WO2023153794A1

WO2023153794A1 - Composition for enhancing ovarian function or preventing or treating ovotoxicity

Info

Publication number: WO2023153794A1
Application number: PCT/KR2023/001830
Authority: WO
Inventors: 이주희; 김동일; 김선아; 유성경
Original assignee: 동국대학교 와이즈캠퍼스 산학협력단
Priority date: 2022-02-14
Filing date: 2023-02-08
Publication date: 2023-08-17

Abstract

The present invention relates to a composition for enhancing ovarian function or preventing or treating ovotoxicity. By means of Magnolia Liliflora Bud, Paeonia Lactiflora, Malt, and Astragalus Membranaceus extracts inhibiting the production of active oxygen, it is confirmed that the present invention exhibits an effect of protecting ovarian cells, inhibiting apoptosis, and activating the PI3K/Akt signaling pathway against ovotoxicity induced by VCD, and the present invention can thereby be used for enhancing ovarian function or preventing, treating, or improving ovotoxicity.

Description

Composition for strengthening ovarian function or preventing or treating dystocia

The present invention relates to a composition for enhancing ovarian function or preventing or treating dystocia.

The ovary is not only a female reproductive organ, but also an organ that maintains female health through the production of various hormones. Female aging can be divided into general aging of the body and aging of reproductive function, among which aging of reproductive function generally means ovarian aging. Ovarian aging is a phenomenon that occurs in all women. In general, from the age of 35, the number of follicles decreases along with the quality of oocytes, resulting in a decrease in fertility. It is known that it can cause not only polycystic ovary syndrome, infertility, and early menopause, but also systemic problems such as dementia, osteoporosis, heart disease, menopausal disease, and metabolic disorders.

Menopausal disease is a representative disease caused by ovarian aging (decreased ovarian function). It occurs when the secretion of estrogen, a female hormone, is reduced. In addition to physical symptoms such as skin aging, depression, poor concentration, insomnia, headache, tinnitus, and nervousness, symptoms can also appear in the mental nervous system.

Ovotoxicity refers to a toxic effect on ovarian tissue and germ cells of the ovary caused by various harmful factors. Anticancer chemotherapeutic agents, polycyclic aromatic hydrocarbons (PAHs), etc. are known as representative infertility factors, and such intoxication triggers ovarian aging, causing premature ovarian failure or reduced fertility.

4-vinylcyclohexene diepoxide (VCD) is a material that has been typically used to manufacture rubber tires, polyesters, and plastics, and has recently attracted attention as a chemical for environmental factors related to reproductive aging. VCD accelerates the natural cell death process, induces the destruction of selective ovarian small pre-antral (primordial and primary) follicles, and inhibits auto phosphorylation by directly interacting with the oocyte-associated c-kit receptor to promote oocyte growth. It impairs cell viability and is known to affect the activity of PI3K protein and Akt, a member of the PI3K family, in the process.

Hormone therapy, which uses herbal medicines and/or functional foods to improve various symptoms caused by ovarian function decline or supplements estrogen whose production is reduced due to ovarian function decline, is currently being used clinically, but ovarian function decline There is no way to fundamentally treat it.

An object of the present invention is Shin ( Magnoliae Flos ) or malt ( Hordei Fructus Germinatus ) To provide a composition for enhancing ovarian function containing an extract as an active ingredient.

Another object of the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and Astragalus ( Astragali Radix ) To provide a pharmaceutical composition for preventing or treating premature ovarian failure or premature menopause comprising at least one extract selected from the group consisting of as an active ingredient.

Another object of the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and Astragalus ( Astragali Radix ) To provide a health functional food composition for preventing or improving premature ovarian failure or early menopause, containing at least one extract selected from the group consisting of as an active ingredient.

Another object of the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and Astragalus ( Astragali Radix ) To provide a pharmaceutical composition for preventing or treating ovotoxicity containing at least one extract selected from the group consisting of an active ingredient.

Another object of the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and Astragalus ( Astragali Radix ) To provide a health functional food composition for preventing or improving ovotoxicity containing at least one extract selected from the group consisting of as an active ingredient.

In order to achieve the above object, the present invention is Shin ( Magnoliae Flos ) or malt ( Hordei Fructus Germinatus ) Provides a composition for enhancing ovarian function containing an extract as an active ingredient.

In addition, the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and astragalus ( Astragali Radix ) Provides a pharmaceutical composition for preventing or treating premature ovarian failure or premature menopause containing at least one extract selected from the group consisting of as an active ingredient .

In addition, the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and astragalus ( Astragali Radix ) A health functional food composition for preventing or improving premature ovarian failure or early menopause containing at least one extract selected from the group consisting of as an active ingredient to provide.

In addition, the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and astragalus ( Astragali Radix ) It provides a pharmaceutical composition for preventing or treating ovotoxicity containing at least one extract selected from the group consisting of as an active ingredient.

In addition, the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and astragalus ( Astragali Radix ) Provides a health functional food composition for preventing or improving ovotoxicity containing at least one extract selected from the group consisting of as an active ingredient do.

According to the present invention, the extracts of Shinyi, Peony, Maltger and Astragalus Astragalus inhibit the production of reactive oxygen species, thereby protecting ovarian cells against VCD-induced ovotoxicity, inhibiting apoptosis, and activating the PI3K/Akt signaling pathway. By confirming that, it can be used for strengthening ovarian function or preventing, treating, or improving dystocia.

Figure 1 is the result of analyzing the DPPH radical and superoxide anions scavenging activity of peony extract.

Figure 2 shows the results of analyzing the cytotoxicity of the extracts of Shinyi (MFMW and MFME), peony (PR), malt (HFG) and Astragalus (AR) on ovarian cells.

Figure 3 shows the ovarian cell protective effect of Shinyi (MFMW and MFME), peony (PR), malt (HFG) and Astragalus (AR) extracts against ovotoxicity induced by 4-vinylcyclohexene diepoxide (hereinafter referred to as VCD) is the result of analyzing

Figure 4 analyzes the inhibitory effect of reactive oxygen species (hereinafter referred to as ROS) production of kidneys (MFMW and MFME), peony (PR), malt (HFG) and astragalus (AR) extracts in ovarian cells treated with VCD is a result

FIG. 5 is a result of analyzing changes in morphology of whole cell morphology or intracellular nuclei through DAPI staining when treatment with MFMW and Astragalus (AR) extracts in VCD-treated ovarian cells.

6 is a result of analyzing the apoptosis inhibitory effects of extracts of MFMW, peony (PR), malt (HFG), and Astragalus (AR) in VCD-treated ovarian cells.

Figure 7 is a result of analyzing the activity change of the PI3K / Akt signaling pathway according to the treatment time of Shinyi (MFMW), peony (PR), malt (HFG) and Astragalus (AR) extracts.

8 shows the results of visual observation of uterine and ovarian tissues in the normal group (Con), control group (V), and experimental group (V+ML and V+MH) in the animal model of premature ovarian failure (Con: vehicle, V: VCD 160mg/ kg/day, V+ML: VCD 160mg/kg + MFMW Low dose 100mg/kg, V+MH: VCD 160mg/kg + MFMW High dose 300mg/kg).

Figure 9 shows the results of measuring the weight index of the uterus + ovary and ovary according to the treatment with Shinyi extract in an animal model of premature ovarian failure (Con vs group: ^## p < 0.01, ^### p < 0.001, VCD vs group: * p < 0.05, ***p < 0.001).

10 shows the results of measuring serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) of each group in an animal model of premature ovarian failure (NS: not significant).

11 is a histopathological analysis result of ovarian tissue through H&E staining.

Hereinafter, the present invention will be described in more detail.

The present invention is Shin ( Magnoliae Flos ) or malt ( Hordei Fructus Germinatus ) Provides a composition for enhancing ovarian function containing an extract as an active ingredient.

Shinyi ( Magnoliae flos ) refers to the buds of magnolias and magnolias, a deciduous shrub. Magnolia is a deciduous tree that has been cultivated for horticultural purposes since ancient times. Flowers bloom before leaves, and the flowering period is between March and April. In oriental medicine, its nature is regarded as warm, and it is effective for headache, back pain and rhinitis, treats sinusitis, and sees that it has medicinal properties as a pain reliever.

Peony ( Paeoniae Radix ) is a dicotyledonous perennial plant belonging to the peony genus and is mainly distributed in Korea, Mongolia, China, and East Siberia. The peony root contains a large amount of benzoic acid, asparagine, and paeoniflorin. It is mainly used to treat amenorrhea, hematemesis, anemia, and bruises. Peony flowers are widely used for horticultural purposes, and recently, peony seeds have been reported to have excellent anticancer and antimutagenic effects because they contain a large amount of physiologically active substances such as trans-resvaratrol, trans-viniferin, and cis-viniferin.

Malt ( Hordei Fructus Germinatus ) is a sprouted and dried barley ( Hordeum vulgare Linn, 大麥) of the Poaceae family. The synonyms of malt include barley malt, barley wall, barley hair, and barley malt (麥蘖). ), grain maek (谷麥), etc., and amylase, a malt enzyme, acts to convert starch into sugar, so it is also known as “malt”. Malt contains diastase, invertase, glucose 6-phosphate dehydrogenase, a-amylase, b-amylase, protease, dextrin It contains a lot of enzymes involved in protein metabolism, such as maltose, glucose, and vitamin B. , it has been reported to be effective in hepatitis, etc.

Astragalus ( Astragali Radix ) is a perennial herb belonging to the leguminous family (Leguminosae), which digs up the roots of Astragalus membranaceus Bunge and peels and dries them. In oriental medicine, it has been used for purposes such as diuresis, tonic, lowering blood pressure, anti-inflammatory, antipyretic, pain relief, etc. Immunity, anticancer, antiviral, antibacterial, diuretic, anti-inflammatory, etc. are very diverse. In particular, the saponin component contained in astragalus is known to exhibit effects such as antioxidant, anti-aging, and skin regeneration.

The extract may be extracted with water, C1 to C4 alcohol, or a mixed solvent thereof.

In addition, the extract may exhibit an ovarian protective effect against ovotoxicity through reactive oxygen species (ROS) production inhibitory activity, and the ovotoxicity is induced by 4-vinylcyclohexene diepoxide (VCD) can

In addition, the extract can inhibit apoptosis of ovarian cells.

In addition, the extract can activate the PI3K/Akt signaling pathway.

The pharmaceutical composition of the present invention is prepared in a unit dose form or in a multi-dose container by formulating using a pharmaceutically acceptable carrier according to a method that can be easily performed by those skilled in the art. It can be prepared by incorporating into

The pharmaceutically acceptable carrier is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.

In the present invention, the content of the additives included in the pharmaceutical composition is not particularly limited and may be appropriately adjusted within the range of content used in conventional formulations.

The pharmaceutical composition may be formulated into an injectable formulation such as an aqueous solution, suspension, or emulsion, a pill, a capsule, a granule, a tablet, a cream, a gel, a patch, a spray, an ointment, a warning agent, a lotion, a liniment agent, a paste agent, and a cataplasma agent. It may be formulated in the form of one or more skin external preparations selected from the group consisting of, but is not limited thereto.

The pharmaceutical composition of the present invention may additionally contain pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, gelatin, alginates, and polyvinyl pyrrolidone. binders such as talc, calcium stearate, lubricants such as hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, surfactants such as polysorbates, cetyl alcohol, glycerol and the like, but are not limited thereto. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method. For oral administration, it may be formulated into tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, and the like. In the case of parenteral administration, it may be formulated as an injection solution, suppository, powder for respiratory inhalation, aerosol for spray, ointment, powder for application, oil, cream, etc.

The dosage of the pharmaceutical composition of the present invention depends on the patient's condition, weight, age, sex, health condition, dietary constitution specificity, the nature of the preparation, the severity of the disease, the administration time of the composition, the administration method, the administration period or interval, the excretion rate and The range may vary depending on the type of drug, and may be appropriately selected by a person skilled in the art. For example, it may range from about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be divided and administered once or several times a day.

The pharmaceutical composition may be administered orally or parenterally (eg, intravenous, subcutaneous, intraperitoneal or topical application) depending on the desired method. The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, administration route, etc. of the pharmaceutical composition, and those skilled in the art can achieve effective treatment for the desired treatment. The dosage can be easily determined and prescribed. Administration of the pharmaceutical composition of the present invention may be administered once a day, or may be divided and administered several times.

The present invention can be generally used as a commonly used food.

The food composition of the present invention can be used as a health functional food. The term "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with the Health Functional Food Act, and "functional" refers to food that is not related to the structure and function of the human body. It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.

The health functional food composition may include conventional food additives, and the suitability as the "food additive" is determined in accordance with the General Rules and General Test Methods of Food Additives approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is judged according to the specifications and standards for the item.

Items listed in the "Food Additive Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as dark pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.

The food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like. For example, among health functional foods in the form of capsules, hard capsules can be prepared by mixing and filling a composition according to the present invention with additives such as excipients in a conventional hard capsule, and soft capsules contain the composition according to the present invention. It can be prepared by mixing with additives such as excipients and filling in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, and the like, if necessary.

Definitions of terms for the excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in literature known in the art, and include those having the same or similar functions. There is no particular limitation on the type of food, and it includes all health functional foods in the usual sense.

In the present invention, the term "prevention" refers to any action that suppresses or delays the symptoms or diseases by administering the composition according to the present invention. In the present invention, the term "treatment" refers to all activities that improve or beneficially change the symptoms of the symptoms or diseases by administration of the composition according to the present invention. In the present invention, "improvement" means any action that improves the bad condition of the symptom or disease by administering or ingesting the composition of the present invention to a subject.

In addition, the present invention is Shin ( Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) and astragalus ( Astragali Radix ) It provides a pharmaceutical composition for preventing or treating ovotoxicity, containing as an active ingredient one or more extracts selected from the group consisting of .

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

[[ 실험예Experimental example 1] 추출물 제조 1] Extract preparation

Shinyi (辛荑, Magnoliae) Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus) Germinatus ) and Astragalus extracts ( Astragali Radix ) were prepared. The four kinds of medicinal plants were purchased from Human Herb Co., Ltd. (Daegu, Korea), selected, and selected ones were used.

1-1. Manufacture of Sinyi Extract

800 mL of distilled water or 70% ethanol was added to 100 g of Shini, soaked for a week, filtered twice with a filter paper with 8 μm pore size, and the filtered solution was concentrated under reduced pressure and freeze-dried, and pulverized to obtain MFMW ( Magnoliae 3.3 g of Flos macerated water and 4.94 g of Magnoliae Flos macerated ethanol (MFME) were obtained.

1-2. Manufacture of peony extract

800 mL of 70% ethanol was added to 100 g of peony, followed by extraction by heating and refluxing for 4 hours. After cooling the extract at room temperature, it was filtered twice with a filter paper having a pore size of 8 μm, and the filtered solution was concentrated under reduced pressure and freeze-dried, and pulverized to obtain 13.0443 g of peony extract.

1-3. malt extract manufacturing

800 mL of 70% ethanol was added to 100 g of malt, followed by extraction by heating under reflux for 4 hours. After filtering twice with a filter paper with a pore size of 8 μm, the filtered solution was concentrated under reduced pressure and freeze-dried, and pulverized to obtain 6.731 g of malt extract.

1-4. Astragalus extract manufacturing

800 mL of 70% ethanol was added to 100 g of Astragalus root, followed by extraction by heating and refluxing for 4 hours. After cooling the extract at room temperature, it was filtered twice with a filter paper having a pore size of 8 μm, and the filtered solution was concentrated under reduced pressure and freeze-dried, and pulverized to obtain 19.1111 g of Astragalus extract.

[[ 실험예Experimental example 2] 항산화 활성 측정 2] Measurement of antioxidant activity

2-1. DPPH Measurement of radical scavenging activity

In order to confirm the antioxidant activity of the peony extract, the scavenging activity for DPPH radicals was measured. 1mL of 1M DPPH solution and 450μL of 50mM Tris-HCl buffer (pH 7.4) were added to 50μl of the sample and mixed. After reacting the mixture at room temperature for 30 minutes, absorbance was measured at a wavelength of 517 nm using a microplate reader (VersaMax, Molecular Devices, USA). The scavenging activity of the DPPH radical was expressed as a concentration showing 50% scavenging ability (EC ₅₀ ).

2-2. superoxide anion ( Superoxide anions) scavenging activity measurement

In order to confirm the antioxidant activity of the peony extract, the scavenging activity for peroxide anion was measured using the NBT reduction method. 10 μL of 30 mM EDTA (ethylene-diamine-tetraacetic acid, pH 7.4), 1 μL of 30 mM hypoxanthine, and 200 μL of 1.42 mM NBT (nitroblue tetrazolium) were added to 30 μL of sample, followed by reaction at room temperature for 3 minutes, followed by oxidation of 1 U/mL xanthine 10 μL of enzyme (xanthine oxidase) was added, and the total volume was adjusted to 300 μL with 50 mM phosphate buffer (pH 7.4). After incubating the reaction solution at room temperature for 20 minutes, the absorbance was measured at a wavelength of 560 nm, and the results were expressed in terms of NBT reduction EC ₅₀ values by superoxide radicals.

[[ 실험예Experimental example 3] 세포 생존율 측정 3] Measurement of cell viability

In order to confirm the cytotoxicity of the four extracts, cell viability was measured by MTT (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) assay method. 1 × 10 ⁴ cells/well of CHO-K1 cells were cultured in a 96-well plate, and after serum starvation for 4 hours, the four extracts were pretreated by concentration, respectively, and VCD 1.5mM was sequentially applied. treated and cultured for 24 hours under conditions of 37°C and 5% CO ₂ . 10μL of MTT solution 2mg/mL per well was added and reacted for 2 hours in an incubator at 37°C and 5% CO ₂ conditions. After completely removing the MTT solution and culture medium, 100μL DMSO (dimethyl sulfoxide, Junsei Chemical Co., Tokyo , Japan) to dissolve formazan crystals formed in cells, and absorbance was measured at 540 nm with an ELISA plate reader (Tecan Austria GmbH, Grodig, Austria). Cell viability relative to the control group was expressed as a percentage.

[[ 실험예Experimental example 4] 4] ROSROS 측정 measurement

In order to confirm whether the ovarian cell-protective efficacy of the four extracts was due to oxidative stress inhibition, the ROS activities of the four extracts were measured. After culturing CHO-K1 cells in a 6-well plate, the above four extracts were pre-treated for 1 hour, treated with VCD 1.5 mM, and further cultured for 18 hours. DCFH-DA (2' 7-dichlorofluorescein diacetate) diluted with PBS (phosphate buffer saline) was added to the medium at a concentration of 1 μM, incubated for 30 minutes, and then the cells were washed twice with PBS. Thereafter, the fluorescence intensity was measured using a CytoFLEX flow cytometer (Beckman Coulter Inc., USA), and the increase rate of DCF fluorescence intensity was calculated as a percentage (%) compared to the control group.

[[ 실험예Experimental example 5] 세포 형태 관찰 5] Observation of cell morphology

In order to confirm whether Shinyi and Astragalus extracts protect CHO-K1 cells from damage caused by VCD through cell morphological changes, CHO-K1 cells were pretreated with Shinyi and Astragalus extracts at various concentrations for 1 hour, respectively, and VCD 1.5 After treatment with mM, and incubation at 37° C. and 5% CO ₂ for 24 hours, the cells were observed under an optical microscope (Nikon, Japan). To observe CHO-K1 cell nuclei, CHO-K1 cells were washed twice with PBS, fixed with 4% paraformaldehyde, and washed twice with PBS. Nuclei were stained by reacting with 2 μg/mL DAPI (4,6-diamidino-2-phenylindole) solution for 15 minutes, and then the cells were observed under a fluorescence microscope (ECLIPSE Ts2-FL; Nikon, Tokyo, Japan).

[[ 실험예Experimental example 6] 6] 웨스턴western 블롯blot (Western blot)(Western blot)

For protein extraction, after washing the cells twice with PBS, RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA) was added and reacted at 4 ° C for 30 minutes and centrifuged at 12,000 × g for 30 minutes to obtain the supernatant. gathered After measuring the protein concentration with a Bicinchoninic acid protein assay kit (Thermo Fisher Scientific), a protein sample was prepared using a sample buffer (Bio-Rad, Hercules, CA, USA). After the same amount of protein was separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the protein was transferred to a PVDF membrane (membrane). In order to block the non-specific binding of the antibody, the membrane was treated with a blocking buffer (5% non-fat milk) for 1 hour, and then washed with a PBST solution containing 0.1% Tween 20. It was treated overnight at 4 ° C using the antibody of each protein to be reported, the secondary antibody was HRP-linked anti-rabbit (anti-rabbit) or anti-mouse (anti-mouse) was used, and enhanced chemiluminoescence solution (Amersham , Piscataway, NJ, USA), the expression level of each protein was observed in a Fusion Solo-2 image image analyzer (Vilber Lourmat, Paris, France), and images were acquired.

[[ 실험예Experimental example 7] 동물 모델 준비 7] Animal model preparation

As experimental animals, 11-18 g B6C3F1 female mice were purchased from the central laboratory animals and used for experiments after being acclimatized to the laboratory environment while supplying sufficient food and water for one week. The laboratory environment maintained a temperature of 22 ± 2 ° C and continued day and night in 12-hour increments until the end of the experiment.

[[ 실험예Experimental example 8] 8] 조기난소부전premature ovarian failure 유발 cause

The control group was intraperitoneally injected with sesame oil (Sigma-Aldrich, USA), and the control (VCD) and experimental groups (VCD+MFMW 100mg/kg, VCD+MFMW 300mg/kg) were injected with VCD (Sigma-Aldrich, USA). , USA) was dissolved in sesame oil and intraperitoneally injected at a concentration of 160 mg/kg/day 5 times a week for a total of 2 weeks to induce premature ovarian failure. Oral administration of tap water to the normal and control groups, and Shinyi extract to the concentration of 100 mg/kg or 300 mg/kg in the experimental group, 5 times a week from one week before VCD treatment, were administered orally simultaneously with VCD for 2 weeks, and VCD treatment was completed. After that, it was orally administered for another week, for a total of 4 weeks.

[[ 실험예Experimental example 9] 체중 및 자궁/난소 무게 측정 9] Body weight and uterine/ovarian weight measurement

After the mice were sacrificed on the last day of the experiment, the entire uterus and ovaries were removed, observed with the naked eye, and their weights were measured.

[[ 실험예Experimental example 10] 10] 간독성liver toxicity 분석 analyze

In order to confirm the hepatotoxicity of the Shinyi extract, on the last day of the experiment, blood was obtained from mice by cardiac sampling method and centrifuged at 3000 rpm for 15 minutes using a centrifuge (Beckman Coulter, Fullerton, CA) to obtain the supernatant. GOT (glutamic oxaloacetic transaminase) and GPT (glutamic pyruvic transaminase), which are indicators of liver damage, were measured in the obtained serum. Asan set GOT and GPT test solutions (Asan Pharmaceutical Co., Ltd., Seoul, Korea) were purchased and used. 100 μL of the substrate solution for measuring GOT and GPT was left at 37° C. for 5 minutes, mixed with 20 μL of serum, and reacted at 37° C. for 60 minutes for GOT and 30 minutes for GPT. Then, it was mixed with 100 μL of colorant solution, left at room temperature for 20 minutes, mixed with 0.4N sodium hydroxide (NaOH) 1mL, left at room temperature for 10 minutes, and the absorbance was measured at 505 nm to calculate the amount of GOT and GPT.

[[ 실험예Experimental example 11] 11] 병리조직학적histopathological 관찰 observe

One ovary and uterus were fixed in 10% formalin, embedded in paraffin, and 5 μm thin sections were made, stained with Hematoxylin-Eoxin (H&E), and general histopathologic findings were observed and analyzed under an optical microscope.

[[ 실험예Experimental example 12] 통계분석 12] Statistical analysis

All experimental results were expressed as mean ± standard error of the mean (SEM), and to test significance, GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA) was used After one way analysis of variance (ANOVA) was performed, it was analyzed by Tukey's multiple comparison test at the p<0.05 level.

[[ 실시예Example 1] 항산화 활성 분석 1] Antioxidant activity assay

According to Experimental Example 2, as a result of analyzing the antioxidant activity of the peony extract through the measurement of DPPH radical and peroxide anion scavenging activity, as shown in FIG. 1, the EC ₅₀ (effective concentration 50%) values were 85.2 μg / mL and It was confirmed that 25.5 μg / mL showed excellent scavenging ability in a concentration-dependent manner. From the above results, it was confirmed that the peony extract had excellent antioxidant activity.

[[ 실시예Example 2] 세포독성 분석 2] Cytotoxicity assay

As a result of analyzing the cytotoxicity of the four extracts according to Experimental Example 3, as shown in FIG. 2, when compared to the control, Shinyi extract had an effect on CHO-K1 cell viability up to 200 μg / mL in both MFMW and MFME It was confirmed that peony, malt and astragalus extracts did not affect CHO-K1 cell viability up to 200 μg/mL compared to the control group.

[[ 실시예Example 3] VCD로 유도된 3] induced by VCD 난독성에obscurity 대한 보호 효과 분석 Analysis of protective effect against

CHO-K1 cells were pretreated with the above four extracts at various concentrations (1, 10, 100, and 200 μg/mL) for 1 hour, and after inducing ovotoxicity with VCD 1.5mM, protective effect against ovotoxicity. As a result of the analysis, as shown in FIG. 3, the Shinyi extract showed a concentration-dependent protective effect against dyslexia in MFMW, and showed a survival rate almost similar to that of the control group not treated with VCD at a concentration of 100 μg / mL or more, MFME showed a protective effect against dyslexia at concentrations of 1 and 10 μg/mL. In addition, it was confirmed that peony, malt and astragalus extracts showed a concentration-dependent protective effect against dyslexia.

[[ 실시예Example 4] 4] ROSROS 생성 억제 효과 분석 Production inhibition effect analysis

As a result of analyzing the ROS generation inhibitory activity of the four extracts according to Experimental Example 4, as shown in FIG. 4, treatment with VCD significantly increased ROS generation, and in all of the four extract treatment groups It was confirmed that ROS generation was significantly reduced in a concentration-dependent manner.

[[ 실시예Example 5] 세포 형태 및 세포핵 관찰 5] Observation of cell morphology and cell nucleus

According to Experimental Example 5, as a result of treating the cells treated with VCD with Shini and Astragalus extracts and observing the cells under an optical microscope, as shown in FIG. 5, apoptosis was observed in the VCD alone treatment group (VCD), whereas , Shinyi and Astragalus extract treated groups (VCD+MFMW and VCD+AR) showed cell growth similar to that of the VCD untreated group (Con) in a concentration-dependent manner, and the protective effect of Shinyi and Astragalus extract against dyslexia induced by VCD was observed. It was confirmed that indicated

In addition, as a result of observing the cell nucleus with DAPI and then observing it under a fluorescence microscope, as shown in FIG. 5, condensation and fragmentation of the nucleus were observed in the VCD alone treatment group (VCD), indicating apoptosis. In the Astragalus extract-treated groups (VCD+MFMW and VCD+AR), nuclear condensation and fragmentation were reduced in a concentration-dependent manner, confirming that VCD-induced apoptosis was inhibited.

[[ 실시예Example 6] VCD로 유도된 세포사멸 억제 효과 분석 6] Analysis of VCD-induced apoptosis inhibitory effect

According to Experimental Example 6, as a result of analyzing the expression change of caspase-3, an apoptosis-related protein (PARP), according to the treatment of the four extracts through Western blot, as shown in FIG. 6, Compared to the control group, PARP cleavage occurred during VCD treatment, resulting in two bands, and in the case of proform caspase-3, the band intensity decreased, confirming that apoptosis was induced. On the other hand, when the 4 types of extracts were pretreated and treated with VCD, it was confirmed that the PARP band was not separated into two, and caspase-3 was not reduced compared to VCD alone. From the above results, it was confirmed that the four extracts inhibited cell death induced by VCD and had a cell protective effect from dystotoxicity.

[[ 실시예Example 7] 7] 난독성obscurity 보호 효능의 기전 확인 Confirmation of mechanism of protective efficacy

In order to investigate the mechanism of the intractability protective efficacy of the four extracts, according to Experimental Example 6, changes in the PI3K / Akt signaling pathway known as the toxicity mechanism of VCD according to the treatment of the four extracts were examined by western As a result of analysis through blotting, as shown in FIG. 7, when the above four extracts (100 μg/mL) were treated, the expression of Akt and mTOR, which are major kinases of PI3K/Akt signaling, were increased in a time-dependent manner. (signal) direction was confirmed to be activated. From the above results, it was confirmed that the above four extracts sequentially activate the PI3K/Akt signaling pathway and, through this, exhibit the efficacy of protecting ovarian cells from VCD.

[[ 실시예Example 8] 자궁 및 난소 조직의 육안 관찰 8] Visual observation of uterine and ovarian tissue

As a result of visual observation of the uterine and ovarian tissues according to Experimental Example 9, as shown in FIG. 8, the normal group (Con) maintains normal size of both the uterine and ovarian tissues, whereas the VCD-treated group (V) confirmed that the tissues of the uterus and ovaries were significantly atrophied. In addition, it was confirmed that the Shinyi extract and VCD treated groups (V+ML and V+MH) exhibited similar uterine and ovarian tissue sizes to those of the normal group.

[[ 실시예Example 9] 자궁 및 난소 전체의 무게와 난소 무게 비교를 통한 난소/자궁 보호 효과 분석 9] Analysis of the ovarian/uterine protection effect by comparing the weight of the entire uterus and ovary with the weight of the ovary

According to Experimental Example 9, as a result of comparing the weight of the entire uterus and ovary with the weight of the ovary, as shown in FIG. 9, the weight of the VCD-treated group (V) decreased significantly compared to the normal group (Con), It was confirmed that the extract and VCD treatment groups (V+ML and V+MH) significantly increased the weight compared to the VCD treatment group, and that the increased weight was almost similar to that of the normal group. From the above results, it was confirmed that the Shinyi extract effectively protects the ovary and uterus from weight loss of the ovary and uterus caused by VCD.

[[ 실시예Example 10] 10] 간독성liver toxicity 평가 evaluation

As a result of analyzing the concentration change of GOT and GPT according to the Shinyi extract treatment according to Experimental Example 10, as shown in FIG. 10, it was confirmed that VCD or Shinyi extract had no hepatotoxicity through no significant difference between the experimental groups. .

[[ 실시예Example 11] 11] 병리조직학적histopathological 분석 analyze

As a result of histopathological analysis through H&E tissue staining according to Experimental Example 11, as shown in FIG. 11, the ovaries of the normal group (Con) had well-developed mature follicles and corpus luteum in primordial follicles of various sizes, and normal While the size was maintained, the size of the ovaries of the VCD-treated group (VCD) significantly atrophied and the number of follicles also significantly decreased, showing histological findings suggesting menopause. On the other hand, in the Shinyi extract and VCD-treated groups (VCD+ML and VCD+MH), ovarian size atrophy or follicle and corpus luteum degeneration were not observed, and it was confirmed that they were almost similar to the normal group. From the above results, it was confirmed that the Shinyi extract could prevent premature ovarian failure caused by VCD.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

Magnoliae Flos ) or malt ( Hordei Fructus Germinatus ) Composition for enhancing ovarian function containing an extract as an active ingredient.
Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei A pharmaceutical composition for preventing or treating premature ovarian failure or premature menopause, comprising at least one extract selected from the group consisting of Fructus Germinatus ) and Astragalus ( Astragali Radix ) as an active ingredient.
Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei A health functional food composition for preventing or improving premature ovarian failure or premature menopause, comprising at least one extract selected from the group consisting of Fructus Germinatus ) and Astragalus ( Astragali Radix ) as an active ingredient.
The composition according to claims 1 to 3, wherein the extract is extracted with water, C1 to C4 alcohol, or a mixed solvent thereof.
The method according to claim 1 to claim 3, wherein the composition characterized in that the extract exhibits an ovarian protective effect against ovotoxicity through reactive oxygen species (ROS) generation inhibitory activity.
The composition according to claim 5, characterized in that the difficulty is induced by 4-vinylcyclohexene diepoxide (VCD).
The composition according to claims 1 to 3, wherein the extract inhibits apoptosis of ovarian cells.
The composition according to claims 1 to 3, wherein the extract activates the PI3K/Akt signaling pathway.
Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei A pharmaceutical composition for preventing or treating ovotoxicity, comprising at least one extract selected from the group consisting of Fructus Germinatus ) and Astragali Radix as an active ingredient.
Magnoliae Flos ), peony ( Paeoniae Radix ), malt ( Hordei Fructus Germinatus ) And Astragalus ( Astragali Radix ) A health functional food composition for preventing or improving ovotoxicity, comprising at least one extract selected from the group consisting of as an active ingredient.