KR20220029479A - Composition for preventing or treating female menopausal symptoms comprising vitex rotundifolia extract - Google Patents

Abstract

The present invention relates to a composition for alleviating female menopausal symptoms containing a Vitex rotundifolia extract. The composition according to an aspect contains vitexicarpin or vitaxin contained in the Vitex rotundifolia extract as an active component, wherein the vitexicarpin or vitaxin can exhibit estrogen-like activity at a low concentration. Therefore, the composition according to an aspect can effectively alleviate menopausal symptoms such as weight gain and uterine atrophy without side effects.

Description

A pharmaceutical composition for preventing or treating female menopausal symptoms comprising an extract of Sunbigi tree {Composition for preventing or treating female menopausal symptoms comprising vitex rotundifolia extract}

The present invention relates to a composition for improving women's menopausal symptoms, comprising an extract of Sunbigi tree.

Menopause refers to the period of transition to menopause due to natural reasons such as aging, menopause, and post-menopause, and the various symptoms and signs experienced during this period are called menopause syndrome. According to statistics from the Health Insurance Review and Assessment Service, the number of patients receiving treatment for menopause and other pre-menopausal disorders has steadily increased from 671,000 in 2015 to 690,000 in 2018, approaching 700,000.

On the other hand, the main cause of such menopausal disorders is a decrease in estrogen secretion, and estrogen replacement therapy has been mainly used as a treatment. However, since estrogen replacement therapy is an artificial administration of hormones into the body, there is a risk of side effects such as uterine bleeding, breast cancer, and uterine cancer. Therefore, there is a need to develop a new treatment for menopause that is effective in alleviating menopausal symptoms while having no side effects using safer natural products.

Republic of Korea Patent Registration No. 10-2284907

One aspect is to provide a pharmaceutical composition for preventing or treating female menopausal symptoms comprising an extract of S.

Another aspect is to provide a food composition for preventing or improving female menopausal symptoms comprising an extract of Sunbigi tree.

Other objects and advantages of the present application will become more apparent from the following detailed description in conjunction with the appended claims and drawings. Content not described in this specification will be omitted because it can be sufficiently recognized and inferred by those skilled in the technical field or similar technical field of the present application.

Each description and embodiment disclosed in this application is also applicable to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

One aspect provides a pharmaceutical composition for preventing or treating female menopausal symptoms comprising an extract of S.

The term "Sunbigi extract" refers to an extract obtained by extracting S. serrata tree (scientific name vitex rotundifolia , Chaste tree, or Beach vitex), which is the leaf, fruit, stem, root and outpost (plant of) may be one or more extracts selected from the group consisting of, preferably, extracts of leaves, fruits or stems, and most preferably may be extracts of fruits, but is not limited thereto.

The extract may include extracts of S. serrata biloba with water, lower alcohols having 1 to 4 carbon atoms, polyhydric alcohols, hydrocarbon-based solvents, or mixed solvents thereof, and preferably those extracted with water, methanol, or ethanol. there is. In addition, the extract may contain any one or more of an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or a crude product or a purified product. In addition, the extract may refer to one or more single components separated from the extract.

According to one embodiment, the S. serrata extract contains about 0.01 to 100, 0.1 to 100, 1 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, , 60 to 100, 70 to 100, 80 to 100, 90 to 100, or 100% by weight of methanol may be obtained by extraction. In addition, according to one embodiment, the extract of S. S. serrata is about 0.01 to 100, 0.01 to 90, 0.01 to 80, 0.01 to 70, 0.01 to 69, 0.01 to 60, 0.01 to 50, 0.1 to 69, 0.1 to 60, 0.1 to 50, 1 to 69, 1 to 60, 1 to 50, 10 to 69, 10 to 60, 10 to 50, 20 to 69, 20 to 60, 20 to 50, 30 to 69 , 30 to 60, or 30 to 50% by weight of ethanol may be obtained by extraction. In addition, according to one embodiment, the extract of the Sunbigi tree may be obtained by hot water extraction of the Sunbigi tree with water (or purified water).

As an extraction method for obtaining the S. serrata extract, conventional extraction methods, for example, hot water extraction, cold extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution method, compression method, etc. can be used. And, preferably, it can be extracted through hot water extraction method or solvent extraction method. In addition, the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method.

The pharmaceutical composition for preventing or treating female menopausal symptoms may be to include the extract of Sunbigi extract at a concentration of about 0.01 μg/mL or more, specifically, about 5 μg/mL or more. More specifically, the pharmaceutical composition for preventing or treating female menopausal symptoms includes about 0.01 to 1000, 0.01 to 500, 0.01 to 200, 0.01 to 100, 0.01 to 99, 0.01 to 90, 0.1 to 1000, 0.1 to 500, 0.1 to 200, 0.1 to 100, 0.1 to 99, 0.1 to 90, 0.1 to 80, 1 to 1000, 1 to 500, 1 to 200, 1 to 100, 5 to 1000, 5 to 500, 5 to 200, 5 to 100, 5 to 70, 5 to 60, 5 to 50, 10 to 70, 10 to 60, 10 to 50, 20 to 70, 20 to 60, 20 to 55, 20 to 50, or 25 to 50 μg/mL may be included.

According to one embodiment, in the case where the extract of S. serratus oleifera is a methanol extract obtained by extracting the S. serratus oleifera with methanol, the pharmaceutical composition for preventing or treating female menopausal symptoms is about 0.01 μg of the M. It may be included in a concentration of /mL or more or about 5 μg/mL or more (specifically, about 5 to 100 μg/mL). When the composition includes the methanol extract of S. serrata at a concentration outside the numerical range, the estrogen-like activity of the composition may decrease.

According to one embodiment, when the S. serrata oleifera extract is an ethanol extract obtained by extracting the S. serrata oleifera with about 0.01 to 69% by weight (specifically, about 30 to 50% by weight) of ethanol, the female menopause The pharmaceutical composition for the prevention or treatment of symptoms may include the ethanol extract of Sunbigi tree at a concentration of about 0.01 to 99 μg/mL (specifically, about 25 to 50 μg/mL). When the composition contains the ethanol extract of S. oleifera at a concentration outside the numerical range, the estrogen-like activity of the composition may decrease.

According to one embodiment, when the S. S. serrata extract is a hot water extract obtained by extracting the S. S. serrata with water (or purified water), the pharmaceutical composition for preventing or treating female menopausal symptoms is may be included in a concentration of about 0.01 μg/mL or more (specifically, about 25 μg/mL or more).

The active ingredient of the S. serrata extract according to an aspect may be vitexicarpin or vitexin, and the vitexicarpin or vitexin is HPLC (High Performance Liquid Chromatography) and can be isolated and obtained.

Accordingly, the extract of S. serrata contained in the pharmaceutical composition for preventing or treating female menopausal symptoms may include bitexicarpine, bitexin, or a combination thereof. Alternatively, the S. serrata extract included in the pharmaceutical composition for preventing or treating female menopausal symptoms may be bitexicarpine, bitexin, or a combination thereof.

The Vitexicarpin may be a compound having a structure of the following Chemical Formula 1:

[Formula 1]

.

.

The molecular formula of bitexicarpine is C ₁₉ H ₁₈ O ₈ , and the molecular weight may be about 370 to 380 g/mol.

The Vitexin may be a compound having a structure of the following Chemical Formula 2:

[Formula 2]

.

.

The molecular formula of the bitexin is C ₂₁ H ₂₀ O ₁₀ , and the molecular weight may be about 430 to 440 g/mol.

According to one embodiment, the pharmaceutical composition for preventing or treating female menopausal symptoms may include the bitexicarpine at a concentration of about 0.01 to 10 μM. Specifically, the pharmaceutical composition for preventing or treating female menopausal symptoms may include the bitexicarpine in a concentration of about 0.01 to 1, 0.01 to 0.7, 0.1 to 0.7, or 0.2 to 0.4 μM. According to one embodiment, when the pharmaceutical composition for preventing or treating female menopausal symptoms includes the vitaxicarpine at a concentration outside the numerical range, the estrogen-like activity of the composition may decrease.

According to one embodiment, the pharmaceutical composition for preventing or treating female menopausal symptoms may include the vitexin at a concentration of about 1 to 1000 μM. Specifically, the pharmaceutical composition for preventing or treating female menopausal symptoms includes the vitexin at a concentration of about 1-500, 10-500, 10-150, 10-100, 10-70, 30-70, or 40-60 μM. may include. According to one embodiment, when the pharmaceutical composition for preventing or treating female menopausal symptoms includes the vitaxin at a concentration outside the numerical range, the estrogen-like activity of the composition may decrease.

According to one embodiment, the pharmaceutical composition for the prevention or treatment of female menopausal symptoms comprises the extract of S. serrata, including the vitaxicarpine or vitexin; Alternatively, by including the vitaxicarpine or vitexin isolated from the serrata serrata extract as an active ingredient, it is possible to effectively alleviate menopausal symptoms such as weight gain and uterine atrophy without side effects. In particular, according to one embodiment, the vitaxicarpine or vitaxin contained in the pharmaceutical composition for preventing or treating female menopausal symptoms may exhibit remarkably excellent estrogen-like activity at a low concentration as described above, and as a natural product-derived component. It may not cause side effects. Therefore, according to one embodiment, the pharmaceutical composition for preventing or treating female menopausal symptoms may exhibit superior estrogen-like activity and superior safety compared to existing menopausal therapeutic agents, and thereby, is useful for preventing or improving menopausal symptoms can be used

The term "climacteric", also referred to as menopause, refers to the period of cessation of menstruation as a sign of loss of female reproductive function. At menopause, the amount or cycle of menstruation becomes irregular due to ovarian dysfunction, and menstruation is abolished due to a decrease in estrogen secretion over several months to three years. cause of

The climacteric symptoms include hot flashes, sweating, dry skin, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, dysuria, urgency, frequent urination, urinary incontinence, difficulty concentrating, short-term memory disorder, anxiety, depression, insomnia, nerves Hypersensitivity, memory loss, muscle pain, arthralgia, uterine atrophy, weight gain, and may be at least one selected from the group consisting of osteoporosis, but is not limited thereto. Preferably, the female menopausal symptoms include uterine atrophy, weight gain , or a combination thereof.

The term "comprising as an active ingredient" means containing an effective amount to the extent that it can exhibit the effect of preventing, treating, and improving female menopausal symptoms.

The pharmaceutical composition may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition. Preferably, the composition comprises tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, transdermal absorption agents, gels, lotions, It may have any one formulation selected from the group consisting of ointments, creams, patches, cataplasms, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages and suppositories, and may be administered orally or It may be in various parenteral formulations, but is not limited thereto.

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 one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may 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 may be administered to mammals such as rats, mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The preferred dosage of the pharmaceutical composition varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for more effective prophylaxis or treatment, it is preferable to administer the extract of S. serrata at about 0.0001 to 200 mg/kg per day, preferably at about 0.001 to 100 mg/kg, and administration is administered once a day. It can be administered or divided into several doses.

Another aspect provides a food composition for preventing or improving female menopausal symptoms comprising an extract of S.

The extract of Sunbigi tree may include bitexicarpine, bitexin, or a combination thereof. Alternatively, the extract of Sunbigi tree may be bitexicarpine, bitexin, or a combination thereof.

The symptoms of female menopause are hot flashes, sweating, dry skin, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, dysuria, urgency, frequent urination, incontinence, difficulty concentrating, short-term memory disorder, anxiety, depression, insomnia, nervousness, memory loss. , myalgia, arthralgia, uterine atrophy, weight gain, and may be at least one selected from the group consisting of osteoporosis, but is not limited thereto, and preferably, the female menopausal symptoms include uterine atrophy, weight gain, or a combination thereof. can be

The term “food composition” may include all foods in a conventional sense, and may be used interchangeably with terms known in the art, such as functional foods and health functional foods.

In the case of using the Sunbigi extract according to an aspect as a food additive, the Sunbigi extract may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment), and may further include a food pharmaceutically acceptable food supplement additive. Since the composition according to an aspect uses an extract derived from a natural product as an active ingredient, there is no big problem in terms of safety and stability, so there is no great limitation on the amount of mixing.

The term "functional food" refers to food manufactured and processed using raw materials or ingredients useful for the human body according to Health Functional Food Act No. 6727, and "functionality" refers to the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological effects.

The term "health functional food" refers to a food manufactured and processed by methods such as extraction, concentration, purification, mixing, etc. of specific ingredients as raw materials or in food raw materials for the purpose of health supplementation.

There is no limitation on the type of food that can be used extract according to one aspect. In addition, the food composition comprising the extract of S. serrata according to one aspect as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in food according to the selection of those skilled in the art. Examples of food that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There is a vitamin complex, and it can be prepared by adding the extract according to one aspect as a main component to juice, tea, jelly and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (eg, formula milk, infant food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplements , seasoned foods (eg soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.) ), beverages (eg, fruit, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the composition according to an aspect is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, natural carbohydrates, and the like as additional ingredients like a conventional beverage. In addition, the composition according to an aspect includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage.

Among the terms or elements mentioned in the food composition, those mentioned in the description of the pharmaceutical composition are understood to be the same as those mentioned in the description of the pharmaceutical composition.

The composition according to one aspect may include an extract of Sunbigi tree comprising bitexicarpine or bitexin; Alternatively, by including bitexicarpine or bitexin isolated from the spruce tree extract as an active ingredient, it is possible to effectively alleviate menopausal symptoms such as weight gain and uterine atrophy without side effects. In particular, the vitaxicarpine or vitaxin included in the composition according to an aspect may exhibit excellent estrogen-like activity at a low concentration, and as a natural product-derived ingredient, the possibility of causing side effects is very low. For this reason, the composition according to an aspect exhibits superior estrogen-like activity and superior safety compared to conventional menopausal drugs, and thus can be usefully used to prevent or improve menopausal symptoms.

1 is a diagram confirming the estrogen-like activity of an extract of Sunbigi extract extracted using 100% methanol.
Figure 2 is a diagram confirming the estrogen-like activity of the extract of Sunbigi extract extracted using 100% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, or hot water.
Figure 3 is a diagram showing the results of HPLC and MS analysis of the extract of Sunbigi tree extracted using 30% ethanol.
Figure 4 is artemetin (Artemetin, 1), vitexicarpin (Vitexicarpin, 2), hesperidin (Hesperidin, 3), luteolin (Luteolin, 4), vitexin (Vitexin, 5) isolated from the extract , and a diagram showing the structural formula of vanillic acid (6).
Figure 5 is artemetin (Artemetin, 1), vitexicarpin (Vitexicarpin, 2), hesperidin (Hesperidin, 3), luteolin (Luteolin, 4), vitexin (Vitexin, 5) isolated from the extract , and a diagram confirming the estrogen-like activity of vanillic acid (6).
FIG. 6 is a diagram showing the weight control effect of an oleifera extract (VFE) on an ovariectomized menopausal animal model (OVX) (A: a graph measuring the change in body weight; B: a graph measuring a total cholesterol level in plasma) .
FIG. 7 is a diagram showing the uterine atrophy control effect of VFE extract on an ovariectomized menopausal animal model (OVX) (A: an excised uterine image (top) and a cross-sectional microscopy image of the uterus (bottom); B: Graph of uterine weight measurement; C: Electrophoretic image (top) and protein quantification graph (bottom) measuring ER-α and ER-β protein expression levels; D: Plasma estradiol concentration measured graph).
8 is a diagram showing the results of confirming whether hepatotoxicity and nephrotoxicity are induced when VFE) is administered to an ovariectomized animal model (OVX) of menopause (A: plasma alanine aminotransferase , a graph measuring the concentration of ALT); B: a graph measuring the concentration of creatine in plasma).
Figure 9 is a diagram confirming the breast cancer cell proliferation inhibitory activity of 100% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, or extracts of Sunbigi tree extract extracted using hot water.

Hereinafter, an aspect will be described in more detail through Preparation Examples and Examples. However, these Preparations and Examples are for illustrative purposes of an aspect, and the scope of an aspect is not limited to these Preparations and Examples, and the Preparations and Examples of an aspect are average knowledge in the art. It is provided to more completely explain an aspect to those who have.

Example 1: Preparation of extract of S. serrata

After purchasing and drying Korean pure bigi fruit (Manhyeongja) at Gyeongdong Market in Jegi-dong, Seoul, the dried sample (30 g) was mixed with 1L of methanol (100%) or ethanol (100%, 70%, 50%, or 30%) After mixing, extraction was repeated three times at 50° C. for 1 hour. Thereafter, the solvent was evaporated in a vacuum at 40° C. to obtain an extract. In addition, after mixing a sample (30 g) of dried Sunbigi fruit with 1 L of water, hot water extraction was performed while stirring to obtain an extract.

Example 2: Evaluation of estrogen-like activity of Sunbigi extract

2-1. Analysis of Estrogen-Like Activity of Methanol Extracts

MCF-7 cell proliferation experiment (E-SCREEN Assay) was performed using the methanol (100%) extract of S. serrata prepared in Example 1. In order to confirm the role of the sample as an estrogen receptor activating ligand, this experiment was conducted under conditions (a medium containing charcoal stripped Fetal Bovine Serum) in which the influence of other hormones was suppressed. Since the MCF-7 cell line abundantly expresses the estrogen receptor, it is used as a method to evaluate the estrogenicity of a sample. To confirm the proliferation of MCF-7 cells by the extract, MCF-7 cells (1 x 10 ⁴ cells/well) were first seeded in a 48-well plate and cultured for 24 hours. Samples were treated at concentrations of 0, 5, 10, 25, 50, and 100 μg/mL, respectively, and phenol red-free DMEM supplemented with 10% charcoal-dextran stripped serum (phenol) red-free DMEM) was dissolved in each well and cultured for 144 hours. As a control, 100 nM of an estrogen receptor antagonist (ICI 182,780) was treated with the sample. Then, Ez-Cytox (DoGEN) solution was added to each well and incubated for 1 hour again. Cell viability was calculated by measuring an optical density (OD) value at 450 nm using a SPARK 10M microplate reader, and the results are shown in FIG. 1 .

1 is a diagram confirming the estrogen-like activity of an extract of Sunbigi extract extracted using 100% methanol.

As shown in FIG. 1 , when MCF-7 cells were treated with extract, MCF-7 cell proliferation was significantly increased compared to the control group (p<0.05), and MCF-7 cells extract and estrogen receptor antagonist (ICI 182,780) ) was confirmed that the MCF-7 cell proliferation of the extract was completely blocked when treated together. Through this, it can be seen that the extract of S. serrata exhibits estrogen-like activity mediated by estrogen receptors.

Through this example, it was confirmed that the extract of S. serratus oleifera exhibits estrogen-like activity mediated by estrogen receptors. In addition, it was confirmed that the methanol (100%) extract of Prunus serrata exhibited excellent estrogen-like activity at a concentration of 0.01 μg/mL or more, specifically, 5 μg/mL or more, or 5 to 100 μg/mL.

2-2. Estrogen-like activity analysis according to the concentration of the extraction solvent

The estrogen-like activity of the ethanol (100%, 70%, 50%, or 30%) extract and the hot water extract prepared in Example 1 were comparatively analyzed. As a positive control group, Queens One Tab (dried red clover extract), which is used as a treatment for improving menopausal symptoms, was used. The specific estrogen-like activity analysis method is the same as the method described in Example 2-1, except for the treatment concentration of the Sunbigi extract, and the results are shown in FIG. 2 . In this example, the treatment concentration of the extract of Sunbigi tree is 0, 25, 50, 100 μg/mL.

Figure 2 is a diagram confirming the estrogen-like activity of the extract of Sunbigi extract extracted using 100% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, or hot water.

As shown in FIG. 2 , it was confirmed that estrogen-like activity was shown in the extracts prepared using 70% ethanol, 50% ethanol, 30% ethanol, or hot water among the extracts of Sunbigi tree. Specifically, when 100% ethanol extract was treated, MCF-7 cell proliferation was decreased at all concentrations, and when 70% ethanol extract was treated to a concentration of 25 μg/mL, MCF-7 cell proliferation slightly increased, whereas 50 μg It was confirmed that MCF-7 cell proliferation was reduced when treated with a concentration of /mL or more. In addition, MCF-7 cell proliferation was significantly increased when 50% ethanol extract and 30% ethanol extract were treated to a concentration of 50 μg/mL, whereas MCF-7 cell proliferation was decreased when treated at a concentration of 100 μg/mL or more. was confirmed. In addition, it was confirmed that the hot water extract significantly increased the proliferation of MCF-7 cells at all concentrations of 25, 50, and 100 μg/mL. In addition, as described above, it was confirmed that the MCF-7 cell proliferation-increasing effect of the serranosis extract was completely blocked when treated with an estrogen receptor antagonist (ICI 182,780), so that the estrogen receptor-mediated estrogen-like effect It was confirmed that the activity was shown.

In particular, the estrogen-like activity exhibited when the ethanol (50% or 30%) extract of Sunbeam tree was treated to a concentration of 50 μg/mL was the same as the estrogen-like activity exhibited by Queen's Expedition, which is used as an existing treatment for improving menopausal symptoms. , and it was confirmed that the hot water extract of Sunbigi tree exhibited the same level of estrogen-like activity as that of Queen's Expedition at all concentrations of 25, 50, and 100 μg/mL.

Through this example, the ethanol (0.01 or more to less than 70%, specifically, 30 to 50%) extract of serrata oleifera is 0.01 μg/mL or more to less than 100 μg/mL, specifically, 25 to 50 μg/mL It was confirmed that at a concentration of mL, excellent estrogen-like activity was exhibited at the same level as that of the existing treatment. In addition, it was confirmed that the hot water extract of Sunbigi tree exhibited excellent estrogen-like activity at a concentration of 0.01 μg/mL or more, specifically, 25 μg/mL or more, at a level similar to that of the existing treatment.

Example 3: Analysis of the active ingredient of the extract of Sunbigi tree

3-1. Component analysis of S. serrata extract

HPLC analysis and Mass Spectrometry (MS) analysis were performed on the ethanol (30%) extract of Prunus oleifera prepared in Example 1, and the content of the compound contained in the extract of P. oleracea was measured. The standard samples used in this analysis, Artemetin (1), Vitexicarpin (2), Hesperidin (3), Luteolin (Luteolin, 4), Vitexin (5), And vanillic acid (Vanillic acid, 6) was obtained by separation from the extract of Sunbigi tree in advance, and was provided by Seoul National University. The standard sample was prepared at a concentration of 1 mg/mL by dissolving it in methanol, and the ethanol (30%) extract prepared in Example 1 was dissolved in methanol to prepare a concentration of 10 mg/mL. All samples were filtered with a 0.45 μm RC-membrane syringe filter (Sartorius, Germany), and then HPLC analysis and MS analysis were performed. HPLC analysis conditions were as follows:

Analytical instruments: Agilent 1260 infinity HPLC system, G1311C quaternary pump, G1329B auto sampler, G1316A column oven, G1315D photodiode array (PDA) detector, Waters micromass ZQ

Analytical column: ZORBAX Eclipse XDB-C18 (150 × 4.6 mm, 3.5 μm, Agilent Technologies)

Analysis temperature: 30℃

Detection wavelength: 280 nm and 254 nm

Solvent A: formic acid in water at a concentration of 0.2%

Solvent B: formic acid in acetonitrile at a concentration of 0.2%

Flow rate: 0.8 mL/min

Gradient conditions: as shown in Table 1 below.

time (min) Solvent A (%) Solvent B (%) 0 92 8 20 92 8 25 75 25 28 73 27 35 63 37 40 60 40 52 60 40 60 40 60 65 0 100

Comparing the HPLC analysis results (retention time and UV spectrum results) and MS analysis results of the ethanol (30%) extract prepared in Example 1 with the HPLC and MS analysis results of the standard sample, the Example 1 It was confirmed whether the standard sample was detected in the ethanol (30%) extract of Sunbigi tree.

Figure 3 is a diagram showing the results of HPLC and MS analysis of the extract of Sunbigi tree extracted using 30% ethanol.

As shown in FIG. 3 , it was confirmed that bitexicarpine (47.074 min) and vanillinic acid (15.869 min) were detected in the standard sample in the ethanol (30%) extract of S. oleracea prepared in Example 1. Other standard samples were contained in a very small amount in the ethanol (30%) extract of Sunbigi tree prepared in Example 1, and it was estimated that it was not confirmed in the state of the extract.

Next, in order to analyze the contents of vitaxicarpine and vanillinic acid contained in the ethanol (30%) extract of S. oleraceae prepared in Example 1, a calibration curve of the standard sample was obtained and analyzed, and in order to derive a calibration curve , 7 concentrations were selected in the concentration range of 0.0078-0.5 mg/mL of vitaxicarpine and vanillinic acid, which are the standard samples, and peak area values for each concentration obtained by HPLC and MS analysis, respectively, were measured. All experiments were repeated three times to obtain data. The derived calibration curves are shown in Table 2 below, and the correlation coefficient of each calibration curve was 0.99 or more. By substituting the values of each peak corresponding to vitaxicarpine and vanillinic acid detected in HPLC and MS analysis results of the ethanol (30%) extract of S. oleraceae prepared in Example 1 into the calibration curve of Table 2 below, the content was determined. analyzed.

As shown in Table 2, the contents of bitexicarpine and vanillinic acid contained in the ethanol (30%) extract of S. oleracea prepared in Example 1 were 1.981 ± 0.019 mg/g and 1.049 ± 0.004 mg/g, respectively. As a result, it was confirmed that the ethanol (30%) extract prepared in Example 1 contained bitexicarpine and vanillinic acid in significantly higher amounts than artemethin, hesperidin, luteolin, and bitexin. .

sample Calibration Curve r ² Content (mg/g) Vitexicarpin y = 4,670,666.3799 x + 28.1243 0.9999 1.981 ± 0.019 Vanillic acid y = 6,577,450.3226 x + 99.4849 0.9994 1.049 ± 0.004

3-2. Analysis of active ingredients of S. serrata extract

Artemetin (1), vitexicarpin (2), hesperidin (3), luteolin (Luteolin, 4), vitexin (5), and vanillin isolated from the extract The estrogen-like activity of each acid (Vanillic acid, 6) was comparatively analyzed. The samples used in this analysis, Artemetin (1), Vitexicarpin (2), Hesperidin (3), Luteolin (Luteolin, 4), Vitexin (5), and Vanillic acid (6) was previously obtained by separating from an extract of Sunbigi tree, and was provided by Seoul National University. Each of the samples was identified as a single component through HPLC analysis, MS analysis, and NMR (nuclear magnetic resonance) analysis, and the structural formulas of components 1-6 are shown in FIG. 4 .

Figure 4 is artemetin (Artemetin, 1), vitexicarpin (Vitexicarpin, 2), hesperidin (Hesperidin, 3), luteolin (Luteolin, 4), vitexin (Vitexin, 5) isolated from the extract , and a diagram showing the structural formula of vanillic acid (6).

The specific estrogen-like activity analysis method is the same as the method described in Example 2-1 except for the treatment concentration of each sample, and the results are shown in FIG. 5 . The treatment concentrations of each of the samples were as follows:

Artemetin (1): 0, 5, 10, 20 μM

Vitexicarpin (2): 0, 0.16, 0.31, 0.63 μM

Hesperidin (3): 0, 5, 10, 20 μM

Luteolin (4): 0, 5, 10, 20 μM

Vitexin (5): 0, 25, 50, 100 μM

Vanillic acid (6): 0, 25, 50, 100 μM.

Figure 5 is artemetin (Artemetin, 1), vitexicarpin (Vitexicarpin, 2), hesperidin (Hesperidin, 3), luteolin (Luteolin, 4), vitexin (Vitexin, 5) isolated from the extract , and a diagram confirming the estrogen-like activity of vanillic acid (6).

As shown in Figure 5, artemetin (Artemetin, 1), vitexicarpin (Vitexicarpin, 2), hesperidin (Hesperidin, 3), luteolin (Luteolin, 4), vitexin ( Among Vitexin, 5), and Vanillic acid, it was confirmed that only vitexicarpine and vitexin exhibited estrogen-like activity. Specifically, when bitexicarpine was treated, MCF-7 cell proliferation was increased at all concentrations of 0.16, 0.31, and 0.63 μM. It was confirmed that the In addition, when bitexin was treated, MCF-7 cell proliferation was increased at all concentrations of 25, 50, and 100 μM, and in particular, it was confirmed that treatment with 50 μM showed the most excellent effect of increasing MCF-7 cell proliferation. . In addition, as described above, the effect of increasing the proliferation of MCF-7 cells exhibited by bitexicarpine or bitexin was completely blocked when treated with an estrogen receptor antagonist (ICI 182,780). It was confirmed that the estrogen-like activity was mediated.

Through this example, it was confirmed that the active ingredients of the extract of Sunbigi tree were vitexicarpine and vitexin. Specifically, it was confirmed that bitexicarpine exhibited excellent estrogen-like activity at a very small and low concentration of 0.1 to 0.7 μM, specifically, 0.2 to 0.4 μM. In addition, it was confirmed that vitexin also exhibits excellent estrogen-like activity at a small concentration of 10 to 150 μM, specifically, 30 to 70 or 40 to 60 μM. In particular, as described above, vitexicarpine exhibited excellent estrogen-like activity at a lower concentration compared to vitaxin, and it was confirmed that the most important active ingredient of the S.

Example 4: Confirmation of the efficacy of improving menopausal symptoms through ovarian resection menopause animal experiment

4-1. Confirmation of weight gain control effect of S. serrata extract

It was confirmed whether or not the extract of S. serrata extract can control or improve weight gain, which is known as one of the symptoms of menopause.

Specifically, all animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Gachon University (GIACUCR2017034, approved on February 23, 2018). Animal experiments were conducted on 36 female Sprague-Dawley rats 5 weeks of age and weighing 130 to 150 g. The rats were maintained in a room at 24±2° C. with a relative humidity of 50-55%, and food and water were provided ad libitum. After one week, all rats were anesthetized by intraperitoneal injection of a mixture of Zoletil ^® (Virbac, Carros, France) and Rompun ^® (TS, Bayer, Leverkusen, Germany) (3:1, 0.2 mg/kg body weight). A sham operation (sham) was performed on 6 mice and bilateral ovariectomy (OVX) was performed on the remaining 30 mice. After surgery, the rats were given a solid diet during a recovery period of 1 week. Finally, the ovariectomized rats were divided into three groups (n = 10):

(1) OVX, (2) OVX + FeQ, (3) OVX + VFE.

The OVX group was an ovariectomized rat, a group to which feramine Q (FeQ), which is a conventional menopausal treatment, and an oleifera extract were not administered at all, and the OVX + FeQ group was an ovariectomized rat, FeQ 100 mg/kg ( rat body weight) was orally administered once daily for 8 weeks, and the OVX + VFE group was ovariectomized rats, and 50 mg/kg (rat body weight) ) refers to the group orally administered once daily for 8 weeks. The FeQ and Sunbigi extract were suspended in 0.5% aqueous methylcellulose solution and administered to animals in a suspension state. For reference, the animals in the sham and OVX groups were administered with an equal volume of 0.5% aqueous methyl cellulose aqueous solution (without FeQ and S. oleifera extract) once daily for 8 weeks. To investigate the effect of S. oleracea extract on the menopause model, the body weight of the animals was evaluated once a week. In addition, at the end of the experiment period, the animals used in the experiment were fasted for 24 hours, and then blood and uterus were collected. The collected blood was centrifuged at 3500 rpm for 15 minutes at 4°C to separate plasma, and the separated plasma was stored at -20°C. Total cholesterol levels were measured from the separated plasma using GENIA (Seong-Nam, Korea).

FIG. 6 is a diagram showing the weight control effect of an oleifera extract (VFE) on an ovariectomized menopausal animal model (OVX) (A: a graph measuring the change in body weight; B: a graph measuring a total cholesterol level in plasma) .

As shown in FIG. 6 , the body weight of the animals was further increased in the ovariectomized group compared to the normal group, and it was confirmed that the weight reduction effect was observed when FeQ or S. In particular, compared to FeQ, it was confirmed that the weight loss effect of the S. In addition, the total cholesterol level in the blood of animals was further increased in the ovariectomized group compared to the normal group, and it was confirmed that the effect of reducing the total cholesterol level was shown when FeQ or S. In particular, in view of the fact that the total cholesterol level reduction effect of the S. S. chinensis extract administration group was similar to the effect of the FeQ administration group, even though the administered dose of the S. oleracea extract was smaller than the administered dose of FeQ, the total cholesterol of the P. It was confirmed that the numerical reduction effect was more excellent.

Through this example, in weight gain, which is known as one of the symptoms of menopause, the S. serrata extract can effectively control or improve weight gain due to menopause by exhibiting more excellent weight loss and cholesterol reduction effects compared to the existing menopausal treatment. confirmed that there is.

4-2. Confirmation of uterine atrophy control effect of S.

It was investigated whether or not extract of serrata oleifera can control or improve uterine atrophy, which is known as one of the symptoms of menopause.

Specifically, the uterus and blood were collected from the animal test group of Example 4-1, that is, the normal group (sham), the ovariectomized group ((1) OVX, (2) OVX + FeQ, (3) OVX + VFE) and analyzed. First, the extracted uterus was cooled with saline, the weight was measured, and images were taken by taking a picture. In addition, some uterine tissue was fixed in 10% neutral buffered formalin for 24 hours and then embedded in paraffin to prepare a 5 μm uterine tissue sample section. After staining the section with hematoxylin and eosin (H&E), optical Endometrial thickness was analyzed by microscopic imaging. Tissue samples were analyzed at 40 and 100 magnifications and photographed using an Olympus microscope (BX43F, Tokyo, Japan).

Next, 30 μg of the protein extracted from the harvested uterus was loaded into each lane of a 10% (mg/ml) SDS-PAGE gel and subjected to gel electrophoresis analysis. The protein band formed by gel electrophoresis was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), blocked with 2% (mg/mL) bovine serum albumin (BSA), and diluted appropriately (1 :1000) mouse monoclonal ER-α primary antibody (Santa Cruz, Dallas, TX, USA, sc-8005), mouse monoclonal ER-β primary antibody (Santa Cruz, Dallas, TX, USA, sc-53494) , and rabbit monoclonal GAPDH primary antibody (Cell Signaling Technology, Danvers, MA, USA). Next, detection was performed with a goat anti-mouse secondary antibody conjugated with peroxidase (Santa Cruz, Dallas, TX, USA), diluted 1:2000. To visualize the immunoreactive band, the membrane was reacted with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL, USA), and then the density of the protein band was quantified using J software images normalized by GAPDH expression. .

In addition, blood collected from the animal test group of Example 4-1, that is, the normal group (sham), the ovariectomized group ((1) OVX, (2) OVX + FeQ, (3) OVX + VFE) was collected at 4 ° C. Plasma was separated by centrifugation at 3500 rpm for 15 minutes, and the estradiol level was measured from the separated plasma using a Quantikine ^® immunoassay kit (R&D Systems, Minneapolis, MN, USA).

FIG. 7 is a diagram showing the uterine atrophy control effect of VFE extract on an ovariectomized menopausal animal model (OVX) (A: an excised uterine image (top) and a cross-sectional microscopy image of the uterus (bottom); B: Graph of uterine weight measurement; C: Electrophoretic image (top) and protein quantification graph (bottom) measuring ER-α and ER-β protein expression levels; D: Plasma estradiol concentration measured graph).

As shown in FIG. 7 , the thickness of the uterus itself, the thickness of the endometrium, and the weight of the uterus of the animals were significantly reduced in the ovariectomy group compared to the normal group. It was confirmed that the thickness and the thickness of the endometrium were significantly increased, and the weight of the uterus also increased ( FIGS. 7A and 7B ). On the other hand, it was confirmed that there was no significant change in the thickness of the uterus itself, the thickness of the endometrium, and the weight of the uterus when FeQ was administered to the ovariectomy group. In addition, the expression levels of estrogen receptor ER-α and ER-β proteins were decreased in the ovariectomized group compared to the normal group. It was confirmed that the increase (Fig. 7C). In particular, it was confirmed that the effect of increasing the expression of ER-α and ER-β proteins of the S. oleifera extract was superior to that of FeQ. In addition, the plasma estradiol concentration was also significantly decreased in the ovariectomized group compared to the normal group, and it was confirmed that the plasma estradiol concentration was restored to the level of the normal group when the ovarian tree extract was administered to the ovariectomized group (Fig. 7D). ). On the other hand, when FeQ was administered to the ovariectomy group, it was confirmed that the concentration of estradiol in plasma was rather decreased.

Through this example, in uterine atrophy, known as one of the menopausal symptoms, the serratus oleifera extract contains the thickness of the uterus itself, the thickness of the endometrium, the weight of the uterus, the ER-α and ER-β protein expression levels, and the estra It was confirmed that by significantly increasing the diol concentration, it was possible to effectively control or improve uterine atrophy due to menopause. In addition, it was confirmed that the uterine atrophy improvement effect of the extract of Sunbigi extract is significantly superior to that of the existing menopausal treatment.

Example 5: Safety evaluation of S. serrata extract

5-1. Toxicity evaluation of S. serrata extract

In order to evaluate the toxicity of the extract, we analyzed whether hepatotoxicity and nephrotoxicity were induced when the extract was administered to an ovariectomized animal model (OVX).

Specifically, the blood collected from the animal test group of Example 4-1, that is, the normal group (sham), the ovariectomized group ((1) OVX, (2) OVX + FeQ, (3) OVX + VFE) was collected from 4 Plasma was separated by centrifugation at 3500 rpm for 15 minutes at ℃, and the concentrations of alanine aminotransferase (ALT) and creatine were measured from the separated plasma using GENIA (Seong-Nam, Korea).

8 is a diagram showing the results of confirming whether hepatotoxicity and nephrotoxicity are induced when VFE) is administered to an ovariectomized animal model (OVX) of menopause (A: plasma alanine aminotransferase , a graph measuring the concentration of ALT); B: a graph measuring the concentration of creatine in plasma).

As shown in FIG. 8 , the concentrations of ALT and creatine in the plasma of animals were further increased in the ovariectomized group compared to the normal group, and the concentrations of ALT and creatine in the plasma were further increased when the oleracea extract was administered to the ovariectomized group It was confirmed that it decreased to a level similar to that of the normal group. On the other hand, it was confirmed that the concentration of ALT in plasma was further increased when FeQ was administered to the ovariectomized group.

Through this example, it was confirmed that the S. serrata extract does not induce hepatotoxicity and renal toxicity, and thus it is safer than the existing menopausal treatment. Therefore, it was confirmed that the extract of Sunbigi tree can be safely applied to menopausal patients for the prevention or treatment of menopausal symptoms.

5-2. Evaluation of breast cancer cell proliferation inhibitory activity of Sunbigi extract (checking the effect of improving the side effects of hormone replacement treatment)

In general, hormone replacement therapy (HRT) is mainly used for menopausal symptoms, but HRT including estradiol proliferates estrogen receptor-positive breast cancer cells and has side effects such as the risk of breast cancer. Therefore, in order to evaluate the risk of breast cancer of the S. oleracea extract having estrogen-like activity, MCF-7 cells, which are representative estrogen receptor-positive breast cancer cells, were treated with the S. S. oleracea extract and then cultured under normal growth conditions. Breast cancer cells was checked for proliferation.

Specifically, unlike in Examples 2 and 3, in this Example, MCF-7 cells were cultured under normal cell culture conditions (Normal Fetal Bovine Serum-containing medium) was cultured. MCF-7 cells (1 x 10 ⁴ cells/well) were incubated in 96-well plates for 24 hours, and samples were added to the cultured cells, i.e., S. ethanol (100%, 70%, 50%, or 30%). ) extract or hot water extract was treated at a concentration of 0, 25, 50, or 100 μg/mL, respectively. After processing the sample and incubating for 24 hours, Ez-Cytox (DoGEN) reagent was added, and the absorbance was measured at 450 nm after incubation for another hour. The results are shown in FIG. 9 .

Figure 9 is a diagram confirming the breast cancer cell proliferation inhibitory activity of 100% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, or extracts of Sunbigi tree extract extracted using hot water.

As shown in FIG. 9 , it was confirmed that none of the extracts extracted from 100% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, or hot water did not induce the proliferation of breast cancer cells. In particular, the 100% ethanol extract significantly inhibited the proliferation of breast cancer cells at all concentrations of 25, 50, and 100 μg/mL, and the 70% and 50% ethanol extract inhibited the proliferation of breast cancer cells at the concentrations of 100 μg/mL. It was confirmed that it was significantly suppressed.

Through this example, it was confirmed that, despite having an estrogen-like activity, the S. serrata extract showed excellent safety because the possibility of side effects such as the occurrence of breast cancer or proliferation of breast cancer cells was very low compared to the conventional HRT. Therefore, it was confirmed that the extract of Sunbigi tree origin is derived from natural products and can be usefully used for the prevention or improvement of menopausal symptoms without side effects.

The above description is for illustration, and those of ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (10)

A pharmaceutical composition for preventing or treating female menopausal symptoms, comprising an extract of S.
The method according to claim 1,
The composition is a pharmaceutical composition for preventing or treating female menopausal symptoms, which comprises the extract of S. serrata at a concentration of 5 μg/mL or more.
The method according to claim 1,
A pharmaceutical composition for the prevention or treatment of female menopausal symptoms, wherein the extract of the S.
4. The method according to claim 3,
The composition comprises the bitexicarpine in a concentration of 0.01 to 10 μM, a pharmaceutical composition for preventing or treating female menopausal symptoms.
The method according to claim 1,
A pharmaceutical composition for the prevention or treatment of female menopausal symptoms, wherein the extract of the Sunbigi tree includes vitexin.
6. The method of claim 5,
The composition comprises the vitexin in a concentration of 1 to 1000 μM, a pharmaceutical composition for preventing or treating female menopausal symptoms.
The method according to claim 1,
The symptoms of female menopause are hot flashes, sweating, dry skin, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, dysuria, urgency, frequent urination, incontinence, difficulty concentrating, short-term memory disorder, anxiety, depression, insomnia, nervousness, memory loss. , Myalgia, arthralgia, uterine atrophy, weight gain, and at least one selected from the group consisting of osteoporosis, a pharmaceutical composition for preventing or treating female menopausal symptoms.
A food composition for preventing or improving women's menopausal symptoms, comprising an extract of Sunbigi tree.
9. The method of claim 8,
The supunbhi tree extract is a food composition for preventing or improving women's menopausal symptoms, comprising bitexicarpine, bitexin, or a combination thereof.
9. The method of claim 8,
The symptoms of female menopause are hot flashes, sweating, dry skin, vaginal atrophy, lower urethral atrophy, vaginitis, cystitis, dysuria, urgency, frequent urination, incontinence, difficulty concentrating, short-term memory disorder, anxiety, depression, insomnia, nervousness, memory loss. , myalgia, arthralgia, uterine atrophy, weight gain, and at least one selected from the group consisting of osteoporosis, a food composition for preventing or improving female menopausal symptoms.

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