and it was confirmed that there was a significant reduction in body weight. In addition, it was confirmed that oral administration of sea buckthorn fruit extract or fractions thereof had the effect of significantly increasing estrogen receptor expression and muscle strength, which had decreased due to ovarian removal.

Therefore, it was found that the sea buckthorn fruit extract or its fraction of the present invention has the effect of preventing, improving or treating female menopausal symptoms by increasing estrogen secretion, especially muscle weakness, weight gain, body fat increase and It was confirmed through specific experiments that symptoms such as increased blood lipid content can be effectively alleviated, improved, or treated.

The term “prevention” of the present invention refers to all actions that suppress or delay the progression of female menopausal symptoms by administering the composition of the present invention.

The term “treatment” of the present invention refers to any action that improves or beneficially changes female menopausal symptoms by administering the composition of the present invention.

The term "improvement" in the present invention means at least reducing the degree of symptoms, for example, parameters related to the condition being treated by administration of a composition comprising the Ecklonia cava extract of the present invention.

The “food composition” includes, in addition to sea buckthorn fruit extract or fractions thereof as an active ingredient, food raw materials usable as foods and food additives listed in the Food Standards and Specifications (“Food Code”) commonly used in food production. May contain food additives.

The food composition does not need to be particularly limited, but may include, for example, carbohydrates and flavoring agents. The carbohydrates include monosaccharides, such as glucose, fructose, etc.; Disaccharides such as maltose, sugar, lactose, etc.; Oligosaccharides or polysaccharides such as dextrin, starch syrup, cyclodextrin, etc.; Sugar alcohols such as xylitol, sorbitol, erythritol, etc. can be used. The flavoring agent may be a natural flavoring agent [thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and a synthetic flavoring agent (saccharin, aspartame, etc.).

In the food composition of the present invention, the content of sea buckthorn fruit extract or fractions thereof does not need to be specifically limited as long as it shows the effect of preventing or improving female menopausal symptoms, for example, 0.01 to 99% by weight, 0.1 to 50% by weight, 0.1% by weight. It may be included in 20% by weight. In the case of a health drink composition, it may be included at a rate of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

In the above food composition, the active ingredient, sea buckthorn fruit extract or fraction thereof, varies depending on the condition, weight, presence, degree, and period of the disease of the ingestor, but may be appropriately selected by a person skilled in the art. For example, the daily dosage may be 0.01 mg/kg to 1.0 g/kg, preferably 0.1 mg/kg to 500 mg/kg, more preferably 1 mg/kg to 300 mg/kg, and the number of administrations may be There is no need to specifically limit it, but it can be adjusted by a skilled artisan within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

The food composition does not need to be particularly limited, but may be, for example, powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation, or beverage formulation.

In addition, the sea buckthorn fruit extract or a fraction thereof can be added to general foods to provide functionality for preventing or improving female menopausal symptoms. There is no need to specifically limit the foods that can be added, but for example, confectionery, bread or rice cake, processed cocoa products or chocolate, meat or Added to processed egg products, processed fish products, tofu or jelly, noodles, tea, coffee, beverages, special purpose foods, sauces, seasoned foods, dressings, kimchi, salted seafood, pickled foods, stewed foods, alcoholic beverages, raisins, and other foods. It can be. In addition, it can be added to dairy products, processed meat products, packaged meat, and egg products as exemplified in the livestock product processing standards and ingredient specifications (“Livestock Products Code”) pursuant to Article 4 of the Livestock Products Sanitation Management Act.

Meanwhile, a food composition containing the sea buckthorn fruit extract or a fraction thereof as an active ingredient can be used as a health functional food for improving female menopausal symptoms.

The above “health functional food” refers to food manufactured (including processing) in accordance with legal standards using raw materials or ingredients with functional properties useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The terminology or scope of the above “health functional food” may vary depending on the country, but it is called “Dietary Supplement” in the United States, “Food Supplement” in Europe, “Health Functional Food” or “Health Functional Food” in Japan. It may fall under “Food for Special Health Use (FoSHU)”, Chinese “health food”, etc.

The above food composition or health functional food may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions of the “Food Additives Code” and general test methods, etc., unless otherwise specified. Follow.

The present invention relates to a pharmaceutical composition for preventing or treating female menopausal symptoms containing sea buckthorn fruit extract or a fraction thereof as an active ingredient.

The “pharmaceutical composition” may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions, etc., in addition to sea buckthorn fruit extract or fractions thereof as active ingredients.

The “carrier” is a compound that facilitates the addition of the compound into cells or tissues. The “excipient” is a compound added to give the active ingredient an appropriate form to formulate it or increase its amount to make it more convenient to use. The “diluent” is a compound diluted in water that not only stabilizes the biologically active form of the compound of interest, but also dissolves the compound.

There is no need to specifically limit the carrier, excipient, and diluent, but for example, lactose, glucose, sugar, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., and oral administration is preferred.

The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route described above. When formulated, one or more buffers (e.g. saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g. EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g. aluminum hydroxide) side), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, and such solid preparations include at least one excipient, for example, in the pharmaceutical composition of the present invention. , starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose It can be prepared by mixing methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or gelatin. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries and processing them into a granule mixture.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water or liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, or preservatives may be included. .

In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention can be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation according to methods known in the art. The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

The pharmaceutical composition of the present invention can provide desirable effects for preventing, improving, or treating female menopausal symptoms when it contains an effective amount of sea buckthorn fruit extract or a fraction thereof. In this specification, 'effective amount' refers to an amount that shows a greater response than the negative control, and preferably refers to an amount sufficient to improve or treat female menopausal symptoms. The sea buckthorn fruit extract or fraction thereof may be included at a concentration of 5 to 250 μg/g, preferably 10 to 200 μg/g, and more preferably 30 to 100 μg/g, based on the total content of the pharmaceutical composition. At this time, if the content of the sea buckthorn fruit extract or its fraction is less than the lower limit, the cell survival rate is excellent, but the effect of improving female menopausal symptoms may not be achieved to the desired extent. Conversely, if the above upper limit is exceeded, the effect of improving female menopausal symptoms may not increase as much as the concentration increases. Meanwhile, as a result of in vitro experiments, when the concentration of the sea buckthorn fruit extract or fraction thereof of the present invention was within the above range, a significant effect was observed in improving female menopausal symptoms, but no side effects such as cytotoxicity were observed. The effective amount of sea buckthorn fruit extract or fractions thereof included in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is commercialized.

The total effective amount of the pharmaceutical composition of the present invention can be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. . The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease.

The appropriate dosage of the pharmaceutical composition varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and is usually A skilled physician can easily determine and prescribe an effective dosage for desired treatment or prevention. According to a preferred embodiment, the preferred daily dosage of the sea buckthorn fruit extract or fraction thereof is 0.01 mg/kg to 1.0 g/kg, preferably 0.1 mg/kg to 500 mg/kg, More preferably It may be 1 mg/kg to 300 mg/kg.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebroventricular injection.

Hereinafter, the present invention will be described in detail through examples, but the present invention is not limited to the following examples.

<Example>

Example 1: Preparation of sea buckthorn fruit ethanol extract (VTE)

Add 100 ml of 70% ethanol per 10 g of sea buckthorn fruit powder, extract at 70°C for 6 hours, filter the extract with filter paper, and then concentrate under reduced pressure until the sugar content reaches 10 to 20 brix. Tree fruit ethanol extract (VTE) was obtained.

Example 2: Preparation of sea buckthorn fruit hexane fraction (VTH)

After completely removing ethanol from the ethanol extract of sea buckthorn fruit prepared in Example 1, an equal amount of hexane was added and mixed, and the hexane fraction was recovered. The hexane fraction obtained by repeating this process three times was concentrated under reduced pressure to obtain a hexane fraction (VTH) from which the solvent was completely removed.

Example 3: Preparation of active fraction (VTHP4) of hexane fraction of sea buckthorn fruit

The sea buckthorn fruit hexane fraction (VTH) prepared in Example 2 was dissolved in methanol, then divided into five sections considering the separation time and peak using HPLC, and the fractions for each section were collected and concentrated to produce five effective types. Hexane fractions (VTHP1 to VTHP5) were obtained.

The separation times for the five effective hexane fractions are shown in Table 1 below. The HPLC used was Waters 1525 Binary HPLC pump, Waters 996 Photodiode Array Detector, and the column used was Agilent Eclipse C18 column (250X21.2 mm). The flow rate of the mobile phase was 0.3 mL/min, the oven temperature was 25°C, and the injection volume was 10 ㎕. Separation conditions are 30-80% MeOH, and samples diluted to a concentration of 500 ug/mL are used.

Among the five effective hexane fractions shown in Table 1 below, VTHP4 was obtained at RT 61-81 minutes.

division VTHP1 VTHP2 VTHP3 VTHP4
(Example 3) VTHP5 RT(min) 5-21 21-41 41-61 61-81 81-100

Example 4: Preparation of sea buckthorn fruit hexane extract (VTHX)

After adding 100 ml of hexane per 10 g of sea buckthorn fruit powder and extracting at 70° C. for 6 hours, the extract was filtered through filter paper and then concentrated under reduced pressure until the sugar content was 10 to 20 brix. Hexane extract (VTHX) was obtained.

<Test example>

Statistical analysis

All experiments were repeated three times, and the results were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS software (ver. 20.0, SPSS Inc., Chicargo, IL, USA). One-way ANOVA was performed to verify the difference between each mean value, and the significant difference was verified using tukey's HSD post-hoc comparison at a significance level of less than 0.05.

Test Example 1: Muscle cell protection effect

1-1: Inhibitory effect on oxidative cell damage in muscle cells

In order to confirm the muscle cell protection effect of the sample according to the embodiment of the present invention, mouse myoblast C2C12 cells were treated with a control group (untreated), a induced control group (H ₂ O ₂ , 1 mM), and sea buckthorn fruit extract. Or fraction samples thereof were divided into treatment groups (VTE, VTH and VTHP4, 25 μg/mL + H ₂ O ₂ , 1 mM).

The H ₂ O ₂ treatment is a substance that causes oxidative cell damage, and the protective effect of sea buckthorn fruit extract or its fractions (VTE, VTH, and VTHP4) on oxidative cell damage was confirmed.

Specifically, C2C12 cells were seeded in a 96-well plate at a concentration of 5 × 10 ⁴ cells/well the day before the experiment, and incubated in Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum (HS) to induce differentiation into muscle cells. medium was used. Each well was treated with sea buckthorn fruit extract or its fraction sample at a concentration of 25 μg/mL, and after 1 hour, all groups except the control group were treated with 1 mM H ₂ O ₂ . After incubation for 48 hours (2 days after differentiation), the medium was replaced with new medium. After another 48 hours (4 days after differentiation), 50 μL of MTT reagent dissolved in PBS at 5 mg/mL was added and incubated at 37°C for 2 hours. After culturing, the absorbance was measured at 460 nm using a micro-plate reader (SpectraMax M2e, Molecular Devices, San Jose, USA) (see Figure 1a).

Figure 1a shows the degree of cell damage in normal and induced control groups after treating mouse myoblast C2C12 cells with sea buckthorn fruit extract or fractions thereof according to an embodiment of the present invention and simultaneously treating them with hydrogen peroxide to induce cell damage. This is a graph compared to .

Looking at Figure 1a, it can be seen that the cell viability of all groups treated with the sea buckthorn fruit extract or fraction thereof sample of the present invention was significantly increased compared to the induced control group (H ₂ O ₂ ). In the case of the induced control group (H ₂ O ₂ ), the cell viability was 65.4 ± 3.81%***, which was lowered to about 65% compared to the normal group (Control, 100%), but the cell viability of the sample administration group (VTE) of Example 1 was 65.4 ± 3.81%***. This was 70.5 ± 4.09*%, which was a significant increase of 7.9% compared to the induced control group (H ₂ O ₂ ), and the cell viability of the sample administered group (VTH) of Example 2 was 74.5 ± 4.63%**, which was a significant increase of 7.9% compared to the induced control group (H 2 O 2 ). ₂ O ₂ ), the cell viability of the sample administration group (VTHP4) of Example 3 was 86.9 ± 3.75%***, a significant increase of 32.9% compared to the induced control group (H ₂ O ₂ ). did.

1-2: Effect of increasing expression of antioxidant enzymes

In order to confirm the antioxidant effect of the sample according to an embodiment of the present invention, mouse myoblast C2C12 cells were treated with a control group (untreated), an induced control group (H ₂ O ₂ , 1 mM), and sea buckthorn fruit extract or its Fraction samples were divided into treatment groups (VTE, VTH, and VTHP4, 25 μg/mL + H ₂ O ₂ , 1 mM).

Specifically, C2C12 cells were seeded in a 12-well plate at a concentration of 2×10 ⁵ cells/well the day before the experiment, treated with 25 μg/mL of sea buckthorn fruit extract or fractions thereof prepared according to Examples 1 to 3, and 1 After the time reaction, the culture medium containing the sea buckthorn fruit extract or its fraction was treated with 1 mM H ₂ O ₂ and cultured in an incubator at 37°C for 48 hours, then replaced with new medium and cultured for 48 hours. proceeded. Muscle cells after 4 days of culture were washed with PBS and harvested by adding Nucleozol (Macherey-Nagel, Germany). Total RNA was extracted through centrifugation, and cDNA was synthesized using the qPCR Mastermix kit (TOYOBO, Japan). Subsequently, mRNA expression of antioxidant enzyme genes (Cu/Zn-SOD and GPx (glutathione peroxidase)) was confirmed through qPCR analysis (see Figure 1b).

Figure 1b shows mouse myoblast C2C12 cells treated with sea buckthorn fruit extract or fractions thereof according to an embodiment of the present invention and simultaneously treated with hydrogen peroxide to induce cell damage, followed by treatment with antioxidant enzymes Cu/Zn-SOD and This is a graph comparing the mRNA expression level of GPx with the normal group and induced control group. The mRNA expression levels of Cu/Zn-SOD and GPx in the above graph are all expressed as relative values to the normal group (Control).

Looking at Figure 1b, in relation to Cu/Zn-SOD, in the case of the induced control group (H ₂ O ₂ ), the expression level of Cu/Zn-SOD was 0.63 ± 0.040**, about 63% compared to the normal group (Control, 1.0). However, the expression level of Cu/Zn-SOD in the VTHP4-treated group of Example 3 of the present invention was 0.81±0.037*, which significantly increased by 27.5% compared to the induced control group. In addition, in relation to GPx, in the case of the induced control group (H ₂ O ₂ ), the expression level of GPx was 0.52 ± 0.026**, which was lowered by about 50% compared to the normal group (Control, 1.0), but in Example 2 of the present invention In the VTH treated group, the expression level of GPx was 0.72 ± 0.023*, which was significantly increased by 37.7% compared to the induced control group, and in the VTHP4 treated group of Example 3, the expression level of GPx was 0.90 ± 0.025**, compared to the induced control group. There was a significant increase of 72.4%.

From these results, it can be seen that the sea buckthorn fruit extract or its fraction of the present invention increases the expression of antioxidant enzymes in muscle cells caused by oxidative damage, thereby suppressing oxidative cell damage.

Test Example 2: Inhibitory effect on adipocyte differentiation

2-1: Inhibitory effect on adipocyte differentiation of sea buckthorn fruit extract or fraction

The C3H10T1/2 cell line was cultured in DMEM medium supplemented with 10% FBS, 1% penicillin, and streptomycin at 37°C in a 5% CO₂ environment. The C3H10T1/2 cells were cultured in a 6-well plate at a concentration of 2.5 Each sample was added at 50 μg/mL, and the cells were differentiated for 9 days, changing the medium every 3 days. As a positive control group, 30 μM of resveratrol was treated.

After completion of differentiation, the medium was removed, the cells were fixed with 10% neutral buffered formalin, and then stained with 0.5% Oil Red O staining. The results of confirming the degree of adipocyte differentiation are shown in Figure 2.

Figure 2 is for confirming the effect of inhibiting adipocyte differentiation after treating C3H10T1/2 cells, a pluripotent stem cell line, with sea buckthorn fruit extract or fractions thereof according to an embodiment of the present invention. Figure 2a shows Oil Red O staining method. This is a photograph to confirm the degree of fat production by staining fat globules produced in adipocytes through the process. Figure 2b shows the degree of adipogenesis by dissolving fat globules stained with Oil Red O and measuring their absorbance compared to the normal group. This is a graph converted to .

Looking at Figure 2b, it can be seen that the degree of adipogenesis in all groups treated with the sea buckthorn fruit extract or fraction thereof sample of the present invention was significantly reduced compared to the normal group (NC). In particular, the degree of adipocyte differentiation in the sample administration group (VTE) of Example 1 was 0.81 ± 0.035*, a significant decrease of 18.9% compared to the normal group, and the degree of adipocyte differentiation in the sample administration group (VTH) of Example 2 was 0.69. ± 0.031**, a significant decrease of 30.2% compared to the normal group, and the degree of adipocyte differentiation in the sample administration group (VTHP4) of Example 3 was 0.61 ± 0.035**, a significant decrease of 38.9% compared to the normal group.

2-2: Comparison of the effects of sea buckthorn fruit, leaves, and seed extracts on inhibiting adipocyte differentiation

An attempt was made to compare the effects of the sea buckthorn fruit extract of the present invention and the sea buckthorn leaf extract or seed extract on adipocyte differentiation inhibition.

For this purpose, the same procedure as Example 1 was performed, but sea buckthorn leaf ethanol extract (Leaf-E) was prepared by extracting sea buckthorn leaves instead of sea buckthorn fruits, and sea buckthorn seed ethanol extract (Seed-E) was prepared by extracting sea buckthorn seeds. . In addition, the same procedure as in Example 4 was performed, but sea buckthorn leaf hexane extract (Leaf-H) was prepared by extracting sea buckthorn leaves instead of sea buckthorn fruits, and sea buckthorn seed hexane extract (Seed-H) was prepared by extracting sea buckthorn seeds.

Thereafter, the results of confirming the degree of adipocyte differentiation using the same method as Test Example 2-1 are shown in Figure 3.

Figure 3 shows sea buckthorn fruit ethanol extract (Fruit-E), sea buckthorn leaf ethanol extract (Leaf-E), and sea buckthorn seed ethanol extract (Seed) according to Example 1 of the present invention in C3H10T1/2 cells, a pluripotent stem cell line. -E), Inhibition of adipocyte differentiation after treatment with sea buckthorn fruit hexane extract (Fruit-H), sea buckthorn leaf hexane extract (Leaf-H), and sea buckthorn seed hexane extract (Seed-H) according to Example 4 To confirm the effect, Figure 3a is a photograph to confirm the degree of adipogenesis by staining fat globules generated in adipocytes through Oil Red O staining, and Figure 3b shows the degree of adipogenesis using Oil Red O staining. This is a graph in which fat globules stained with Oh are melted and their absorbance is measured and converted to that of the normal group.

Looking at Figure 3b, it can be seen that the degree of adipogenesis in all groups treated with sea buckthorn fruit extract samples of the present invention was significantly reduced compared to the normal group (NC). In particular, the degree of adipocyte differentiation in the sample administration group (Fruit-E) of Example 1 was 0.72 ± 0.049**, which was significantly reduced by about 28% compared to the normal group, and the sample administration group (Fruit-H) of Example 4 had a significant decrease of about 28% compared to the normal group. The degree of adipocyte differentiation was 0.62 ± 0.058†, a significant decrease of about 38% compared to the normal group. In particular, the degree of adipocyte differentiation in the sample administration group (Fruit-H) of Example 4 was similar to that of sea buckthorn leaf ethanol extract (Leaf-E), sea buckthorn seed ethanol extract (Seed-E), and sea buckthorn leaf hexane extract (Leaf-H). ), and a significant decrease compared to sea buckthorn seed hexane extract (Seed-H).

Test Example 3: Effect of increasing estrogen receptor activity

3-1: Comparison of the effects of sea buckthorn fruit, leaves, and seed extracts on increasing estrogen receptor activity

We sought to compare the estrogen receptor activation effects of the sea buckthorn fruit extract of the present invention and the sea buckthorn leaf extract or seed extract. To evaluate estrogen activity, cell proliferation effect was measured using MCF-7 cells.

Specifically, an estrogen-free solution prepared by diluting 5% dextran-coated charcoal-stripped FBS (TCB, USA) and 1% penicillin-streptomycin in phenol-free DMEM. The culture medium was used in the experiment.

24 hours before the experiment, the MCF-7 cell plate was cultured by replacing it with estrogen-free culture medium, and then the corresponding MCF-7 cells were distributed in a 96-well plate at a concentration of 5 × 10 ³ cells/well and incubated in 5% CO ₂ at 37°C. Samples were processed after pre-culturing in an incubator for 24 hours. The final DMSO concentration of the sample was treated to be 0.1%, and E2 (estradiol) was used as a positive control. After sample treatment and reaction for 120 hours, cell viability was measured using MTT reagent. Treat each well with 30 ㎕ of MTT reagent, react for 2 hours and 30 minutes in an incubator at 5% CO ₂ at 37°C, measure the absorbance at 570 nm using a microplate reader, and convert to relative cell proliferation rate. , shown in Figure 4 and Table 2 below.

division MCF-7 cell proliferation rate (%) Control group (NC) 100±3.79 Positive control group (E2) 141±4.81 ^† Example 1 (Fruit-E) 107±3.72 ^* Leaf-E 110±4.01 ^* Seed-E 102±5.15 Example 4 (Fruit-H) 125±4.12 ^† Leaf-H 97±3.34 Seed-H 93±5.11

The *, **, and † indicate significant differences at p<0.05, p<0.01, and p<0.1, respectively, compared to the control group.

Looking at Figure 4 and Table 2 above, it can be seen that when treated with sea buckthorn fruit extract according to an example of the present invention, the proliferation of MCF-7 cells significantly increased compared to the control group. In particular, when the sea buckthorn fruit hexane extract of Example 4 was treated, the sea buckthorn fruit ethanol extract, sea buckthorn leaf ethanol extract, sea buckthorn seed ethanol extract, sea buckthorn leaf hexane extract, and sea buckthorn seed hexane extract of Example 1 were used. It can be seen that the proliferation of MCF-7 cells significantly increased compared to the untreated case.

For reference, in order to confirm whether each sample exerts an MCF-7 cell proliferation effect by acting on the estrogen receptor, the sample was treated with ICI 182,780, an estrogen receptor inhibitor, and it was specifically confirmed that the cell proliferation effect was significantly inhibited. Accordingly, it can be inferred that the MCF-7 cell proliferation effect confirmed in Figure 4 and Table 2 is due to each sample activating the estrogen receptor.

3-2: Effect of increasing estrogen receptor activity of sea buckthorn fruit extract or fraction

We sought to compare the estrogen receptor activation effect of sea buckthorn fruit extract or fractions thereof according to an embodiment of the present invention.

Thereafter, the results of confirming the estrogen receptor activation effect in the same manner as Test Example 2-1 are shown in Figure 5 and Table 3 below.

division MCF-7 cell proliferation rate (%) Control group (NC) 100±2.84 Positive control group (E2) 138±3.76 ^† Example 1 (VTE) 107±2.89 ^* Example 2 (VTH) 113±3.26 ^** Example 3 (VTHP4) 131±3.85 ^† Example 4 (VTHX) 128±3.72 ^†

The *, **, and † indicate significant differences at p<0.05, p<0.01, and p<0.1, respectively, compared to the control group.

Looking at Figure 5 and Table 3, it can be seen that the proliferation of MCF-7 cells was significantly increased compared to the control group when treated with the sea buckthorn fruit extract or fraction according to an example of the present invention. In particular, when the active fraction of the hexane fraction of the sea buckthorn fruit of Example 3 and the hexane extract of the sea buckthorn fruit of Example 4 were treated, not only the control group but also the ethanol extract of the sea buckthorn fruit of Example 1 and the sea buckthorn fruit of Example 2 were treated. It can be seen that the proliferation of MCF-7 cells significantly increased compared to the case of treatment with the hexane fraction.

In addition, it can be inferred that the MCF-7 cell proliferation effect confirmed in Figure 5 and Table 3 is due to each sample activating the estrogen receptor.

Test Example 4: Female menopausal symptom induction animal model (1) production and sample administration

4-1: Production of female menopausal symptom inducing animal model (1)

In order to confirm the effect of the sea buckthorn fruit extract or fraction thereof of the present invention in improving female menopausal symptoms, an animal model in which female menopausal symptoms were induced was created.

The animals used in the experiment were 9-week-old ICR female mice. The mice were divided into a normal group without surgery, a group in which laparotomy was performed without ovaries (sham), and an ovariectomy group in which the ovaries were surgically removed. was classified. An animal model in which female menopausal symptoms were induced was prepared by removing the ovaries of mice using the above method.

Specifically, 9-week-old ICR female mice were purchased from Orient Bio, and 46 mice with a 1-week acclimatization period were bred for 12 weeks to induce female menopausal symptoms after ovariectomy surgery. During the experimental period, five experimental animals were raised in one cage, and environmental conditions were adjusted to room temperature of 24 ± 2 ℃, relative humidity of 55 ± 10%, and lighting time of 12 hours. Feed and water were allowed to be consumed freely. Ovariectomy was performed by administering respiratory anesthesia using CO ₂ gas, removing hair from the skin, making an incision, removing the ovaries, and suturing them. The experimental samples were administered orally once a day for a total of 12 weeks.

4-2: Test group classification and sample administration

Normal group (Sham): Experimental group in which laparotomy was performed without ovaries removed, 6 animals

Control group (OVX): Saline administration group after ovariectomy, 10 animals

Experimental group 1 (VTE): Oral administration of 100 mg/kg of ethanol extract of sea buckthorn fruit of Example 1 after ovariectomy, 10 animals

Experimental group 2 (VTH): Oral administration of 100 mg/kg of hexane fraction of sea buckthorn fruit of Example 2 after ovariectomy, 10 animals

Experimental group 3 (VTHP4): Oral administration of 50 mg/kg of the active fraction of the hexane fraction of sea buckthorn fruit of Example 3 after ovariectomy, 10 animals

Test Example 5: Body fat reduction and muscle loss recovery effect by administration of sea buckthorn fruit extract or fraction

Changes in body fat mass and lean body mass (muscle mass) of sea buckthorn fruit extract or fractions thereof according to an example of the present invention on an animal model inducing female menopausal symptoms were measured and are shown in Table 4 below. At this time, the fat content of the mice was analyzed using dual energy X-ray absorptiometry on the whole body of anesthetized mice in each experimental group using INALYZER (MEDIKORS).

division Total body weight (g) Fat mass (% of body mass) Lean body mass (% of body mass) Initial Final Gap Inital Final Gap Initial Final Gap Sham 47.12
±4.72 48.22
±4.65 1.1
±0.11 ^** 40.42
±3.01 ^** 40.65
±3.56 ^** 0.23
±0.97 59.58
±3.01 ^** 59.35
±3.56 ^** -0.23
±0.97 OVX 45.68
±4.37 48.85
±4.39 3.17
±0.43 46.91
±1.26 47.91
±1.75 1.00
±0.79 53.09
±1.26 52.09
±1.75 -1.00
±0.79 VTE 44.33
±4.36 46.82
±4.27 ^* 2.49
±0.30 47.24
±1.83 47.02
±1.90 -0.22
±1.19 ^* 52.76
±1.83 52.98
±1.90 0.22
±1.19 ^* VTH 45.85
±7.47 48.16
±7.44 2.31
±0.66 ^* 47.48
±1.20 46.58
±1.28 ^* -0.90
±0.47 ^* 52.52
±1.20 53.42
±1.28 ^* 0.90
±0.47 ^* VTHP4 44.37
±6.61 46.07
±6.17 ^* 1.7
±0.47 ^** 47.13
±1.92 46.01
±1.69 ^* -1.12
±0.95 ^** 52.87
±1.92 53.99
±1.69 ^* 1.12
±0.95 ^**

In Table 4, * and ** indicate significant differences (Student's t-test) at p<0.05 and p<0.01, respectively, compared to the control group (OVX; oophorectomy group).

5-1: Body fat reduction effect

Looking at Table 4 above, it was confirmed that the control group (OVX, oophorectomy group) in which female menopause was induced by removing the ovaries increased body weight and fat mass compared to the normal group (sham) in which only open surgery was performed without removing the ovaries. , it can be seen that obesity, a female menopausal symptom induced by the removal of the ovaries, was induced.

However, it was confirmed that the increase in body weight and fat mass was significantly suppressed in the group administered the sea buckthorn fruit extract or fractions thereof according to Examples 1 to 3 compared to the control group (OVX). In particular, the group administered sea buckthorn fruit extract or fractions thereof according to Examples 1 to 3 showed a decrease in fat mass after the end of the experiment.

From the above results, in mice in which female menopausal symptoms were induced by removing the ovaries, body weight and fat mass increased and obesity was induced, but in the group administered sea buckthorn fruit extract or fractions thereof according to Examples 1 to 3, body weight gain was suppressed and fat mass was reduced. rather decreased, confirming that the induction of obesity in mice was suppressed. That is, it was demonstrated that the sea buckthorn fruit extract or fractions thereof according to Examples 1 to 3 are effective in preventing and improving obesity induced by female menopause.

5-2: Muscle loss recovery effect

Looking at Table 4 above, it was confirmed that the control group (OVX, oophorectomy group) in which female menopause was induced by removing the ovaries had a decrease in lean body mass (muscle mass) compared to the normal group (sham) in which only open surgery was performed without removing the ovaries. By doing so, it can be seen that muscle loss, a symptom of female menopause induced by removal of the ovaries, was induced.

However, it was confirmed that lean body mass (muscle mass) significantly increased in the group administered sea buckthorn fruit extract or fractions thereof according to Examples 1 to 3 compared to the control group (OVX).

From the above results, in mice in which female menopausal symptoms were induced by removing the ovaries, lean body mass (muscle mass) was reduced and muscle loss was induced, but in the group administered sea buckthorn fruit extract or fractions thereof according to Examples 1 to 3, lean mass (muscle mass) was induced. Muscle mass) significantly increased, confirming that muscle loss in mice was recovered. That is, it was demonstrated that the sea buckthorn fruit extract or fractions thereof according to Examples 1 to 3 are effective in preventing and improving muscle loss induced by female menopause.

Test Example 6: Changes in blood lipid composition due to administration of sea buckthorn fruit extract or fraction

In order to evaluate the effect of the sea buckthorn fruit extract or its fractions according to Examples 1 to 3 on blood lipid composition in an animal model inducing female menopausal symptoms, blood was collected from each experimental group of mice and used with the Accute TBA-40FR kit (Toshiba Medical Systems Co., Japan), triglyceride, total cholesterol (T-cholesterol), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and free fatty acid in the blood according to the manufacturer's protocol. The content was measured.

Figure 6 shows changes in blood lipid components due to administration of the sea buckthorn fruit extract or fractions thereof of the present invention in an ovariectomized animal model, specifically, blood triglyceride content, blood cholesterol content, blood HDL content, and blood LDL content. and a graph showing fatty acid content in blood.

Looking at Figure 6, the group treated with sea buckthorn fruit fraction (VTHP4) of Example 3 of the present invention has blood triglyceride content, total cholesterol (T-cholesterol) content, HDL content, and LDL compared to the control group (OVX). It can be seen that all contents have decreased significantly.

Test Example 7: Female menopausal symptom induction animal model (2) production and sample administration

7-1: Production of female menopausal symptom inducing animal model (2)

In order to confirm the effect of the sea buckthorn fruit extract or fraction thereof of the present invention in improving female menopausal symptoms, an animal model in which female menopausal symptoms were induced was created.

The animals used in the experiment were 7-week-old Sprague Dawley rats, and were classified into a normal group that did not undergo surgery, a group that underwent laparotomy without removing the ovaries (sham), and an ovariectomy group in which the ovaries were surgically removed. did. An animal model in which female menopausal symptoms were induced was prepared by removing the ovaries of rats using the above method.

Specifically, 7-week-old Sprague Dawley rats were purchased from Orient Bio, and 70 rats that had an acclimatization period of 1 week were subjected to ovariectomy at the 8th week of rearing to induce female menopausal symptoms. During the experimental period, five experimental animals were raised in one cage, and environmental conditions were adjusted to room temperature of 24 ± 2 ℃, relative humidity of 55 ± 10%, and lighting time of 12 hours. Feed and water were allowed to be consumed freely. Ovariectomy was performed by administering respiratory anesthesia using CO ₂ gas, removing hair from the skin, making an incision, removing the ovaries, and suturing them. Oral administration of the samples began at the 11th week of rearing, and the experimental samples were administered orally once a day for a total of 12 weeks. The body weight of the animal model was measured weekly.

7-2: Test group classification and sample administration

Normal group (Sham): Experimental group in which laparotomy was performed without ovaries removed, 10 animals.

Control group (OVX): Saline administration group after ovariectomy, 10 animals

Positive control group (E2): Oral administration of 17β-estradiol 0.1 mg/kg after ovariectomy, 10 animals

Experimental group 1 (VTE): Oral administration of 100 mg/kg of ethanol extract of sea buckthorn fruit of Example 1 after ovariectomy, 10 animals

Experimental group 2 (VTH): Oral administration of 100 mg/kg of hexane fraction of sea buckthorn fruit of Example 2 after ovariectomy, 10 animals

Experimental group 3 (VTHP4): Oral administration of 50 mg/kg of the active fraction of the hexane fraction of sea buckthorn fruit of Example 3 after ovariectomy, 10 animals

Experimental group 4 (VTHX): Oral administration of 100 mg/kg of sea buckthorn fruit hexane extract of Example 4 after ovariectomy, 10 animals

The classification and sample dosage of the experimental groups are briefly shown in Table 5 below.

experimental group Treatment and administration conditions population dosage Sham Normal control (open surgery is performed) + excipient administration 10 5mL/kg OVX OVX (oophorectomy) + excipient administration 10 5mL/kg E2 OVX + 17β-estradiol 0.1 mg/kg 10 5mL/kg VTE OVX + 100 mg/kg of composition of Example 1 10 5mL/kg VTH OVX + 100 mg/kg of composition of Example 2 10 5mL/kg VTHP4 OVX + 50 mg/kg of composition of Example 3 10 5mL/kg VTHX OVX + 100 mg/kg of composition of Example 4 10 5mL/kg

Test Example 8: Effect of improving weight gain by administration of sea buckthorn fruit extract or fraction

During female menopause, symptoms of weight gain generally appear due to a decrease in estrogen, and obesity is caused by high-fat diet intake. Body weight was measured after oral administration of the sea buckthorn fruit extract or fraction of the example for 12 weeks to an animal model inducing female menopausal symptoms and is shown in Figure 7 and Table 6.

division Weight (g) Sham 314.7±13.98 ^** OVX 401.7±26.33 E2 349.5±24.35 ^* VTE 394.2±19.62 VTH 379.5±21.46 VTHP4 355.2±18.73 ^* VTHX 358.3±19.87 ^*

The * and ** indicate significant differences at p<0.05 and p<0.01, respectively, compared to the OVX group.

Looking at Figure 7 and Table 6, it can be seen that the body weight of the OVX rat (negative control) from which the ovaries were removed was significantly increased compared to the normal Sham rat from which the ovaries were not removed. On the other hand, in the VTHP4 administered group according to Example 3 of the present invention and the VTHX administered group according to Example 4, the weight gain in the menopausal induced animal model was found to be significantly suppressed.

These results mean that the sea buckthorn fruit extract or fraction of the present invention has a significant effect in suppressing weight gain due to menopause. Statistical analysis also confirmed the significance of efficacy (*, **: p<0.05, p<0.01 vs. OVX negative control).

Test Example 9: Effect of increasing estrogen receptor expression by administration of sea buckthorn fruit extract or fraction

After measuring the weight of femoral muscle tissue isolated from an animal model inducing female menopausal symptoms, RNA was extracted using Nucleozol. The amount of extracted RNA was confirmed using NanoDrop. In addition, cDNA was synthesized by reverse transcription with remix ReverTra Ace qPCR RT master, and then real-time PCR was performed using sequence-specific primers for each gene and SYBR Green. Then, the expression level of each mRNA was analyzed using the relative quantification method of the target gene using the GAPDH gene as a control, and is shown in Figures 8a, 8b, and Table 8. The sequence information of the gene-specific primers used to confirm the expression level of the estrogen receptor is shown in Table 7 below.

sequence number denomination base sequence SEQ ID NO: 1 ER alpha_F_rat GCA CAT TCC TTC CTT CCG TC SEQ ID NO: 2 ER alpha_R_rat CTC GTT CCC TTG GAT CTG GT SEQ ID NO: 3 ER beta_F_rat ACA GTC CTG CTG TGA TGA AC SEQ ID NO: 4 ER beta_R_rat ACT AGT AAC AGG GCT GGC AC

division mRNA relative expression level ERα ERβ Sham 1.00±0.17 1.00±0.22 ^* OVX 0.72±0.19 0.50±0.18 E2 0.94±0.15 1.73±0.27 ^** VTE 0.78±0.16 0.8±0.21 VTH 1.03±0.19 1.01±0.23 ^* VTHP4 0.95±0.18 1.51±0.18 ^** VTHX 1.19±0.22 1.71±0.25 ^**

The * and ** indicate significant differences at p<0.05 and p<0.01, respectively, compared to the OVX group.

Looking at FIGS. 8A, 8B, and Table 8, it can be seen that the expression level of estrogen receptors is significantly reduced in OVX rats (negative control) from which ovaries have been removed, compared to normal Sham rats without ovaries removed. On the other hand, it can be confirmed that the expression of estrogen receptors in the group administered the sea buckthorn fruit extract or fraction of the present invention increased compared to the OVX rats (negative control group). In particular, the expression of estrogen receptors in the group administered sea buckthorn fruit extract in Example 4 increased to a level similar to that of the positive control group.

Test Example 10: Effect of improving muscle strength reduction by administration of sea buckthorn fruit extract or fraction

10-1: Improvement effect in reducing grip strength

Changes in muscle strength of sea buckthorn fruit extract or fractions thereof according to an example of the present invention on an animal model inducing female menopausal symptoms were measured and are shown in Figure 9 and Table 9 below. At this time, the muscle strength (grip strength) of the animal model was measured using a Grip strength meter (animal grip strength system, San Diego, USA, MAI 125-01) by having the rat hold a bar with the front toes and then holding the origin of the tail and keeping the body horizontal. The value that appears on the gauge was measured when the bar was pulled at a constant speed and released.

division Grip strength (g) Sham 725.65±51.61 ^** OVX 583.2±50.16 E2 688.8±48.94 ^* VTE 600.7±37.99 VTH 632.6±43.74 VTHP4 693.2±44.68 ^* VTHX 672.4±41.82 ^*

The * and ** indicate significant differences at p<0.05 and p<0.01, respectively, compared to the OVX group.

Looking at FIG. 9 and Table 9 below, it can be seen that grip strength is significantly reduced in OVX rats (negative control group) from which ovaries have been removed compared to normal Sham rats without ovaries removed. On the other hand, in the VTHP4 according to Example 3 of the present invention and the VTHX administered group according to Example 4 of the present invention, the decrease in grip strength in the menopausal induced animal model was found to be significantly suppressed.

These results mean that the sea buckthorn fruit extract or fraction of the present invention has a significant effect in suppressing the decrease in muscle strength due to menopause. Statistical analysis also confirmed the significance of efficacy (*, **: p<0.05, p<0.01 vs. OVX negative control group).

10-2: Effect of increasing the expression level of muscle-related biomarkers

After measuring the weight of femoral muscle tissue isolated from an animal model inducing female menopausal symptoms, RNA was extracted using Nucleozol. The amount of extracted RNA was confirmed using NanoDrop. In addition, cDNA was synthesized by reverse transcription with remix ReverTra Ace qPCR RT master, and then real-time PCR was performed using sequence-specific primers for each gene and SYBR Green. Then, the expression level of each mRNA was analyzed using the relative quantification method of the target gene using the GAPDH gene as a control, and is shown in Figures 10a, 10b, and Table 11. The sequence information of the gene-specific primers used to confirm the expression level of muscle-related biomarkers is shown in Table 10 below.

sequence number denomination base sequence SEQ ID NO: 5 MyoD_F_rat GGA GAC ATC CTC AAG CGA TGC SEQ ID NO: 6 MyoD_R_rat GCA CCT GGT AAA TCG GAT TG SEQ ID NO: 7 MurF1_F_rat GTG AAG TTG CCC CCT TAC AA SEQ ID NO: 8 MurF1_R_rat TGG AGA TGC AAT TGC TCA GT

division mRNA relative expression level MurF1 MyoD Sham 1.00±0.18 ^* 1.00±0.13 ^* OVX 0.57±0.19 0.607±0.12 E2 2.01±0.38 ^** 1.03±0.18 ^* VTE 0.58±0.27 0.482±0.09 VTH 0.97±0.22 ^* 0.463±0.11 VTHP4 1.9±0.34 ^** 0.96±0.17 ^* VTHX 1.7±0.28 ^** 0.89±0.14 ^*

The * and ** indicate significant differences at p<0.05 and p<0.01, respectively, compared to the OVX group.

Looking at Figures 10a, 10b, and Table 11, it can be seen that the expression level of muscle-related biomarkers is significantly reduced in OVX rats (negative control group) from which ovaries have been removed, compared to normal Sham rats without ovaries removed. . On the other hand, it can be confirmed that the expression of muscle-related biomarkers was significantly increased in the group administered the sea buckthorn fruit extract or fraction of the present invention compared to the OVX rats (negative control group). In particular, the expression of muscle-related biomarkers in the VTHP4-administered group in Example 3 and the VTHX-administered group in Example 4 increased to a level similar to that of the positive control group.

Through these results, the present inventors found that the sea buckthorn fruit extract or its fraction of the present invention protects muscle cells, recovers muscle loss caused by decreased estrogen secretion, reduces body fat increased due to decreased estrogen secretion, and reduces blood lipid components. It was specifically confirmed that the change was good. Accordingly, it was found that the sea buckthorn fruit extract or its fraction of the present invention can be usefully used in the prevention or treatment of various female menopausal symptoms, including muscle loss, muscle weakness, obesity, and blood lipid disorders.

Below, a formulation example of a composition containing the sea buckthorn fruit extract or a fraction thereof of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.

Formulation Example 1: Preparation of powder

20 mg of sea buckthorn fruit fraction of Example 3

lactose 100mg

Talc 10mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

Formulation Example 2: Preparation of tablets

10 mg of sea buckthorn fruit fraction of Example 3

corn starch 100mg

lactose 100mg

Magnesium stearate 2mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

Formulation Example 3: Preparation of capsules

10 mg of sea buckthorn fruit fraction of Example 3

crystalline cellulose 3mg

lactose 14.8mg

magnesium stearate 0.2mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.

Formulation Example 4: Manufacturing of health functional food

1,000 mg of sea buckthorn fruit fraction of Example 3

vitamin mixture Appropriate amount

Vitamin A Acetate 70 ㎍

Vitamin E 1.0mg

Vitamin B1 0.13 mg

Vitamin B2 0.15mg

Vitamin B6 0.5mg

Vitamin B12 0.2 μg

Vitamin C 10mg

biotin 10 μg

Nicotinic acid amide 1.7mg

folic acid 50 μg

Calcium Pantothenate 0.5mg

mineral mixture Appropriate amount

Ferrous sulfate 1.75mg

zinc oxide 0.82mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15mg

potassium phosphate dibasic 55mg

Potassium citrate 90mg

calcium carbonate 100mg

Magnesium chloride 24.8mg

The composition ratio of the above vitamin and mineral mixture is a mixture of ingredients suitable for health functional foods in a preferred embodiment, but the mixing ratio may be modified arbitrarily, and the above ingredients are mixed according to a normal health functional food manufacturing method. Next, granules can be prepared and used to manufacture a health functional food composition according to a conventional method.

Formulation Example 5: Preparation of beverage formulation

1,000 mg of sea buckthorn fruit fraction of Example 3

citric acid 1,000 mg

oligosaccharide 100g

plum concentrate 2g

taurine 1g

Add purified water Total 900 mL

After mixing the above ingredients according to a typical beverage production method, stirring and heating at 85° C. for about 1 hour, the resulting solution was filtered, placed in a sterilized 2 L container, sealed, sterilized, and stored in the refrigerator. Then, the solution according to the present invention was mixed. A functional beverage composition manufacturing example was used.

Although the present invention has been described in terms of the above-mentioned preferred embodiments, various modifications and variations can be made without departing from the gist and scope of the invention. Additionally, the appended claims cover such modifications or variations as fall within the subject matter of the present invention.

Claims (12)

A food composition for preventing or improving female menopausal symptoms comprising sea buckthorn fruit extract or a fraction thereof as an active ingredient. According to paragraph 1,
The sea buckthorn fruit extract is characterized in that it is an extract of one or more solvents selected from water, alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, hexane, benzene, chloroform, glycerin, butylene glycol, and propylene glycol. A food composition for preventing or improving female menopausal symptoms. According to paragraph 2,
A food composition for preventing or improving female menopausal symptoms, wherein the solvent is 20 to 80% by volume of methanol, ethanol, butanol, or propanol. According to paragraph 2,
A food composition for preventing or improving female menopausal symptoms, wherein the solvent is hexane. According to paragraph 1,
A food composition for preventing or improving female menopausal symptoms, wherein the sea buckthorn fruit fraction is a hexane fraction of sea buckthorn fruit. According to paragraph 1,
A food composition for preventing or improving female menopausal symptoms, wherein the female menopausal symptoms are caused by decreased secretion of estrogen. According to paragraph 1,
The above female menopausal symptoms include facial flushing, dry skin, arrhythmia, edema, sweating, urgent urination, pain in urination, frequent urination, urinary incontinence, cystitis, atrophy of the lower urethra, decreased muscle mass, muscle weakness, muscle pain, joint pain, osteoporosis, sensitivity to cold, vaginal dryness, and vaginal atrophy. , fatty liver, abdominal obesity, increased body fat, weight gain, blood lipid disorder, hyperlipidemia, difficulty concentrating, short-term memory disorder, memory loss, headache, heart palpitations, dizziness, sweating during sleep, sleep disorders, depression, anxiety, and nervousness. A food composition for preventing or improving female menopausal symptoms, characterized in that it contains one or more types selected from among. According to paragraph 1,
A food composition for preventing or improving female menopausal symptoms, wherein the female menopausal symptoms are one or more selected from the group consisting of decreased muscle mass, decreased muscle strength, fatty liver, abdominal obesity, increased body fat, weight gain, blood lipid disorder, and hyperlipidemia. According to paragraph 1,
The food composition is a food composition for preventing or improving female menopausal symptoms, characterized in that it is a powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation, or beverage formulation. According to paragraph 1,
The food composition is a food composition for preventing or improving female menopausal symptoms, characterized in that it is a health functional food. A pharmaceutical composition for preventing or treating female menopausal symptoms comprising sea buckthorn fruit extract or a fraction thereof as an active ingredient. According to clause 11,
The pharmaceutical composition is a pharmaceutical composition for preventing or treating female menopausal symptoms, characterized in that the pharmaceutical composition is an oral preparation.