KR20210154001A - Composition containing the extracts or fractions of Agastache rugosa for the prevention and treatment of postmenopausal syndrome - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating postmenopausal syndrome, comprising an extract of Agastache rugosa or a fraction thereof as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating postmenopausal syndrome comprising an extract of Agastache rugosa or a fraction thereof as an active ingredient; a food composition for preventing or improving postmenopausal syndrome; and a method for preventing or treating postmenopausal syndrome, comprising administering the composition to individuals other than humans. The Agastache rugosa extract of the present invention has no resistance even when taken for a long time, can be safely applied without side effects, and can be advantageously used as a composition for preventing or treating postmenopausal syndrome by promoting estrogen activity and osteoblast differentiation.

Description

A composition for preventing or treating female menopause syndrome containing baechohyang extract {Composition containing the extracts or fractions of Agastache rugosa for the prevention and treatment of postmenopausal syndrome}

The present invention relates to a composition for preventing, improving or treating female menopause syndrome, comprising an extract of baechohyang (Agastache rugosa) or a fraction thereof as an active ingredient. More specifically, a pharmaceutical composition for the prevention or treatment of female menopause syndrome comprising a baechohyang extract or a fraction thereof as an active ingredient; a food composition for preventing or improving female menopause syndrome; And it relates to a method for preventing or treating female menopause syndrome, comprising administering the composition to a subject other than a human.

Due to recent advances in medicine and improvement in diet, the elderly population is continuously increasing worldwide. Accordingly, the problem of various metabolic diseases is rapidly emerging due to aging, examples of which include obesity, diabetes, and cardiovascular diseases such as stroke. In particular, women experience a sudden hormonal imbalance due to menopause and experience the menopause syndrome. Menopause syndrome includes menopausal syndromes such as hot flashes, insomnia, and depression, and chronic diseases such as osteoporosis and high blood pressure. In particular, osteoporosis is a disease in which bone strength is weakened and fractures occur easily due to a decrease in bone density and a decrease in bone quality. It is also known that the decrease in hormones after menopause leads to a metabolic imbalance, leading to rapid weight gain. Therefore, estrogen decline due to menopause is closely related to bone health and metabolic diseases as well as hormone-dependent diseases.

Estrogen affects the human body by changing the expression of genes through the estrogen receptor as a signal transmitter. Estrogen receptors include estrogen receptor-α, estrogen receptor-β, and G protein-coupled receptor 30 (GPR30) located in the cell membrane. These estrogen receptors are distributed in various tissues such as the lungs, brain, and bone marrow as well as tissues related to female reproduction, such as the uterus, breast, and ovaries. will be. Hormone replacement therapy, which artificially administers estrogen, is widely used for the treatment of the menopausal syndrome, but there is a controversy over its safety as it raises the possibility of causing uterine cancer, breast cancer or gallbladder disease when used for a long period of time. Therefore, research to find natural estrogen derived from natural products with fewer side effects is being actively conducted.

Osteoporosis, one of the menopausal syndromes, is a disease caused by the breakdown of bone homeostasis. For normal bone formation, there must be a balance between the action of osteoclasts that absorb bone and osteoblasts that form bone. will induce Since hormone replacement therapy does not have a significant effect on osteoporosis, the currently used osteoporosis treatment mainly consists of bisphosphate, which weakens the function of osteoclasts. .

On the other hand, baechohyang is a dicotyledonous plant, a perennial herb in the family Lamiaceae, distributed in Korea, Japan, Taiwan, and China. The flower is lip-shaped, blooms in July-September, turns purple, hangs in the inflorescence of yunsan, and has a fragrance. The inflorescence is ear-shaped and is 5-15cm long. The calyx is divided into 5 pieces, and the corolla is 8-10mm long, the upper lip-shaped petals are small, the lower lip is large, and it is divided into 5 pieces. 2 out of 4 stamens are long. The fruit is a fissionaceae and is flat and oval in the shape of an egg. It is known that young shoots are used as vegetables and are cultivated for ornamental purposes. There is no known information about the efficacy of baechohyang for alleviating women's menopausal syndrome.

Accordingly, the present inventors made intensive research efforts to develop a new composition for preventing or treating menopausal syndrome, and as a result, it was confirmed that the baechohyang extract or a fraction thereof had estrogenic activity, and it was confirmed that there was an effect of increasing osteoblast activity and inhibiting osteoclast formation. , the present invention was completed.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating female menopause syndrome comprising a baechohyang extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving female menopause syndrome comprising a baechohyang extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating female menopause syndrome, comprising administering a composition comprising a baechohyang extract or a fraction thereof as an active ingredient to individuals other than humans.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating female menopause syndrome comprising a baechohyang extract or a fraction thereof as an active ingredient.

As used herein, the term "baechohyang ( Agastache rugosa )" is a dicotyledonous perennial herb in the order Lamiaceae, also called bangah, bangae, and the like. In China, it is written as Kwakhyang (藿香), and in Korea, mature baechohyang is called Kwakhyang and used as medicine. It is known to be effective for digestion, stomach pain, pain relief, vomiting, abdominal pain, and colds, but there has been no reported effect on relieving menopausal syndrome for women.

Baechohyang in the present invention can use all of its leaves, stems, or roots to produce an extract, and specifically, although not limited thereto, each of the above-ground parts including leaves and stems are used. In addition, the baechohyang can be used without limitation, such as collected, cultivated, or commercially available ones.

The baechohyang extract refers to all forms by additional processing of the extract as well as the baechohyang crude extract.

The baechohyang extract may be baechohyang itself or a pulverized product thereof, or an extract obtained through an extraction process. The extraction process may be carried out according to a conventional method used in the art.

The baechohyang extract of the present specification may be prepared in the following steps, but is not limited thereto.

1) extracting dried baechohyang by adding an extraction solvent;

2) filtering the extract of step 1); and

3) preparing an extract by concentrating the filtered extract of step 2) under reduced pressure; and

4) preparing a fraction by additionally extracting the extract of step 3) with an organic solvent.

The extraction solvent may be water, alcohol, or a mixture thereof, specifically, a solvent selected from C1 to C4 lower alcohols or mixed solvents thereof, and more specifically, extraction with methanol or ethanol, but limited thereto it is not going to be The amount of the extraction solvent may be 2 to 10 times the dry weight of baechohyang, but is not limited thereto.

The extraction method may use an extraction method such as hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction, and specifically, may be extracted one to five times by an ultrasonic extraction method. The extraction temperature may be specifically 10° C. to 100° C., and more specifically, room temperature, but is not limited thereto. The extraction time may be specifically 1 to 7 days, more specifically 3 to 7 days, but is not limited thereto.

The method for preparing the extract is a method using an extraction apparatus such as supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction or ultrasonic extraction, or a method using an adsorption resin including XAD and HP-20, such as a conventional extraction in the art. A method may be used, and specifically, extraction may be performed under reflux or extraction at room temperature with heating, but is not limited thereto. The number of extractions may be specifically 1 to 5 times, and more specifically, extraction may be repeated 3 times, but is not limited thereto.

The fraction of baechohyang may be extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid. The organic solvent having 1 to 6 carbon atoms may be obtained by sequential fractionation of n-hexane, methylene chloride, ethyl acetate, n-butanol, and water, but is not limited thereto. The organic solvent having 1 to 6 carbon atoms is alcohol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, having 1 to 6 carbon atoms. chloride), hexane (hexane), cyclohexane (cyclohexane) and petroleum ether (petroleum ether) may be at least one selected from the group consisting of.

Specifically, in the present invention, it may be methylene chloride or ethyl acetate, but is not limited thereto.

The fluid may use a subcritical or supercritical fluid, which is any one of cosolvents including water, carbon dioxide, acetone, and water.

The fractionation may be repeated one or more times, for example, 1 to 5 times. Separation of the fraction may be performed by a known method such as filtration. In addition, the fractionation may include the step of concentrating and / or drying the obtained fraction. The concentration may be reduced pressure concentration. The drying may be reduced pressure drying, boiling drying, spray drying, room temperature drying or freeze drying.

The fraction of baechohyang is a hexane fraction, methyl chloride fraction, ethyl acetate fraction and butanol fraction, which are fractionated by sequentially adding hexane, methyl chloride, ethyl acetate and butanol after suspending the baechohyang extract in water. At least one selected from the group consisting of an ethyl acetate fraction and a butanol fraction. It may include fractions. In this case, the baechohyang extract may be an ethanol extract, and the baechohyang extract may include a methylene chloride fraction and an ethyl acetate fraction.

The extract of baechohyang may be included in an amount of 0.001 to 80% by weight based on the total weight of the composition. By including the extract of baechohyang in the above range in the composition, it has an appropriate composition ratio with other ingredients and may exhibit the prevention, improvement or treatment effect of bone disease, and may be preferable in terms of economy and efficiency. Specifically, the extract of baechohyang is based on the total weight of the composition 0.005 to 78% by weight, or 0.01 to 76% by weight, or 0.05 to 74% by weight, or 0.1 to 72% by weight, or 0.5 to 70% by weight, or 1.0 to 68% by weight, or 1.0 to 66% by weight, or 1.0 to 64% by weight, or 1.0 to 62% by weight, or 1.0 to 60% by weight, or 1.0 to 50% by weight, or 1.0 to 40% by weight, or 1.0 to 30% by weight. It may be included in an amount of weight %, or 1.0 to 20% by weight, or 1.0 to 10% by weight.

As used herein, the term "active ingredient" refers to a component capable of exhibiting the desired activity alone or in combination with a carrier having no activity by itself.

As used herein, the term “female menopause syndrome” may refer to menopausal syndrome. Menopause syndrome is the vasomotor symptoms of estrogen deficiency and includes hot flashes, sleep disturbance, depression, palpitations, sweating, cold sweat, and pain in bones and muscles.

In addition, the female menopause syndrome includes cardiovascular diseases such as hypertension, osteoporosis, and obesity (Nat Rev Dis Primers. 2015 Apr 23; 1:15004).

The composition according to the present invention may have estrogenic activity.

In one embodiment of the present invention, the pERE-vitallogenin-Luc plasmid (pERE-vitallogenin-Luc plasmid) is transfected MCF-7 (human breast cancer cell line) to determine whether the baechohyang extract has estrogenic activity. ) to determine the degree of activation of the estrogen response site (Estrogen Responsive Element; ERE) by measuring the luciferase activity (FIG. 1).

In addition, the composition according to the present invention may promote the differentiation of osteoblasts or the activity of osteogenic proteins, and inhibit the generation of osteoclasts.

In another embodiment of the present invention, after inducing the differentiation of osteoblasts into MC3T3E1 cells, which are preosteoblasts in mice, in order to determine whether the baechohyang extract or fraction has the effect of promoting differentiation of osteoblasts (osteocyte), the present invention By treatment with the extract or fraction of baechohyang of the present invention, the activity change of alkaline phosphatase (ALP), a marker for early differentiation of osteoblasts, was measured (FIG. 2), and bone by treatment with baechohyang extract or fraction in MC3T3-E1 osteoblasts. Production protein (Collagen, type I alpha 1(Col1A1)), osteoclast formation inhibitory protein (osteoprotegerin(OPG)), osteoblast differentiation inducing protein (Runt-related transcription factor 2(Runx2), Osterix (Osx)), osteoblasts The expression levels of signaling proteins (Transforming growth factor beta (TGFβ), Bone morphogenetic protein 2/4 (BMP2/4)) for differentiation induction were measured ( FIG. 3 ), and a mouse model induced by ovariectomy to induce menopause. Changes in bone density by treatment with baechohyang extract were measured by micro-CT (FIG. 4).

When the above experimental results are taken together, the baechohyang extract and its fractions have a remarkable estrogenic activity and an effect of promoting differentiation of osteoblasts. That is, it was confirmed that the baechohyang extract and fraction of the present invention activates the estrogen response site, induces the growth of the human breast cancer MCF-7 cell line under hormone-deficient conditions, and has the effect of promoting differentiation of osteoblasts.

Therefore, the composition comprising the extract or fraction of baechohyang of the present invention exhibiting estrogenic activity can be usefully used as a composition for preventing and treating female menopause syndrome caused by estrogen deficiency.

The term "prevention" in the present invention refers to any action that suppresses or delays symptoms of female menopause syndrome by administration of a composition comprising a baechohyang extract or a fraction thereof according to the present invention.

The term "treatment" in the present invention refers to any action in which the symptoms of the female menopause syndrome are improved or beneficially changed by administration of a composition comprising a baechohyang extract or a fraction thereof according to the present invention.

The term "pharmaceutical composition" in the present invention means one prepared for the purpose of preventing or treating a disease, and may be formulated in various forms according to each conventional method and used. For example, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be used in the form of external preparations, suppositories, and sterile injection solutions. Specifically, it may be used in a form suitable for eye drop administration, for example, formulated as eye drops, creams, ointments, gels or lotions.

The composition of the present invention may be prepared in the form of a pharmaceutical composition for preventing or treating wounds further comprising an appropriate carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions, wherein the carrier is a non-natural carrier ( non-naturally occurring carriers).

In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the upper leaf extract and its fractions, for example, starch, calcium carbonate , sucrose or lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to simple diluents such as water and liquid paraffin, which are commonly used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered orally, parenterally, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous, etc. according to a desired method, and the active ingredient of the pharmaceutical composition depends on the age of the subject to be administered. , gender, weight, pathology and its severity, route of administration, or judgment of the prescriber. Determination of dosages based on these factors is within the level of the skilled artisan, and its daily dose is, for example, from 0.1 mg/g/day to 100 mg/g/day, more specifically from 5 mg/g/day to 50 mg/day. g/day, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose. The above dosage does not limit the scope of the present invention in any way.

Another aspect of the present invention for achieving the above object provides a food composition for preventing or improving female menopause syndrome comprising a baechohyang extract or a fraction thereof as an active ingredient.

At this time, the description of "baechohyang", "female menopause syndrome", and "prevention" is the same as described above.

As used herein, the term “improvement” refers to any action in which symptoms of menopausal syndrome are improved or beneficially changed by administering the composition.

The food composition of the present invention provides various types of food additives or functional foods. Specifically, the composition may be processed into leached tea, liquid tea, beverage, fermented milk, cheese, yogurt, juice, probiotics or health supplements, etc., and may be used in the form of other various food additives.

The food composition of the present invention may further include a food pharmaceutically acceptable carrier.

There is no particular limitation on the type of food to which the composition containing the baechohyang extract or fractions thereof of the present invention can be added, for example, various beverages, gum, tea, vitamin complexes, health supplements, and the like. Other ingredients that do not interfere with the treatment effect of menopause may be added to the food composition, and the type thereof is not particularly limited. For example, it may contain various herbal extracts, food-logically acceptable food supplements or natural carbohydrates as additional ingredients, such as conventional food.

The food supplement additive is added to the production of health functional food of each formulation, and those skilled in the art can appropriately select and use it. For example, various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents , stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, and the like, but the types are not limited by the above examples.

At this time, the content of the extract or fraction contained in the food is not particularly limited thereto, but may be included in an amount of 0.01 to 100% by weight, specifically 1 to 80% by weight, based on the total weight of the food composition.

When the food is a beverage, it may be included in a ratio of 1 to 30 g, specifically 3 to 20 g, based on 100 ml. In addition, the composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu). In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine. In addition, preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene ( BHT), etc.), colorant (tar pigment, etc.), color developer (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweetener (dulcin, cyclmate, saccharin, sodium, etc.) , flavorings (vanillin, lactones, etc.), bulking agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam suppressants, solvents, food additives such as improving agents ) can be added. The additive is selected according to the type of food and used in an appropriate amount.

The food composition of the present invention can be prepared by a method commonly used in the art, and during the preparation, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, there are no side effects that may occur when taking the drug for a long time by using food as a raw material, and it can be excellent in portability.

Another aspect of the present invention for achieving the above object provides a method for preventing or treating female menopause syndrome, comprising administering a composition comprising a baechohyang extract or a fraction thereof as an active ingredient to individuals other than humans. .

At this time, the description of the "baechohyang", "active ingredient", and "prevention" is the same as described above.

As used herein, the term "administration" means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. . Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered intranasally, but is not limited thereto.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into 2-3 times. It can also be used alone or in combination with other drug treatments. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

As used herein, the term "individual" refers to all animals, such as rats, mice, and livestock, that have or may develop wrinkles or female menopausal syndrome.

The baechohyang extract or a fraction thereof of the present invention has no resistance even when taken for a long period of time, can be safely applied without side effects, and can be usefully used as a composition for preventing or treating female menopause syndrome by promoting estrogen activity and osteoblast differentiation. .

1 is a graph showing the luciferase activity-inducing effect induced by the estrogen response site (Estrogen Responsive Element: ERE) by the treatment of baechohyang extract.
Figure 2 is a graph showing the results of measuring the increase in the activity of alkaline phosphatase (alkaline phosphatase (ALP)), which is an early differentiation marker of osteoblasts by treatment with baechohyang extract or fractions in MC3T3-E1 osteoblasts.
3 is a bone-generating protein (Collagen, type I alpha 1 (Col1A1)), osteoclast-inhibiting protein (osteoprotegerin (OPG)), osteoblast differentiation-inducing protein (Runt) by treatment with baechohyang extract or fractions in MC3T3-E1 osteoblasts. -related transcription factor 2 (Runx2), Osterix (Osx)), signal transduction protein for inducing osteoblast differentiation (Transforming growth factor beta (TGFβ), Bone morphogenetic protein 2/4 (BMP2/4)) This is a picture showing the result.
4 is a photograph showing the results of micro-CT measurement of changes in bone density by treatment with baechohyang extract in menopausal rat model induced by ovariectomy.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Preparation of baechohyang extract and fractions

Baechohyang used in this example was purchased from Gyeongdong Market. The baechohyang outpost was ground to an appropriate size, put in an extraction container, and an appropriate amount of 80% ethanol was added. These were extracted three times at room temperature for 12 hours each repeatedly using a stirrer, and then filtered through filter paper to obtain an 80% ethanol extract of baechohyang. Specifically, 510 mL of 80% ethanol was added to 50.94 g of dry weight of the above-ground part of baechohyang, extracted three times, and then concentrated under reduced pressure to obtain 7.66 g of an 80% ethanol extract (yield 15.0%).

In addition, the baechohyang fraction was dissolved by adding 100 mL of water to 1.82 g of baechohyang 80% ethanol extract, then adding the same volume of hexane to fractionate the hexane layer with a separatory funnel, and the process was repeated 3 times in total. Thereafter, the same process was carried out using methylene chloride, ethyl acetate, and butanol, and the final residue was used as a residue fraction. Each fraction was again concentrated under reduced pressure, and the yield was 213.69 mg of a hexane fraction, 160.24 mg of a methylene chloride fraction, 24.57 mg of an ethyl acetate fraction, 499.69 mg of a butanol fraction, and 596.04 mg of a residue fraction.

Example 2. Measurement of Estrogen Responsive Element Activation Effect by Baechohyang Extract or Fraction

The estrogen activation effect of the baechohyang extract prepared in Example 1 was measured using MCF-7 breast cancer cell line (American Type of Cell Culture, ATCC). MCF-7 cells ^{were seeded at a density of 5×10 4} cells/well and incubated at 37° C., 5% CO _{2 for 24 hours.} Thereafter, pERE-vitallogenin-Luc plasmid (pERE-vitallogenin-Luc plasmid) was transfected. After culturing for 24 hours, baechohyang extract dissolved in DMSO and genistein isolated from soybean as a control group were treated at a concentration of 10 μM. At this time, only DMSO was treated as a control. After 24 hours of treatment, the cells were lysed and then the activity of luciferase was measured using a dual-Luciferase assay system, and the activity of the control group was compared.

As a result, as can be seen in Figure 1, when each concentration of baechohyang extract (AR) was treated, it was confirmed that the luciferase activity was significantly increased compared to the control group.

Example 3. Increase in activity of alkaline phosphatase (ALP), which is a marker for early differentiation of osteoblasts in baechohyang extracts and fractions

Whether the baechohyang extract and fractions prepared in Example 1 increase ALP activity was confirmed as follows.

After washing MC3T3-E1 osteoblasts with PBS (phosphate buffered saline) solution, the cell membrane was dissolved using 0.2% Triton X-100 and centrifuged at 2,500×g, 4°C for 15 minutes to collect the supernatant, SensoLyte p -nitrophenyl phosphate (p-NPP) ALP activity was measured using an ALP activity assay kit (AnaSpec, Inc., Fremont, CA, USA). The positive control group was treated with 10 μM of genistein. ALP activity was expressed by comparing differentiated cells to 100%.

As a result, as can be seen in FIG. 2 , ALP activity was increased in the group treated with baechohyang extract (AR EX), and a significant difference was observed when compared with the control group. In addition, a significant difference from the control group was shown in the group treated with the methylene chloride fraction (AR MC), and the ethyl acetate fraction (AR EA) among the fractions of baechohyang.

Through this, it was confirmed that the baechohyang extract and fractions have excellent ability to promote the differentiation of osteoblasts by increasing ALP activity, which is an early differentiation marker of osteoblasts, and it was confirmed that there is an efficacy for treating osteoporosis, which is one of the representative diseases of menopause syndrome.

Example 4. Increase in expression of osteoblast differentiation marker genes of baechohyang extract and fractions

Whether the baechohyang extract and fractions prepared in Example 1 increase the expression of genes involved in osteoblast differentiation was confirmed as follows.

More specifically, after culturing MC3T3-E1 osteoblasts (American Type Culture Collection (ATCC), Manassas, VA, USA) in α-MEM (α-modified Eagle's Medium) medium containing 10% fetal bovine serum, 100,000 cells/well were placed in a 6-well microtiter plate. After the cells were grown to 90%, 50 μg/ml L-ascorbic acid (Sigma, St. Louis, MO, USA) and 10 mM β-glycerol phosphate disodium salt hydrate (Sigma) were added to the culture medium prepared in Example 1 above. The baechohyang extract and fractions were treated with osteoblasts to induce differentiation of osteoblasts for 3 days. The group not treated with baechohyang extract and fractions was used as a control group, and western blot was performed to determine the expression levels of TGFβ, BMP2/4, RUNX2, OSX, OPG, and Col1A1, the major proteins involved in osteoblast differentiation and bone formation. did Total protein was harvested and quantified from the differentiated cells using RIPA reagent (Sigma-Aldrich, St. Louis, MO, USA), and then western blot analysis was performed as follows. First, after electrophoresis on an SDS-PAGE gel, an antibody attached to each specific protein was reacted with a polyvinylidene difluoride membrane to measure protein expression. To compare the total amount of protein, it was confirmed that the same amount of protein was taken by using the amount of β-actin.

As a result, as can be seen in Figure 3, in the group treated with baechohyang extract (AR20), bone-generating protein (Collagen, type I alpha 1 (Col1A1)), osteoclast-inhibiting protein (osteoprotegerin (OPG)), osteoblast differentiation Inducing protein (Runt-related transcription factor 2 (Runx2)) was increased. In addition, in the group treated with the ethyl acetate fraction (EA40) of baechohyang, bone-generating protein (Collagen, type I alpha 1(Col1A1)), osteoclast-inhibiting protein (osteoprotegerin(OPG)), osteoblast differentiation-inducing protein (Runt- related transcription factor 2 (Runx2), Osterix (Osx))) increased, and expression of signaling proteins (Transforming growth factor beta (TGFβ), Bone morphogenetic protein 2/4 (BMP2/4)) for inducing osteoblast differentiation It was also confirmed that the amount increased.

Through this, it was confirmed that the baechohyang extract and fractions in MC3T3-E1 osteoblasts promote bone formation and have the effect of inhibiting the generation of osteoclasts.

Example 5. Increase of bone density in ovariectomized menopausal animal model of baechohyang extract

Whether the baechohyang extract prepared in Example 1 increases bone density in an animal model of menopausal osteoporosis through ovarian resection was confirmed as follows.

More specifically, 7-week-old C57BL/6J female mice were purchased, acclimatized for 1 week, and bilateral ovariectomy was performed through anesthesia. In addition, the negative control group (Sham) was sutured without removing the ovaries after resection of the abdominal cavity, and after a recovery period of 2 weeks, 50 mg/kg of baechohyang extract was orally ingested for 10 weeks, and the positive control group (E+P) was 0.1 By ingestion of mg/kg β-estradiol and 1 mg/kg progesterone, changes in bone density through hormone regulation were promoted. After 12 weeks, the changes in bone density were confirmed by measuring the thigh bones of mice in each group through Micro CT.

As a result, as can be seen in FIG. 4 , in the case of the ovarian resection group (OVX), the bone density was significantly decreased, and in the case of the positive control group, it was confirmed that the bone density increased compared to the ovariectomy group. On the other hand, it was confirmed that the baechohyang extract treatment group (AR 50 mg/kg) significantly increased bone density compared to the ovarian resection group or the positive control group.

Through this, it was confirmed that the baechohyang extract promotes the differentiation of osteoblasts, suppresses the decrease in bone density, and has the efficacy of treating osteoporosis, which is one of the representative diseases of the menopause syndrome.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Claims (10)

A pharmaceutical composition for the prevention or treatment of female menopause syndrome, comprising a baechohyang extract or a fraction thereof as an active ingredient.
According to claim 1, wherein the female menopause syndrome is selected from the group of diseases including hot flashes, sleep disorders, depression, palpitations, sweating, menopausal syndrome including bone and muscle pain and osteoporosis, the pharmaceutical composition.
The pharmaceutical composition of claim 1, wherein the baechohyang extract is extracted with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid.
The pharmaceutical composition of claim 1, wherein the baechohyang extract is an ethanol extract.
The pharmaceutical composition of claim 1, wherein the baechohyang fraction is a methylene chloride fraction or an ethylene acetate fraction.
According to claim 1, wherein the composition, based on the total weight of the composition, 0.001 to 80% by weight of the baechohyang extract comprising the extract, the pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the composition has estrogenic activity.
The pharmaceutical composition of claim 1, wherein the composition promotes the differentiation of osteoblasts, inhibition of osteoclast generation, or activity of osteogenic proteins.
A food composition for the prevention or improvement of female menopause syndrome, comprising a baechohyang extract or fraction as an active ingredient.
A method for preventing or treating female menopause syndrome, comprising administering a baechohyang extract or fraction to individuals other than humans.

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