WO2017095011A1 - Composition for preventing and treating premature ovarian failure, containing evodia rutaecarpa bentham extract having protective activity against ovotoxicity - Google Patents

Composition for preventing and treating premature ovarian failure, containing evodia rutaecarpa bentham extract having protective activity against ovotoxicity Download PDF

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Publication number
WO2017095011A1
WO2017095011A1 PCT/KR2016/011302 KR2016011302W WO2017095011A1 WO 2017095011 A1 WO2017095011 A1 WO 2017095011A1 KR 2016011302 W KR2016011302 W KR 2016011302W WO 2017095011 A1 WO2017095011 A1 WO 2017095011A1
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Prior art keywords
extract
sewage
vcd
ovotoxicity
preventing
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PCT/KR2016/011302
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French (fr)
Korean (ko)
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김동일
이주희
김희정
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동국대학교 경주캠퍼스 산학협력단
한국보건산업진흥원
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Priority claimed from KR1020160129640A external-priority patent/KR101860639B1/en
Application filed by 동국대학교 경주캠퍼스 산학협력단, 한국보건산업진흥원 filed Critical 동국대학교 경주캠퍼스 산학협력단
Priority to US16/305,129 priority Critical patent/US10835564B2/en
Publication of WO2017095011A1 publication Critical patent/WO2017095011A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/754—Evodia

Definitions

  • the present invention relates to a composition for preventing or treating egg toxicity, and more particularly, to a composition for preventing or treating egg toxicity including sewage extract or a composition for preventing and treating premature ovarian failure including the same.
  • Sexual reproductive aging may be due to various environmental factors as well as natural aging. Ovarian dysfunction caused by reproductive aging occurs before the age of 40 is called early ovarian failure.
  • Premature ovarian failure is a clinical condition in which ovarian follicles do not respond or disappear due to various factors. Premature ovarian failure occurs in about 1% of women before age 40, with 10-28% of primary amenorrhea and 4-18% of secondary amenorrhea due to premature ovarian failure. Unlike menopause, in which the function of the ovary is completely stopped, pregnancy may be possible in 5-10%, but the prognosis cannot be estimated. The causes of early ovarian failure are caused by genetic factors, ovarian damage caused by autoimmunity, and due to chemotherapy, radiation therapy, ovarian surgery, etc. Although the factor is the etiology, it is often difficult to determine the mechanism and cause of occurrence in most patients.
  • This early ovarian dysfunction can lead to early and more severe complaints of more serious menopause-related symptoms than women with natural menopause, due to early menopause. This can be a significant leading cause of poor health-related quality of life, including cardiovascular disease and osteoporosis, as well as emotional well-being and sexual self-impairment, given life expectancy after menopause.
  • VCD 4-vinylcyclohexene diepoxide
  • VCD is a material typically used to make rubber tires, polyesters and plastics.
  • these VCDs have attracted attention as chemicals of environmental factors related to reproductive aging.
  • VCD accelerates the process of natural cell death, thereby selectively destroying ovarian small pre-antral (primordial and primary) follicles.
  • VCD is a material that selectively destroys primordial follicles in the white paper model, and directly interacts with oocyte-associated c-kit receptors, thereby inhibiting auto phosphorylation, impairing oocyte viability. This process is known to affect kit distribution, affecting downstream PI3K protein activity and PI3K family Akt.
  • the present invention has been made to solve the above-mentioned problems in the prior art, the present inventors have been researched to find an effective herbal extract material for early ovarian failure, as a result of the sewage extract is induced by 4-vinylcyclohexene diepoxide (VCD) There is a significant protective effect on the egg toxicity, thereby confirming the effect of preventing or treating premature ovarian failure, to complete the present invention based on this.
  • VCD 4-vinylcyclohexene diepoxide
  • a pharmaceutical composition for preventing or treating ovotoxicity comprising a sesame oil extract as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating ovotoxicity comprising a sewage oil extract as an active ingredient.
  • the egg toxicity may be induced by 4-vinylcyclohexene diepoxide (VCD).
  • VCD 4-vinylcyclohexene diepoxide
  • the sewage extract may inhibit apoptosis of ovarian cells.
  • the sewage extract may activate the PI3K / Akt signaling pathway.
  • the present invention provides a pharmaceutical composition for preventing or treating premature ovarian failure, comprising sewage extract as an active ingredient.
  • the present invention also provides a health functional food composition for improving early ovarian dysfunction comprising the sesame oil extract as an active ingredient.
  • the present invention also provides a method for preventing or treating premature ovarian failure comprising administering the composition to a subject.
  • the present invention provides a use or prevention of premature ovarian failure of sewage extract.
  • the sesame oil extract according to the present invention increases the DPPH free radical scavenging activity in a concentration-dependent manner, and thus has excellent antioxidant activity, inhibits apoptosis of ovarian cells, and activates the PI3K / Akt signaling pathway to activate 4-vinylcyclohexene diepoxide ( It has a significant protective effect against egg toxicity induced by VCD) and can be used as a medicine and health functional food useful for improving, preventing, suppressing or treating egg toxicity and premature ovarian failure. Therefore, relevant applications include infertility, treatment or prevention of infertility, premature ovarian failure and premenopausal disorders.
  • 1 is a flow chart schematically showing a process for preparing a sewage extract.
  • Figure 2 is a result of comparing the DPPH radical scavenging activity of the sesame oil extract and five medicinal herbs (soils, lobar, acid, Mokyang and Yubaekpi) extract.
  • Figure 3 is a result of comparing the superoxide anions scavenging activity of the sesame oil extract and five medicinal herbs (soil, larvae, pesticides, Mokyang and baekbaekpi) extract.
  • Figure 6 is a result confirming the protective effect of the concentration of sewage extract for VCD induced ovotoxicity (ovotoxicity).
  • Figure 7 shows the results confirmed the cytotoxicity of sewage extract alone against CHO-K1 cells.
  • Figure 8 shows the effect of sewage extract on apoptosis of VHO induced CHO-K1 cells.
  • 9 and 10 are the results confirm the PI3K / Akt signal pathway activity changes according to the treatment of sewage milk extract.
  • 11 is a schematic diagram of an animal experiment for evaluating the antitoxic effect of sesame oil extract.
  • Figure 13 is a diagram showing the weight index of the uterus and ovary according to the sewage extract treatment.
  • Figure 14 is a diagram showing the weight index of the ovary according to the sewage extract treatment.
  • Figure 15 is the result of measuring the concentration of Serum antimullerian hormone (AMH) secreted by sewage extract treatment.
  • AMH Serum antimullerian hormone
  • the present invention provides a pharmaceutical composition for the prevention or treatment of ovotoxicity comprising a sewage extract as an active ingredient.
  • ovotoxicity means that the ovary is damaged by a chemical cause, and the damage includes both loss of ovarian function and apoptosis of ovarian cells.
  • the sewage oil extract as an active ingredient in the composition of the present invention can be obtained by a conventional extraction method, and commercially available one can be purchased and used.
  • Conventional extraction methods may include, but are not limited to, hot water extraction, cold needle extraction, reflux extraction, ultrasonic extraction, and the like.
  • sewage extract may be obtained by the following method.
  • the pulverized mixture is refluxed with water, C 1 to C 4 alcohol or a mixed solvent of water and C 1 to C 4 alcohol at 50 to 100 ° C. for 2 to 5 hours, preferably 4 hours.
  • the volume of the solvent is 1 to 10 times, preferably 5 to 8 times the weight of the crushed sewage oil.
  • the extract is filtered, the filtrate is concentrated, and the concentrate is lyophilized to obtain a sewage extract in powder form.
  • VCD 4-vinylcyclohexene diepoxide
  • pretreatment of sewage extract to CHO-K1 cells, which are hamster ovary-derived chemotactic cells and then treated with VCD, confirming the protective effect of the sewage extract from egg toxicity caused by VCD.
  • CHO-K1 cells which are hamster ovary-derived chemotactic cells
  • the sewage oil extract according to the present invention shows an excellent protective effect on germ cell toxicity by VCD, which is a representative ovarian toxic substance, improving, preventing, suppressing or treating premature ovarian failure, infertility, infertility, premenopausal disorders, etc. It can be used as a useful medicine and health food.
  • the present invention provides a pharmaceutical composition for preventing or treating premature ovarian failure, comprising sewage extract as an active ingredient.
  • prevention means any action that inhibits early ovarian failure or delays the onset by administration of a pharmaceutical composition according to the present invention.
  • treatment means any action that improves or advantageously changes the symptoms of premature ovarian failure by administration of the pharmaceutical composition according to the present invention.
  • Early ovarian dysfunction a disease targeted in the present invention, means that ovarian function is stopped by various factors before the age of 40 in women with normal development, and includes depletion of follicles or dysfunction of follicles. .
  • the pharmaceutical composition of the present invention may further contain at least one known active ingredient having a therapeutic effect of refractoriness or premature ovarian failure along with sesame oil extract.
  • composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of conventional formulations, external preparations, suppositories, and sterile injectable solutions.
  • Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts.
  • the pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general May be administered 0.0001 to 150 mg, preferably 0.001 to 100 mg daily or every other day or divided into 1 to 3 times a day per kg body weight.
  • the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
  • the present invention provides a health functional food composition for improving premature ovarian failure comprising sewage extract as an active ingredient.
  • the term “improvement” refers to any action that at least reduces the parameters associated with the condition being treated, such as the extent of symptoms.
  • the composition of the present invention may be added to a dietary supplement for the purpose of preventing or improving premature ovarian failure.
  • the sewage oil extract of the present invention When used as a food additive, the sewage oil extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.
  • the mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • the sewage oil extract of the present invention in the preparation of food or beverage is added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw material.
  • the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
  • Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and includes all of the dietary supplements in the conventional sense.
  • the health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage.
  • Natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • sweetening agent natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used.
  • the proportion of the natural carbohydrate is generally about 0.01-0.20 g, preferably about 0.04-0.10 g per 100 ml of the composition of the present invention.
  • the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage.
  • the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.
  • the present invention provides a method for preventing or treating premature ovarian failure, infertility, infertility, or premenopausal menopausal disorders comprising administering a sewage extract to a subject.
  • the subject is a human or non-human mammal, and non-human mammals include, but are not limited to, mice, rats, dogs, cats, horses, cattle, sheep, goats, pigs, rabbits, and the like.
  • Scavenging activity for DPPH radicals was determined according to methods known in the art. More specifically, first, 50 ml of the sample was mixed with 1 ml of 1M DPPH solution and 450 ⁇ l of 50 mM Tris-HCl buffer (pH 7.4). The mixture was allowed to react at room temperature for 30 minutes, and then 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 (IC 50 ) showing 50% scavenging activity.
  • IC 50 concentration showing 50% scavenging activity.
  • the scavenging activity for peroxide anion was measured using NBT reduction method. More specifically, 10 ⁇ l of 30 mM EDTA (pH 7.4), 1 ⁇ l of 30 mM hypoxanthine and 200 ⁇ l of 1.42 mM NBT were added to 30 ⁇ l of the sample, followed by 3 minutes of reaction at room temperature, and then 10 ⁇ l of 1 U / ml xanthine oxidase was added. The total dose was adjusted to 300 ⁇ l with 50 mM phosphate buffer (pH 7.4). After incubating the reaction solution for 20 minutes at room temperature, the absorbance was measured at 560 nm, and the result was expressed in terms of NBT reduction IC 50 value by superoxide radical.
  • Cell viability was analyzed by 3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide (MTT) assay. After incubating 2 ⁇ 104 cells / well of CHO-K1 cells in a 48 well plate, serum starvation was performed for 4 hours, followed by various concentrations of VCD or pretreatment of each medicine at 100 ⁇ g / ml, and VCD (1.5 mM) was treated sequentially and incubated at 37 ° C., 5% CO 2 for 24 hours.
  • MTT 3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide
  • the cells were washed three times with PBS, RIPA buffer was added for 30 minutes at 4 °C and centrifuged at 12,000 ⁇ g for 30 minutes to collect the supernatant. After the same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to a PVDF membrane (membrane). The membrane was treated with blocking buffer (5% non-fat milk) for 1 hour and then washed with PBST solution containing 0.1% Tween 20 to block nonspecific binding of antibodies. The antibodies of each protein were treated overnight at 4 ° C., and the secondary antibody was HRP-linked anti-rabbit or anti-mouse, and developed using ECL chemiluminescence detection reagents, followed by image analysis system. Was observed.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • the experimental animals were purchased from 11-18 g B6C3F1-based mouse mice from the central experimental animals and used for experiments after being adapted to the laboratory environment with sufficient feed and water for one week.
  • the laboratory environment was maintained for 12 hours of day and night, maintaining the temperature of 22 ⁇ 2 °C until the end of the experiment.
  • the experimental diet was prepared by mixing the feed of sewage with 100 mg / kg / day or 300 mg / kg / day.
  • the control group was intraperitoneally injected with sesame oil, and the control group (VCD group) and the experimental group (EF group) were intraperitoneally injected with VCD in sesame oil at a concentration of 160 mg / kg / day.
  • the normal group and the control group were fed a normal diet for 4 weeks, and the experimental group was fed an experimental diet containing 100 mg / kg or 300 mg / kg of sewage extract for 4 weeks from one week before the VCD treatment.
  • the blood was collected from the mouse by a blood collection method, and the supernatant was collected by centrifugation at 3000 rpm for 15 minutes using a centrifuge (Beckman Coulter, Fullerton, Calif.).
  • the AMH content was measured using Mouse AMH (Anti-Mullerian Hormone) ELISA kit (Elabscience).
  • Antioxidant efficacy of the sewage oil, earth and sand, larvae, medicinal herbs, Mokhyang and Yubaekpi extract prepared in Example 1-1 was measured and compared with each other.
  • the DPPH radical scavenging activity of each extract was measured through the method described in Example 1-2. As a result, as shown in FIG. 2, EC 50 (effective concentration 50%) of the six medicinal extracts was confirmed.
  • the EC 50 value of the sesame oil extract was 93.1 ⁇ M, which was the lowest among the six herbs.
  • Example 4 i induced by VCD determine the protective effect of the extract ohsuyu for toxicity (ovotoxicity)
  • Example 1-1 After pretreatment of each of the sewage, sedimentary, larvae, medicinal herb, Mokyang, and milky skin extracts prepared in Example 1-1 at 100 ⁇ g / ml for 2 hours before treatment with 1.5 mM of VCD on CHO-K1 cells Screening was performed to evaluate the protective efficacy of the extracts from egg toxicity by VCD. As a result, as shown in Figure 5, it was confirmed that the sewage oil extract has the best protection effect from the VCD compared to the other five extracts.
  • CHO-K1 cells were treated with sewage extract alone at different concentrations, and the cytotoxicity of the sewage extract itself was evaluated through the MTT assay according to Examples 1-4. As a result, as shown in Figure 7, compared with the control, it was confirmed that up to 500 ⁇ g / ml does not affect the CHO-K1 cell viability.
  • Example 7 ohsuyu cell death (Apoptosis) inhibition induced by VCD according to extract check processing effect
  • Example 9 (in vivo) premature ovarian failure preventing effect of the extract using an animal model make ohsuyu
  • the VCD group was significantly decreased compared to the normal group, and the VCD + sewage-treated group increased significantly in a concentration-dependent manner compared to the VCD group.
  • the weight was also about the same as the normal group.
  • the sewage alone administration did not affect the weight. From the above results, it was found that the administration of sewage milk effectively protects the ovaries and the uterus from the weight loss of the ovaries and uterus due to VCD.
  • Anti-Mullerian Hormone is a hormone that is released only from the ovarian follicles and is a direct indicator of ovarian reserve.
  • the VCD group showed no significant difference compared to the normal group, but the AMH concentration showed a tendency to decrease, and the VCD + sewage-treated group In AMH concentration was confirmed to be similar to the normal group.
  • the sewage-only group did not affect the concentration of AMH. From the above results, it was found that the administration of sewage milk had an ovarian protective effect from the decrease of AMH concentration due to VCD.
  • the sesame oil extract according to the present invention increases the DPPH free radical scavenging activity in a concentration-dependent manner, and thus has excellent antioxidant activity, inhibits apoptosis of ovarian cells, and activates the PI3K / Akt signaling pathway to activate 4-vinylcyclohexene diepoxide ( It has a significant protective effect against egg toxicity induced by VCD) and can be used as a medicine and health functional food useful for improving, preventing, suppressing or treating egg toxicity and premature ovarian failure. Therefore, relevant applications include infertility, treatment or prevention of infertility, premature ovarian failure and premenopausal disorders.

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating ovotoxicity and, more specifically, to a pharmaceutical composition for preventing or treating ovotoxicity, containing an Evodia rutaecarpa Bentham extract. According to the present invention, the Evodia rutaecarpa Bentham extract concentration-dependently increases DPPH free radical scavenging activity so as to have excellent antioxidant activity, inhibits apoptosis of ovarian cells, and activates a PI3K/Akt signaling pathway so as to have a significant protective effect against ovotoxicity to be caused by 4-vinylcyclohexene diepoxide (VCD), thereby being usable as a medicine and a dietary supplement, which are useful for alleviating, preventing, inhibiting or treating ovotoxicity, premature ovarian failure and the like. Therefore, subfertility treatment or prevention, premature ovarian failure, a menopausal disorder in the premenopausal period, and the like can be considered application examples related thereto.

Description

난독성 보호 활성을 갖는 오수유 추출물을 포함하는 조기난소부전 예방 및 치료용 조성물Composition for preventing and treating premature ovarian dysfunction comprising sesame oil extract having protective effect on egg toxicity
본 발명은 난독성 예방 또는 치료용 조성물에 관한 것으로서, 보다 구체적으로는 오수유 추출물을 포함하는 난독성 예방 또는 치료용 조성물 또는 이를 포함하는 조기난소부전 예방 및 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating egg toxicity, and more particularly, to a composition for preventing or treating egg toxicity including sewage extract or a composition for preventing and treating premature ovarian failure including the same.
성의 생식노화는 자연적인 노화뿐만 아니라 다양한 환경적 요인에 의할 수 있다. 이러한 생식노화에 의한 난소의 기능부전이 40세 이전에 발생할 경우 조기난소부전이라 한다. Sexual reproductive aging may be due to various environmental factors as well as natural aging. Ovarian dysfunction caused by reproductive aging occurs before the age of 40 is called early ovarian failure.
조기난소부전은 여러 가지 요인에 의해 난소에 있는 난포들이 반응을 않거나 소멸되어 나타나는 임상적 상태이다. 조기난소부전은 40세 이전의 여성의 약 1%에서 발생하며, 1차성 무월경의 10~28%, 2차성 무월경의 4~18%가 조기난소부전으로 인해 발생하는 것으로 알려져 있다. 난소의 기능이 완전히 정지된 폐경과는 달리 5~10%에서 임신이 가능한 경우가 있으나 예후를 가늠하기는 불가하다. 이러한 조기 난소부전의 원인으로는 유전적 요인, 자가 면역에 의한 난소 손상이 원인이 되어 발생하기도 하고 항암치료, 방사선 치료, 난소 수술 등에 의한 의인성 원인이나 바이러스 감염, 혹은 약물, 흡연 등에 의한 환경적 요인을 병인으로 들고 있으나, 대부분의 환자에서 발생 기전과 원인을 규명하기 어려운 경우가 많다. 이러한 조기난소부전은 이른 폐경으로 말미암아 자연적인 폐경 경과의 여성에 비해 더욱 심각한 폐경관련 증상을 조기에 그리고 더욱 위중하게 호소할 수 있다. 이것은 폐경 이후의 여명 (life expectancy)을 고려할 때 정서적 안녕감과 성적 자아 훼손은 물론 심혈관계 질환 및 골다공증 등 건강관련 삶의 질 저하의 심각한 선행 원인이 될 수 있다. Premature ovarian failure is a clinical condition in which ovarian follicles do not respond or disappear due to various factors. Premature ovarian failure occurs in about 1% of women before age 40, with 10-28% of primary amenorrhea and 4-18% of secondary amenorrhea due to premature ovarian failure. Unlike menopause, in which the function of the ovary is completely stopped, pregnancy may be possible in 5-10%, but the prognosis cannot be estimated. The causes of early ovarian failure are caused by genetic factors, ovarian damage caused by autoimmunity, and due to chemotherapy, radiation therapy, ovarian surgery, etc. Although the factor is the etiology, it is often difficult to determine the mechanism and cause of occurrence in most patients. This early ovarian dysfunction can lead to early and more severe complaints of more serious menopause-related symptoms than women with natural menopause, due to early menopause. This can be a significant leading cause of poor health-related quality of life, including cardiovascular disease and osteoporosis, as well as emotional well-being and sexual self-impairment, given life expectancy after menopause.
한의학에서는 조기난소부전과 관련하여 "年未者而經水斷"으로 표현되지만 광범위하게는 폐경 (閉經)의 범주 안에서 찾아볼 수 있다. <소문 (素問)> 「상고천진론」에 '칠칠 임맥허 태충맥쇠소 천계갈 지도불통 고형괴이무자야'라고 하여 여성은 일반적으로 49세에 폐경이 되는데, 이는 자연스러운 노화과정으로 인한 것이며, 이와 달리 조기난소부전에 의해 초래되는 폐경은 「부청주남녀과」에서 '여자칠칠 천계절, 미급기년이선경단자'라 하여 폐경기에 달하기 전에 월경이 폐지 (閉止)되는 병적 상황을 말하였다. In Chinese medicine, it is expressed as "年 未 者 而 經 水 斷" in relation to premature ovarian failure, but it can be found in the scope of menopause. <Rumor> According to `` Shingo Tianjin theory '', `` chilchiim yigehe taechung changso cheonggyegal unrefined solid mass mujayaya '' women generally menopause at age 49, due to the natural aging process, In contrast, menopause caused by premature ovarian dysfunction was referred to as `` Women's Seven Seasons, Pre-Agemental Menstruation Terminal '' by the Bucheongju Women's and Men's Division, which refers to a pathological situation in which menstruation is abolished before reaching menopause.
한편, 4-vinylcyclohexene diepoxide (VCD)는 고무 타이어, 폴리에스테르, 플라스틱을 제조하는데 전형적으로 사용되어온 물질이다. 그런데 이러한 VCD는 생식노화와 관련된 환경적 요인의 화학물질로서 주목받아왔다. VCD는 자연 세포 사멸 과정을 가속화함으로써 선택적으로 ovarian small pre-antral (primordial and primary) follicles의 파괴를 유발한다. 또한 VCD는 백서 모델에서 선택적으로 원시 난포를 파괴하는 물질로 직접적으로 oocyte-associated c-kit receptor에 상호작용하여 인산화작용 (auto phosphorylation)을 억제하게 되어 난모세포의 생존능력을 손상시킨다. 이 과정에서 kit의 distribution 문제가 생기면서 하위 (downstream)에 있는 PI3K protein의 활성과 PI3K family인 Akt에도 영향을 주는 것으로 알려져 있다. 4-vinylcyclohexene diepoxide (VCD), on the other hand, is a material typically used to make rubber tires, polyesters and plastics. However, these VCDs have attracted attention as chemicals of environmental factors related to reproductive aging. VCD accelerates the process of natural cell death, thereby selectively destroying ovarian small pre-antral (primordial and primary) follicles. In addition, VCD is a material that selectively destroys primordial follicles in the white paper model, and directly interacts with oocyte-associated c-kit receptors, thereby inhibiting auto phosphorylation, impairing oocyte viability. This process is known to affect kit distribution, affecting downstream PI3K protein activity and PI3K family Akt.
따라서, 이러한 난독성, 보다 구체적으로 VCD에 의해 유도되는 난독성 및 이를 통한 조기난소부전을 치료하기 위한 치료제 물질을 개발하기 위하여 다양한 연구들이 진행되고 있고, 특히 인체에 무해하여 장기간 안전하게 복용할 수 있는 생약재에 대한 연구가 활발히 진행되고 있으나 (일본 공개특허 JP2005-298450), 아직 미비한 실정이다.Therefore, various studies have been conducted to develop a therapeutic agent for treating such ovarian toxicity, more specifically, ovarian toxicity induced by VCD and early ovarian failure. Research on herbal medicines is being actively conducted (Japanese Patent Laid-Open No. JP2005-298450), but it is still inadequate.
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로서, 본 발명자들은 조기난소부전에 효과적인 생약재 추출물 소재를 발굴하기 위해 연구 노력한 결과, 오수유 추출물이 4-vinylcyclohexene diepoxide (VCD)에 의해 유도되는 난독성에 유의적인 보호 효과가 있으며, 이에 따라 조기난소부전 예방 또는 치료 효과가 있음을 확인하고, 이에 기초하여 본 발명을 완성하게 되었다.The present invention has been made to solve the above-mentioned problems in the prior art, the present inventors have been researched to find an effective herbal extract material for early ovarian failure, as a result of the sewage extract is induced by 4-vinylcyclohexene diepoxide (VCD) There is a significant protective effect on the egg toxicity, thereby confirming the effect of preventing or treating premature ovarian failure, to complete the present invention based on this.
따라서, 본 발명의 목적은 오수유 추출물을 유효성분으로 포함하는 난독성 (ovotoxicity) 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating ovotoxicity comprising a sesame oil extract as an active ingredient.
본 발명의 다른 목적은 오수유 추출물을 유효성분으로 포함하는 조기난소부전 예방, 개선 또는 치료용 약학적 조성물 또는 건강기능식품조성물을 제공하는 것이다. It is another object of the present invention to provide a pharmaceutical composition or a health functional food composition for preventing, improving or treating premature ovarian failure, which comprises sewage oil extract as an active ingredient.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기 목적을 달성하기 위하여, 본 발명은 오수유 추출물을 유효성분으로 포함하는 난독성 (ovotoxicity) 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating ovotoxicity comprising a sewage oil extract as an active ingredient.
본 발명의 일 구현예로, 상기 난독성은 4-vinylcyclohexene diepoxide (VCD)에 의해 유도될 수 있다.In one embodiment of the present invention, the egg toxicity may be induced by 4-vinylcyclohexene diepoxide (VCD).
본 발명의 다른 구현예로, 상기 오수유 추출물은 난소 세포의 세포사멸 (apoptosis)을 억제할 수 있다.In another embodiment of the present invention, the sewage extract may inhibit apoptosis of ovarian cells.
본 발명의 또 다른 구현예로, 상기 오수유 추출물은 PI3K/Akt 신호전달 경로를 활성화시킬 수 있다.In another embodiment of the present invention, the sewage extract may activate the PI3K / Akt signaling pathway.
또한, 본 발명은 오수유 추출물을 유효성분으로 포함하는 조기난소부전 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating premature ovarian failure, comprising sewage extract as an active ingredient.
또한, 본 발명은 오수유 추출물을 유효성분으로 포함하는 조기난소부전 개선용 건강기능식품 조성물을 제공한다.The present invention also provides a health functional food composition for improving early ovarian dysfunction comprising the sesame oil extract as an active ingredient.
또한, 본 발명은 상기 조성물을 개체에 투여하는 단계를 포함하는 조기난소부전의 예방 또는 치료 방법을 제공한다.The present invention also provides a method for preventing or treating premature ovarian failure comprising administering the composition to a subject.
또한, 본 발명은 오수유 추출물의 조기난소부전 예방 또는 치료 용도를 제공한다.In addition, the present invention provides a use or prevention of premature ovarian failure of sewage extract.
본 발명에 따른 오수유 추출물은 농도 의존적으로 DPPH 자유 라디칼 소거 활성을 증가시키므로 항산화 활성이 우수하고, 난소 세포의 세포사멸 (apoptosis)을 억제하며, PI3K/Akt 신호전달 경로를 활성화시켜 4-vinylcyclohexene diepoxide (VCD)에 의해 유도되는 난독성에 대한 유의적인 보호효과를 가지는바, 난독성, 조기난소부전 등의 개선, 예방, 억제 또는 치료에 유용한 의약품 및 건강기능식품으로 사용될 수 있다. 따라서 관련 적용례로서 불임, 난임의 치료 혹은 예방, 조기난소부전 및 폐경 전 갱년기장애 등을 고려할 수 있다. The sesame oil extract according to the present invention increases the DPPH free radical scavenging activity in a concentration-dependent manner, and thus has excellent antioxidant activity, inhibits apoptosis of ovarian cells, and activates the PI3K / Akt signaling pathway to activate 4-vinylcyclohexene diepoxide ( It has a significant protective effect against egg toxicity induced by VCD) and can be used as a medicine and health functional food useful for improving, preventing, suppressing or treating egg toxicity and premature ovarian failure. Therefore, relevant applications include infertility, treatment or prevention of infertility, premature ovarian failure and premenopausal disorders.
도 1은 오수유 추출물을 제조하는 과정을 개략적으로 도시한 순서도이다.1 is a flow chart schematically showing a process for preparing a sewage extract.
도 2는 오수유 추출물과 5가지 약재 (토사자, 애엽, 산약, 목향 및 유백피) 추출물의 DPPH radical 소거활성을 비교한 결과이다.Figure 2 is a result of comparing the DPPH radical scavenging activity of the sesame oil extract and five medicinal herbs (soils, lobar, acid, Mokyang and Yubaekpi) extract.
도 3은 오수유 추출물과 5가지 약재 (토사자, 애엽, 산약, 목향 및 유백피) 추출물의 Superoxide anions 소거활성을 비교한 결과이다.Figure 3 is a result of comparing the superoxide anions scavenging activity of the sesame oil extract and five medicinal herbs (soil, larvae, pesticides, Mokyang and baekbaekpi) extract.
도 4는 CHO-K1 세포에 대한 VCD의 세포독성 확인한 결과이다.4 shows the results of confirming the cytotoxicity of VCD against CHO-K1 cells.
도 5는 VCD로 유도된 난독성 (ovotoxicity)에 대한 오수유 추출물의 보호 효과를 확인한 결과이다.5 is a result confirming the protective effect of the sewage extract on VCD induced ovotoxicity (ovotoxicity).
도 6은 VCD로 유도된 난독성 (ovotoxicity)에 대한 오수유 추출물의 농도별 보호 효과를 확인한 결과이다.Figure 6 is a result confirming the protective effect of the concentration of sewage extract for VCD induced ovotoxicity (ovotoxicity).
도 7은 CHO-K1 세포에 대한 오수유 추출물 단독의 세포독성을 확인한 결과이다.Figure 7 shows the results confirmed the cytotoxicity of sewage extract alone against CHO-K1 cells.
도 8은 VCD로 유도된 CHO-K1 세포의 세포사멸 (Apoptosis)에서 오수유 추출물이 미치는 영향을 나타낸 것이다.Figure 8 shows the effect of sewage extract on apoptosis of VHO induced CHO-K1 cells.
도 9 및 도 10은 오수유 추출물 처리에 따른 PI3K/Akt 신호경로 활성변화를 확인한 결과이다.9 and 10 are the results confirm the PI3K / Akt signal pathway activity changes according to the treatment of sewage milk extract.
도 11은 오수유 추출물의 난독성 예방 효과를 평가하기 위한 동물실험 모식도이다.11 is a schematic diagram of an animal experiment for evaluating the antitoxic effect of sesame oil extract.
도 12는 대조군 (CON), VCD, VCD+오수유 (EF), 및 오수유 (EF)의 처리에 따른 자궁 및 난소 조직의 육안 관찰 결과이다.12 is a visual observation result of uterine and ovarian tissue following treatment of the control group (CON), VCD, VCD + sewage (EF), and sewage (EF).
도 13은 오수유 추출물 처리에 따른 자궁 및 난소의 무게 지수를 나타내는 도이다.Figure 13 is a diagram showing the weight index of the uterus and ovary according to the sewage extract treatment.
도 14는 오수유 추출물 처리에 따른 난소의 무게 지수를 나타내는 도이다.Figure 14 is a diagram showing the weight index of the ovary according to the sewage extract treatment.
도 15는 오수유 추출물 처리에 따라 분비된 Serum antimullerian hormone (AMH)의 농도를 측정한 결과이다.Figure 15 is the result of measuring the concentration of Serum antimullerian hormone (AMH) secreted by sewage extract treatment.
도 16은 H&E 염색을 통한 난소 조직의 병리조직학적 분석 결과이다.16 shows the results of histopathological analysis of ovarian tissue through H & E staining.
본 발명은 오수유 추출물을 유효성분으로 포함하는 난독성 (ovotoxicity) 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention or treatment of ovotoxicity comprising a sewage extract as an active ingredient.
본 발명에서, "난독성 (ovotoxicity)"이란 화학적 원인에 의해 난소가 손상되는 것을 의미하며, 상기 손상은 난소의 기능이 소실되거나 난소 세포가 세포사멸 (apoptosis)하는 것을 모두 포함한다.In the present invention, "ovotoxicity" means that the ovary is damaged by a chemical cause, and the damage includes both loss of ovarian function and apoptosis of ovarian cells.
본 발명의 조성물에서 유효성분인 오수유 추출물은 통상적인 추출 방법에 의해 얻을 수 있고, 시판되는 것을 구입하여 사용할 수 있다. 통상적인 추출 방법은 열수 추출, 냉침 추출, 환류 추출, 초음파 추출 등을 포함할 수 있으나, 이에 한정되지 않는다.The sewage oil extract as an active ingredient in the composition of the present invention can be obtained by a conventional extraction method, and commercially available one can be purchased and used. Conventional extraction methods may include, but are not limited to, hot water extraction, cold needle extraction, reflux extraction, ultrasonic extraction, and the like.
본 발명에서는 오수유 추출물을 하기와 같은 방법으로 얻을 수 있다. 먼저, 오수유를 물로 깨끗이 세척하고 건조한 후 혼합하고 분쇄한다. 분쇄된 혼합물을 물, C1~C4의 알콜 또는 물과 C1~C4의 알콜의 혼합용매로 50~100 ℃에서 2~5 시간, 바람직하게는 4 시간 동안 환류추출한다. 이때, 용매의 부피는 분쇄한 오수유 중량의 1~10 배, 바람직하게는 5~8 배로 한다. 이후, 추출액을 여과하고 여과액을 농축한 후 농축액을 동결건조하여 분말 형태의 오수유 추출물을 얻는다.In the present invention, sewage extract may be obtained by the following method. First, the effluent is washed with water, dried, mixed and ground. The pulverized mixture is refluxed with water, C 1 to C 4 alcohol or a mixed solvent of water and C 1 to C 4 alcohol at 50 to 100 ° C. for 2 to 5 hours, preferably 4 hours. At this time, the volume of the solvent is 1 to 10 times, preferably 5 to 8 times the weight of the crushed sewage oil. Thereafter, the extract is filtered, the filtrate is concentrated, and the concentrate is lyophilized to obtain a sewage extract in powder form.
본 발명에서, 4-vinylcyclohexene diepoxide (VCD)는 자연 세포 사멸 과정을 가속화함으로써 선택적으로 ovarian small pre-antral (primordial and primary) follicles의 파괴를 유발하는 것으로 알려져 있는데, 본 발명자들은 이러한 VCD에 의해 유도되는 난독성에서의 오수유 추출물의 효과를 관찰하였다.In the present invention, 4-vinylcyclohexene diepoxide (VCD) is known to induce the destruction of ovarian small pre-antral (primordial and primary) follicles selectively by accelerating the process of natural cell death. The effect of sesame oil extract on egg toxicity was observed.
본 발명의 일 실시예에서는, 햄스터 난소 유래의 주화세포인 CHO-K1 세포에 오수유 추출물을 전처리한 후 VCD를 처리하여, VCD에 의한 난독성으로부터 상기 오수유 추출물의 보호 효능을 확인한 결과, 종래에 불임 치료에 사용되어온 약재 추출물들에 비하여 현저히 우수한 보호 효과가 있을 뿐만 아니라 (실시예 4 참조), VCD로 유도된 세포사멸 (Apoptosis)도 억제시킴을 확인하였다 (실시예 7 참조).In one embodiment of the present invention, pretreatment of sewage extract to CHO-K1 cells, which are hamster ovary-derived chemotactic cells, and then treated with VCD, confirming the protective effect of the sewage extract from egg toxicity caused by VCD. In addition to the remarkably excellent protective effect compared to the medicinal extracts that have been used for treatment (see Example 4), it was confirmed that also inhibits apoptosis induced by VCD (see Example 7).
본 발명의 다른 실시예에서는, 난독성 보호 효능의 기전을 알아보기 위해 PI3K/Akt 신호경로 활성화를 확인한 결과, 오수유 추출물이 PI3K/Akt의 활성을 증가시키는 것을 확인하였다 (실시예 8 참조).In another embodiment of the present invention, as a result of confirming the PI3K / Akt signal pathway activation in order to determine the mechanism of protective protection efficacy, it was confirmed that sewage extract increased the activity of PI3K / Akt (see Example 8).
따라서, 본 발명에 따른 오수유 추출물은 대표적인 난소 독성 물질인 VCD에 의한 생식세포 독성에 대한 우수한 보호효과를 나타내는바, 조기난소부전, 불임, 난임, 폐경 전 갱년기장애 등의 개선, 예방, 억제 또는 치료에 유용한 의약품 및 건강기능식품으로 사용될 수 있다.Therefore, the sewage oil extract according to the present invention shows an excellent protective effect on germ cell toxicity by VCD, which is a representative ovarian toxic substance, improving, preventing, suppressing or treating premature ovarian failure, infertility, infertility, premenopausal disorders, etc. It can be used as a useful medicine and health food.
이에, 본 발명의 다른 양태로서, 본 발명은 오수유 추출물을 유효성분으로 포함하는 조기난소부전 예방 또는 치료용 약학적 조성물을 제공한다.Accordingly, in another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating premature ovarian failure, comprising sewage extract as an active ingredient.
본 발명에서 사용되는 용어, "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 조기난소부전을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" means any action that inhibits early ovarian failure or delays the onset by administration of a pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어, "치료"란 본 발명에 따른 약학적 조성물의 투여에 의해 조기난소부전에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. As used herein, the term "treatment" means any action that improves or advantageously changes the symptoms of premature ovarian failure by administration of the pharmaceutical composition according to the present invention.
본 발명에서 대상으로 하는 질병인 "조기난소부전"은 정상적인 발달을 보인 여성에게서 40세 이전에 여러 가지 요인에 의해 난소의 기능이 정지된 것을 의미하며, 난포의 고갈이나 난포의 기능부진도 포함된다."Early ovarian dysfunction", a disease targeted in the present invention, means that ovarian function is stopped by various factors before the age of 40 in women with normal development, and includes depletion of follicles or dysfunction of follicles. .
본 발명의 약학적 조성물은 오수유 추출물과 함께 난독성 또는 조기난소부전 치료 효과를 갖는 공지의 유효성분을 1종 이상 더 함유할 수 있다.The pharmaceutical composition of the present invention may further contain at least one known active ingredient having a therapeutic effect of refractoriness or premature ovarian failure along with sesame oil extract.
본 발명의 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 또한 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of conventional formulations, external preparations, suppositories, and sterile injectable solutions.
상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등이 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical compositions of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is determined by the condition and weight of the patient, Depending on the extent, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명의 약학적 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에서 활성 성분의 흡수도, 불활성율 및 배설속도, 질병종류, 병용되는 약물에 따라 달라질 수 있으며, 일반적으로는 체중 1㎏ 당 0.0001 내지 150 mg, 바람직하게는 0.001 내지 100 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general May be administered 0.0001 to 150 mg, preferably 0.001 to 100 mg daily or every other day or divided into 1 to 3 times a day per kg body weight. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
또한, 본 발명의 또 다른 양태로서, 본 발명은 오수유 추출물을 유효성분으로 포함하는 조기난소부전 개선용 건강기능식품 조성물을 제공한다.In addition, as another aspect of the present invention, the present invention provides a health functional food composition for improving premature ovarian failure comprising sewage extract as an active ingredient.
본 발명에서 사용되는 용어 "개선"이란 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다. As used herein, the term "improvement" refers to any action that at least reduces the parameters associated with the condition being treated, such as the extent of symptoms.
본 발명의 조성물은 조기난소부전의 예방 또는 개선을 목적으로 건강기능식품에 첨가될 수 있다. 본 발명의 오수유 추출물을 식품 첨가물로 사용할 경우, 상기 오수유 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 오수유 추출물은 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The composition of the present invention may be added to a dietary supplement for the purpose of preventing or improving premature ovarian failure. When the sewage oil extract of the present invention is used as a food additive, the sewage oil extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, the sewage oil extract of the present invention in the preparation of food or beverage is added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw material. However, in the case of long-term intake for health and hygiene or for health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and includes all of the dietary supplements in the conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당 및 과당과 같은 모노사카라이드, 말토오스 및 수크로오스와 같은 디사카라이드, 덱스트린 및 시클로덱스트린과 같은 폴리사카라이드, 및 자일리톨, 소르비톨 및 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100㎖당 일반적으로 약 0.01~0.20g, 바람직하게는 약 0.04~0.10g 이다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. Natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, preferably about 0.04-0.10 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01~0.20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.
또한, 본 발명의 다른 양태로서, 본 발명은 오수유 추출물을 개체에게 투여하는 단계를 포함하는 조기난소부전, 불임, 난임, 또는 폐경 전 갱년기장애의 예방 또는 치료 방법을 제공한다. 상기 개체는 인간 또는 비-인간을 포함하는 포유류이며, 비-인간 포유류는 마우스, 랫트, 개, 고양이, 말, 소, 양, 염소, 돼지, 토끼 등을 포함하나 이에 한정되지 않는다.In another aspect of the present invention, the present invention provides a method for preventing or treating premature ovarian failure, infertility, infertility, or premenopausal menopausal disorders comprising administering a sewage extract to a subject. The subject is a human or non-human mammal, and non-human mammals include, but are not limited to, mice, rats, dogs, cats, horses, cattle, sheep, goats, pigs, rabbits, and the like.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예]EXAMPLE
실시예 1. 실험 준비 및 실험 방법Example 1 Experiment Preparation and Experimental Method
1-1. 약재 추출물 제조1-1. Medicinal Herb Extract Manufacturer
오수유와 종래에 불임 치료에 사용되어 온 토사자, 애엽, 산약, 목향, 유백피를 실험에 사용하였고, 상기 약재는 (주)옴니허브의 한약재를 구입하여 선별하고 정선한 것을 사용하였다. 오수유, 토사자, 애엽, 산약, 목향, 유백피 각 100 g에 증류수 800 ml를 가한 뒤, 4 시간 동안 가열 환류 추출하였다. 추출물을 상온으로 식힌 뒤에 8 μm pore size filter paper로 두 번 filtering한 후, 걸러진 용액은 감압 농축 및 동결 건조하고, 이를 분말화하여 하기 표 1과 같이 각 약재의 추출물을 얻었다 (도 1 참조).Earth and sand, sesame, medicinal herb, Mokyang, and Yubaekpi, which have been used for treating infertility and conventional infertility, were used in the experiments. The medicinal herbs were selected and selected by purchasing herbal medicines of Omnihub Co., Ltd. 800 ml of distilled water was added to 100 g of sewage oil, earth and sand, young leaves, powder, moth, and milky skin, and the mixture was heated and refluxed for 4 hours. After the extract was cooled to room temperature and filtered twice with 8 μm pore size filter paper, the filtered solution was concentrated under reduced pressure and lyophilized, and powdered to obtain an extract of each medicinal herb as shown in Table 1 below (see FIG. 1).
Herbal nameHerbal name 오수유Osuyu 토사자Tosa 애엽Love 산약Medicine 목향elecampane 유백피Milky skin
Latin nameLatin name Evodiae Fructus(EF) Evodiae Fructus (EF) Cuscutae Semen (CS) Cuscutae Semen (CS) Artemisiae Argyi Folium(AAF) Artemisiae Argyi Folium (AAF) Dioscoreae Rhizoma (DR) Dioscoreae Rhizoma (DR) Aucklandiae Radix (AR) Aucklandiae Radix (AR) Ulmi cortex(UC) Ulmi cortex (UC)
Yield(g)Yield (g) 16.5/10016.5 / 100 15.7/10015.7 / 100 8.5/608.5 / 60 8.4/1008.4 / 100 53.4/10053.4 / 100 10.9/10010.9 / 100
Yield(%))Yield (%)) 16.516.5 15.715.7 14.214.2 8.48.4 53.453.4 10.910.9
1-2. DPPH 라디칼 (radical) 소거 활성 측정1-2. DPPH radical scavenging activity measurement
DPPH 라디칼에 대한 소거 활성은 종래에 공지된 방법에 따라 측정하였다. 보다 구체적으로, 먼저 시료 50 μl에 1M DPPH 용액 1 ml 및 50 mM Tris-HCl buffer (pH 7.4) 450 μl를 가하여 혼합하였다. 혼합물을 실온에서 30 분간 반응시킨 다음, microplate reader (VersaMax, Molecular Devices, USA)를 이용하여 파장 517 nm에서 흡광도를 측정하였다. DPPH 라디칼의 소거 활성은 50 %의 소거능을 보이는 농도 (IC50)로 표시하였다.Scavenging activity for DPPH radicals was determined according to methods known in the art. More specifically, first, 50 ml of the sample was mixed with 1 ml of 1M DPPH solution and 450 µl of 50 mM Tris-HCl buffer (pH 7.4). The mixture was allowed to react at room temperature for 30 minutes, and then 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 (IC 50 ) showing 50% scavenging activity.
1-3. 과산화물 음이온 (Superoxide anions) 소거 활성 측정1-3. Determination of Superoxide Anions Scavenging Activity
항산화 활성의 또 다른 지표로서 대표적인 활성산소종의 하나인, 과산화물 음이온에 대한 소거 활성을 NBT 환원법을 이용하여 측정하였다. 보다 구체적으로, 시료 30 μl에 30 mM EDTA (pH 7.4) 10 μl, 30 mM hypoxanthine 1 μl, 1.42 mM NBT 200 μl를 가한 다음 실온에서 3분 반응시킨 후에, 1 U/ml xanthine oxidase 10 μl를 첨가하고 50 mM phosphate buffer (pH 7.4)로 총 용량을 300 μl로 맞췄다. 반응용액을 실온에서 20 분간 배양시킨 후, 560 nm 파장에서 흡광도를 측정하였고, 결과는 과산화물 라디칼 (superoxide radical)에 의한 NBT reduction IC50 값으로 환산하여 표시하였다.As another indicator of antioxidant activity, the scavenging activity for peroxide anion, one of the representative reactive oxygen species, was measured using NBT reduction method. More specifically, 10 μl of 30 mM EDTA (pH 7.4), 1 μl of 30 mM hypoxanthine and 200 μl of 1.42 mM NBT were added to 30 μl of the sample, followed by 3 minutes of reaction at room temperature, and then 10 μl of 1 U / ml xanthine oxidase was added. The total dose was adjusted to 300 μl with 50 mM phosphate buffer (pH 7.4). After incubating the reaction solution for 20 minutes at room temperature, the absorbance was measured at 560 nm, and the result was expressed in terms of NBT reduction IC 50 value by superoxide radical.
1-4. MTT assay1-4. MTT assay
세포 생존률은 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay 방법으로 분석하였다. 48 well plate에 2×10⁴ cells/well의 CHO-K1 세포를 배양한 후 4시간 동안 혈청 고갈 (serum starvation) 후, VCD의 다양한 농도 처리 또는 각 약재들을 100 μg/ml로 전처리하고, VCD (1.5 mM)를 순차적으로 처리하여 37℃, 5% CO2의 조건에서 24 시간 동안 배양하였다. 웰 당 30 μl의 MTT solution (2 mg/ml)을 첨가하여 37℃, 5% CO2 배양기에서 3 시간 동안 반응시킨 후, MTT 용액과 배양액을 완전히 제거 후 150 μl dimethyl sulfoxide (DMSO, Sigma-Aldrich, USA)로 세포 내에 형성된 formazan 결정체를 용해하여 ELISA plate reader (DYNEX, Opsys MR,USA)로 595 nm에서 흡광도를 측정하였다. 대조군에 대한 세포생존율을 백분율로 표시하였다.Cell viability was analyzed by 3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide (MTT) assay. After incubating 2 × 10⁴ cells / well of CHO-K1 cells in a 48 well plate, serum starvation was performed for 4 hours, followed by various concentrations of VCD or pretreatment of each medicine at 100 μg / ml, and VCD (1.5 mM) was treated sequentially and incubated at 37 ° C., 5% CO 2 for 24 hours. After adding 30 μl of MTT solution (2 mg / ml) per well for 3 hours at 37 ° C., 5% CO 2 incubator, completely removing MTT solution and culture medium, and then 150 μl dimethyl sulfoxide (DMSO, Sigma-Aldrich). , USA) and the formazan crystals formed in the cells were dissolved and absorbance was measured at 595 nm with an ELISA plate reader (DYNEX, Opsys MR, USA). Cell viability relative to the control is expressed as a percentage.
1-5. 웨스턴 블롯 (Western blot)1-5. Western blot
전기영동을 위한 단백질의 추출을 위하여, 세포를 PBS로 3회 세척한 후, RIPA buffer를 넣어 4 ℃에서 30 분간 반응시키고 12,000 × g에서 30 분간 원심분리하여 상층액을 모았다. 동량의 단백질을 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)로 분리시킨 후, 단백질을 PVDF 멤브레인 (membrane)에 트랜스퍼 (transfer)하였다. 이 멤브레인을 항체의 비특이적 결합을 차단하기 위하여 blocking buffer (5% non-fat milk)를 1 시간 처리 한 후, 0.1 % Tween 20을 함유한 PBST 용액으로 세척하였다. 각각의 보고자 하는 단백질의 항체를 사용하여 4 ℃에서 밤새 처리하였으며, 2차 항체는 HRP-linked anti-rabbit 혹은 anti-mouse를 사용하였고, ECL chemiluminescence detection reagents를 이용하여 발색한 후 Image analyzing system을 사용하여 관찰하였다.In order to extract the protein for electrophoresis, the cells were washed three times with PBS, RIPA buffer was added for 30 minutes at 4 ℃ and centrifuged at 12,000 × g for 30 minutes to collect the supernatant. After the same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to a PVDF membrane (membrane). The membrane was treated with blocking buffer (5% non-fat milk) for 1 hour and then washed with PBST solution containing 0.1% Tween 20 to block nonspecific binding of antibodies. The antibodies of each protein were treated overnight at 4 ° C., and the secondary antibody was HRP-linked anti-rabbit or anti-mouse, and developed using ECL chemiluminescence detection reagents, followed by image analysis system. Was observed.
1-6. 실험 동물1-6. Experimental animals
실험 동물은 11~18 g의 B6C3F1계 암컷 마우스를 중앙실험동물로부터 구입하여 1주일 동안 사료와 물을 충분히 공급하면서 실험실 환경에 적응시킨 후 실험에 사용하였다. 실험실 환경은 온도 22±2 ℃를 유지하면서 12 시간 단위로 낮과 밤이 계속되는 상황을 실험 종료 시까지 유지하였다.The experimental animals were purchased from 11-18 g B6C3F1-based mouse mice from the central experimental animals and used for experiments after being adapted to the laboratory environment with sufficient feed and water for one week. The laboratory environment was maintained for 12 hours of day and night, maintaining the temperature of 22 ± 2 ℃ until the end of the experiment.
1-7. 사료 제조1-7. Feed manufacturing
마우스 한 마리가 하루에 섭취하는 사료량을 5g으로 가정하여 오수유 추출물의 섭취 농도를 100mg/kg/day 또는 300mg/kg/day로 하여 사료에 섞어 실험 식이를 제조하였다 (피드랩, 한국). On the assumption that the amount of feed consumed by one mouse per day was 5 g, the experimental diet was prepared by mixing the feed of sewage with 100 mg / kg / day or 300 mg / kg / day.
1-8. 조기난소부전 유발1-8. Premature ovarian failure
VCD를 sesame oil (Sigma, USA)에 녹인 후, 160 mg/kg/day의 농도로 15 일 동안 매일 1회 복강주사를 하여 조기난소부전을 유발시켰고, 정상군은 동일한 양의 sesame oil을 복강주사 하였다. After dissolving VCD in sesame oil (Sigma, USA), intraperitoneal injection was performed once daily for 15 days at 160 mg / kg / day to induce premature ovarian failure, and the normal group received the same amount of sesame oil intraperitoneally. It was.
1-9. 실험군의 선정1-9. Selection of Experiment Group
정상군 (control group)은 sesame oil을 복강주사 하고, 대조군 (VCD group) 및 실험군 (EF group)은 VCD를 sesame oil에 160 mg/kg/day의 농도로 복강주사 하였다. 정상군과 대조군은 4 주간 정상식이를 섭취시켰고, 실험군은 VCD 처리 일주일 전부터 4 주간 100mg/kg 또는 300mg/kg 농도의 오수유 추출물을 함유한 실험 식이를 섭취시켰다.The control group was intraperitoneally injected with sesame oil, and the control group (VCD group) and the experimental group (EF group) were intraperitoneally injected with VCD in sesame oil at a concentration of 160 mg / kg / day. The normal group and the control group were fed a normal diet for 4 weeks, and the experimental group was fed an experimental diet containing 100 mg / kg or 300 mg / kg of sewage extract for 4 weeks from one week before the VCD treatment.
1-10. 체중 및 자궁과 난소 무게 측정1-10. Body weight and uterus and ovary weight measurement
실험 시작일 (day 1)에 측정한 체중을 기준으로, 매주 체중 변화를 관찰하였다. 실험 마지막 날 마우스를 희생시킨 후, 자궁 및 난소 전체를 적출하여 무게를 측정하였고, 한쪽 난소만 따로 떼어 난소 무게를 측정하여 희생 직전의 체중에 대한 비율로 결과를 처리하였다. Based on the weight measured on the day of the experiment (day 1), the weight change was observed every week. After the sacrifice of the mouse on the last day of the experiment, the uterus and the whole ovary was removed and weighed, and only one ovary was separated to measure the weight of the ovary, and the results were treated as a ratio to the weight immediately before sacrifice.
1-11. 병리조직학적 관찰1-11. Histopathological observation
한쪽 난소 및 자궁은 10 % 포르말린 (formalin)에 고정하여 파라핀 포매하고 5 μm의 박절편을 만들어, Hematoxylin-Eoxin (H&E) 염색하여 광학현미경하에서 일반적인 병리조직학적 소견을 관찰하고, 분석하였다.One ovary and uterus were fixed in 10% formalin, embedded with paraffin, 5 μm slices were made, and stained with Hematoxylin-Eoxin (H & E) to observe general pathological findings under an optical microscope.
1-12. 혈중 AMH 함량 측정1-12. Determination of AMH content in blood
실험 마지막 날 마우스로부터 심장 채혈 방법으로 혈액을 얻어 원심분리기 (Beckman Coulter, Fullerton, CA)를 이용하여, 3000 rpm에서 15 분간 원심분리하여 상층액을 취하였다. 얻어진 혈청에서 Mouse AMH (Anti-Mullerian Hormone) ELISA kit (Elabscience)를 이용하여 AMH 함량을 측정하였다.On the last day of the experiment, the blood was collected from the mouse by a blood collection method, and the supernatant was collected by centrifugation at 3000 rpm for 15 minutes using a centrifuge (Beckman Coulter, Fullerton, Calif.). In the obtained serum, the AMH content was measured using Mouse AMH (Anti-Mullerian Hormone) ELISA kit (Elabscience).
실시예 2. 오수유 추출물의 항산화 효능 확인Example 2. Confirmation of Antioxidant Efficacy of Sewage Extract
상기 실시예 1-1을 통해 제조된 오수유, 토사자, 애엽, 산약, 목향 및 유백피 추출물의 항산화 효능을 측정하여 서로 비교하였다.Antioxidant efficacy of the sewage oil, earth and sand, larvae, medicinal herbs, Mokhyang and Yubaekpi extract prepared in Example 1-1 was measured and compared with each other.
먼저, 상기 실시예 1-2에 기재된 방법을 통해 각 추출물의 DPPH 라디칼 소거 활성을 측정하였고, 그 결과, 도 2에 나타낸 바와 같이, 6가지 약재 추출물의 EC50 (effective concentration 50%)을 확인하였을 때 오수유 추출물의 EC50 값이 93.1 μM로 6가지 약재 중 가장 낮게 나타났다.First, the DPPH radical scavenging activity of each extract was measured through the method described in Example 1-2. As a result, as shown in FIG. 2, EC 50 (effective concentration 50%) of the six medicinal extracts was confirmed. The EC 50 value of the sesame oil extract was 93.1 μM, which was the lowest among the six herbs.
다음으로, 상기 실시예 1-3에 기재된 방법을 통해 각 추출물의 과산화물 음이온 소거 활성을 측정하였고, 그 결과, 도 3에 나타낸 바와 같이, DPPH 라디칼 소거 활성 결과와 마찬가지로 오수유 추출물의 EC50이 가장 낮음을 확인할 수 있었다.Next, the peroxide anion scavenging activity of each extract was measured by the method described in Examples 1-3, and as a result, as shown in FIG. 3, the EC 50 of the sewage extract was the lowest as in the DPPH radical scavenging activity. Could confirm.
실시예 3. CHO-K1 세포에 대한 VCD 독성 확인Example 3. Confirmation of VCD Toxicity on CHO-K1 Cells
CHO-K1 세포에 대한 VCD의 세포독성을 상기 실시예 1-4에 따른 MTT assay로 평가하였으며, 그 결과, VCD에 의해 CHO-K1 세포의 생존율이 약 50%가 되는 농도 (1.5 mM)를 결정하고 (도 4 참조), 이를 하기 실험에 적용하였다.Cytotoxicity of VCD against CHO-K1 cells was evaluated by the MTT assay according to Examples 1-4 above, and as a result, the concentration (1.5 mM) at which the survival rate of CHO-K1 cells was about 50% was determined by VCD. (See FIG. 4), which was applied to the following experiment.
실시예 4. VCD로 유도된 난독성 ( ovotoxicity )에 대한 오수유 추출물의 보호 효과 확인 Example 4 i induced by VCD determine the protective effect of the extract ohsuyu for toxicity (ovotoxicity)
CHO-K1 세포에 VCD 1.5 mM을 처리하기 전 2시간 동안 상기 실시예 1-1을 통해 제조된 오수유, 토사자, 애엽, 산약, 목향 및 유백피 추출물 각각을 100 μg/ml의 농도로 전처리 한 후, VCD에 의한 난독성으로부터 상기 추출물들의 보호 효능을 평가하는 스크리닝을 실시하였다. 그 결과, 도 5에 나타낸 바와 같이, 다른 5가지의 추출물에 비하여 오수유 추출물이 VCD로부터 보호 효능이 가장 우수함을 확인할 수 있었다.After pretreatment of each of the sewage, sedimentary, larvae, medicinal herb, Mokyang, and milky skin extracts prepared in Example 1-1 at 100 μg / ml for 2 hours before treatment with 1.5 mM of VCD on CHO-K1 cells Screening was performed to evaluate the protective efficacy of the extracts from egg toxicity by VCD. As a result, as shown in Figure 5, it was confirmed that the sewage oil extract has the best protection effect from the VCD compared to the other five extracts.
실시예 5. VCD로 유도된 난독성 ( ovotoxicity )에 대한 오수유 추출물의 농도 별 보호 효과 확인 Example 5. confirmation concentrations protective effect of the extract on the ohsuyu I toxicity (ovotoxicity) guided to the VCD
CHO-K1 세포에 오수유 추출물을 다양한 농도 (5, 10, 50, 100, 300, 500 μg/ml)로 2 시간 전처리하고, VCD 1.5 mM로 난독성을 유발시킨 후, 난독성에 대한 보호효과를 확인하였다. 그 결과, 도 6에 나타낸 바와 같이, 오수유 추출물은 농도의존적으로 난독성에 대한 보호 효능을 보였으며, 100 μg/ml 이상의 농도에서 VCD를 처리하지 않은 대조군과 거의 유사한 생존율을 보임을 확인할 수 있었다.Pretreatment with sewage oil extract at various concentrations (5, 10, 50, 100, 300, 500 μg / ml) in CHO-K1 cells for 2 hours, induces egg toxicity with 1.5 mM of VCD, and then protects against egg toxicity. Confirmed. As a result, as shown in Figure 6, the sewage oil extract showed a protective effect against egg toxicity in a concentration-dependent manner, and showed a survival rate similar to that of the control group not treated with VCD at a concentration of 100 μg / ml or more.
실시예 6. CHO-K1 세포에 대한 오수유 추출물 단독의 세포독성 측정Example 6 Cytotoxicity Measurements of Sewage Extracts Alone on CHO-K1 Cells
CHO-K1 세포에 오수유 추출물을 농도별로 단독 처리한 후 상기 실시예 1-4에 따른 MTT assay을 통해 오수유 추출물 자체의 세포독성을 평가하였다. 그 결과, 도 7에 나타낸 바와 같이, 대조군과 비교할 때, 500 μg/ml까지는 CHO-K1 세포 생존율에 영향을 미치지 않음을 확인하였다. CHO-K1 cells were treated with sewage extract alone at different concentrations, and the cytotoxicity of the sewage extract itself was evaluated through the MTT assay according to Examples 1-4. As a result, as shown in Figure 7, compared with the control, it was confirmed that up to 500 μg / ml does not affect the CHO-K1 cell viability.
실시예 7. 오수유 추출물 처리에 따른 VCD로 유도된 세포사멸 ( Apoptosis ) 억제 효과 확인 Example 7. ohsuyu cell death (Apoptosis) inhibition induced by VCD according to extract check processing effect
CHO-K1 세포에서 VCD 1.5 mM을 처리했을 때, 세포사멸 (Apoptosis)이 일어나고, 오수유 추출물 (100 μg/ml)의 전처리로 VCD로 인한 세포사멸이 억제되는지 여부를 확인하기 위해, 오수유 추출물 처리에 따른 Apoptosis-related protein (PARP, caspase-3)의 발현 변화를 상기 실시예 1-5에 따른 웨스턴 블롯을 통해 확인하였다.When treated with 1.5 mM of VCD in CHO-K1 cells, apoptosis occurs, and pretreatment with sewage extract (100 μg / ml) is used to determine whether apoptosis due to VCD is inhibited. Expression changes of the apoptosis-related protein (PARP, caspase-3) were confirmed by Western blot according to Example 1-5.
그 결과, 도 8에 나타낸 바와 같이, 대조군과 비교할 때, VCD를 처리한 경우, PARP cleavage가 일어나 band가 2개로 나타나고, 프로폼 (proform)의 caspase-3의 경우, band 강도 (intensity)가 감소한 바, 세포사멸이 유도된 것을 확인할 수 있었다. 반면, 오수유 추출물을 전처리하고 VCD를 처리한 경우, PARP band가 2개로 분리되지 않았고, caspase-3의 경우도 VCD 단독 처리보다 감소하지 않음을 확인할 수 있었다. 상기 결과로부터, 오수유 추출물이 VCD로 유도된 세포사멸 즉, 난독성으로부터 세포 보호 효과가 있음을 알 수 있었다.As a result, as shown in Figure 8, compared with the control group, when treated with VCD, PARP cleavage occurs and appears as two bands, in the case of proform caspase-3, the band intensity (intensity) is reduced It was confirmed that apoptosis was induced. On the other hand, pretreatment with sewage extract and treatment with VCD did not separate PARP bands into two, and caspase-3 did not decrease compared to treatment with VCD alone. From the above results, it was found that sewage extract had a protective effect against VCD-induced cell death, that is, egg toxicity.
실시예 8. 오수유 추출물의 난독성 (ovotoxicity) 보호 효능의 기전 확인Example 8 Confirmation of Mechanism of Ovotoxicity Protective Efficacy of Fructus Extracts
오수유 추출물의 난독성 보호 효능의 기전을 알아보기 위해, 오수유 추출물 처리에 따른 VCD의 독성 기전으로 알려져 있는 PI3K/Akt 신호전달경로에서의 변화를 상기 실시예 1-5에 따른 웨스턴 블롯을 통해 확인하였다.In order to investigate the mechanism of the protective effect of the toxic oil of the sesame oil extract, the change in the PI3K / Akt signaling pathway known as the virulence mechanism of the VCD according to the treatment of the sesame oil extract was confirmed through the Western blot according to Example 1-5. .
그 결과, 도 9 및 도 10에 나타낸 바와 같이, PI3K/Akt 신호전달의 주요한 키나아제 (kinase)인 Akt, mTOR, 및 GSK-3β의 발현이 오수유 추출물 (100 μg/ml)을 처리한 시간-의존적으로 하위 시그널 (signal) 쪽으로 활성화되는 것을 확인하였다. 즉, 오수유 추출물 처리에 의해 전체적으로 PI3K/Akt 신호 경로를 순차적으로 활성화 시킨다는 것을 확인할 수 있었다. 상기 결과로부터, 오수유 추출물이 PI3K/Akt 신호전달경로의 활성화를 통하여 VCD로부터 세포 보호 효능을 가진다는 것을 알 수 있었다.As a result, as shown in FIGS. 9 and 10, the expression of Akt, mTOR, and GSK-3β, the major kinases for PI3K / Akt signaling, was time-dependent when treated with sewage extract (100 μg / ml). We confirmed that it is activated towards the lower signal side. In other words, it was confirmed that the scavenger extract treatment sequentially activated the PI3K / Akt signaling pathway as a whole. From the above results, it was found that sewage extract had a protective effect against VCD through activation of PI3K / Akt signaling pathway.
실시예 9. ( in vivo ) 동물모델을 이용한 오수유 추출물의 조기난소부전 예방 효과 확인 Example 9. (in vivo) premature ovarian failure preventing effect of the extract using an animal model make ohsuyu
상기 실시예들의 결과에 기초하여, 오수유 추출물의 조기난소부전 예방 효과를 실시예 1-6 내지 1-12의 방법으로 동물모델을 이용하여 확인하였으며, 상기 실험의 모식도를 도 11에 나타내었다.On the basis of the results of the above examples, the early ovarian failure prevention effect of the sewage oil extract was confirmed using the animal model in the method of Examples 1-6 to 1-12, and a schematic diagram of the experiment is shown in FIG.
9-1. 자궁 및 난소 조직의 육안 관찰9-1. Visual observation of the uterus and ovarian tissue
자궁 및 난소 조직을 육안으로 관찰한 결과, 도 12에 나타낸 바와 같이, 정상군은 난소 및 자궁 조직 모두 정상적인 크기를 유지하고 있는 반면, VCD군은 현저히 위축되어 있는 것을 관찰할 수 있었다. 또한, VCD+오수유 투여군은 정상군과 비슷한 정도의 크기가 관찰되었으며, 이는 오수유 단독 투여군에서도 마찬가지로 특이적인 육안적 소견은 발견되지 않았다. As a result of visual observation of the uterus and ovarian tissue, as shown in FIG. 12, it was observed that the normal group maintained normal size of both the ovary and uterine tissue, while the VCD group was significantly contracted. In addition, the VCD + sewage-treated group was observed to have a similar size to that of the normal group, and no specific gross findings were found in the sewage-treated group alone.
9-2. 난소 및 자궁 전체의 무게와 난소 무게 비교를 통한 난소/자궁 보호 효과 확인 9-2. Confirmation of ovarian / uterine protection by comparing the weight of the ovary and entire uterus
난소 및 자궁 전체의 무게와 난소 무게를 비교한 결과, 도 13 및 도 14에 나타낸 바와 같이, VCD군이 정상군에 비해서 유의성있게 감소하였으며, VCD+오수유 투여군은 VCD군에 비해서 유의성있게 농도 의존적으로 증가되었고, 무게 또한 정상군과 거의 비슷함을 알 수 있었다. 반면, 오수유 단독 투여군에서는 무게에 영향을 미치지 않음을 확인하였다. 상기 결과로부터, 오수유의 투여는 VCD로 인한 난소 및 자궁의 무게 감소로부터 난소와 자궁을 효과적으로 보호한다는 것을 알 수 있었다.As a result of comparing the weight of the ovary and the entire uterus with the weight of the ovary, as shown in Figs. 13 and 14, the VCD group was significantly decreased compared to the normal group, and the VCD + sewage-treated group increased significantly in a concentration-dependent manner compared to the VCD group. The weight was also about the same as the normal group. On the other hand, it was confirmed that the sewage alone administration did not affect the weight. From the above results, it was found that the administration of sewage milk effectively protects the ovaries and the uterus from the weight loss of the ovaries and uterus due to VCD.
9-3. 혈중 AMH 함량 측정을 통한 난소 보호 효과 확인9-3. Confirmation of ovarian protection by measuring blood AMH content
항뮬러관 호르몬 (Anti-Mullerian Hormone, AMH)은 난소의 난포에서만 분비되는 호르몬으로 난소예비력 (ovarian reserve)을 잘 반영하는 직접적인 평가지표이다. Anti-Mullerian Hormone (AMH) is a hormone that is released only from the ovarian follicles and is a direct indicator of ovarian reserve.
이에, 마우스의 혈청에서 ELISA kit을 이용하여 혈중 AMH 농도를 측정한 결과, 도 15에 나타낸 바와 같이, 정상군에 비해 VCD군은 유의성을 나타내지 않았지만, AMH 농도가 감소하는 경향을 보였고, VCD+오수유 투여군에서는 AMH 농도가 정상군과 유사한 것을 확인할 수 있었다. 또한 오수유 단독 투여군은 AMH의 농도에 영향을 미치지 않음을 알 수 있었다. 상기 결과로부터, 오수유의 투여가 VCD로 인한 AMH 농도 감소로부터 난소 보호 효과가 있음을 알 수 있었다.Accordingly, as a result of measuring the AMH concentration in the serum of the mouse using the ELISA kit, as shown in FIG. 15, the VCD group showed no significant difference compared to the normal group, but the AMH concentration showed a tendency to decrease, and the VCD + sewage-treated group In AMH concentration was confirmed to be similar to the normal group. In addition, it was found that the sewage-only group did not affect the concentration of AMH. From the above results, it was found that the administration of sewage milk had an ovarian protective effect from the decrease of AMH concentration due to VCD.
9-4. 병리조직학적 분석을 통한 조기난소부전 예방 효과 확인9-4. Confirmation of early ovarian failure prevention effect through pathological analysis
H&E 조직염색을 통하여 병리조직학적 분석 결과, 도 16에 나타낸 바와 같이, 정상군의 난소는 다양한 크기의 원시난포에서 성숙난포 및 황체가 잘 발달되어 있었으며, 정상적인 크기를 유지하고 있었으나, VCD군의 난소는 크기가 현저히 위축되었고, 난포의 수 또한 현저히 감소하는 등 폐경을 암시하는 조직학적 소견을 보였다. 반면, VCD+오수유 투여군은 300 mg/kg의 고농도 투여군에서 난소 크기의 위축 또는 난포 및 황체의 퇴화가 관찰되지 않고 정상군과 거의 유사함을 확인할 수 있었다. 상기 결과로부터, 오수유 투여가 VCD로 유발되는 조기난소부전을 예방할 수 있음을 알 수 있었다. As a result of histopathological analysis through H & E tissue staining, as shown in FIG. 16, the mature ovaries of the normal group had well developed mature follicles and corpus luteum in various sizes of the ovarian follicles, and maintained the normal size, but the ovaries of the VCD group. Was markedly reduced in size and markedly reduced in number of follicles, suggesting histological findings. On the other hand, the VCD + sewage oil-treated group was found to be almost similar to the normal group without ovarian atrophy or follicle and corpus luteum degradation in the high-dose group of 300 mg / kg. From the above results, it was found that effusion administration can prevent premature ovarian failure caused by VCD.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명에 따른 오수유 추출물은 농도 의존적으로 DPPH 자유 라디칼 소거 활성을 증가시키므로 항산화 활성이 우수하고, 난소 세포의 세포사멸 (apoptosis)을 억제하며, PI3K/Akt 신호전달 경로를 활성화시켜 4-vinylcyclohexene diepoxide (VCD)에 의해 유도되는 난독성에 대한 유의적인 보호효과를 가지는바, 난독성, 조기난소부전 등의 개선, 예방, 억제 또는 치료에 유용한 의약품 및 건강기능식품으로 사용될 수 있다. 따라서 관련 적용례로서 불임, 난임의 치료 혹은 예방, 조기난소부전 및 폐경 전 갱년기장애 등을 고려할 수 있다. The sesame oil extract according to the present invention increases the DPPH free radical scavenging activity in a concentration-dependent manner, and thus has excellent antioxidant activity, inhibits apoptosis of ovarian cells, and activates the PI3K / Akt signaling pathway to activate 4-vinylcyclohexene diepoxide ( It has a significant protective effect against egg toxicity induced by VCD) and can be used as a medicine and health functional food useful for improving, preventing, suppressing or treating egg toxicity and premature ovarian failure. Therefore, relevant applications include infertility, treatment or prevention of infertility, premature ovarian failure and premenopausal disorders.

Claims (8)

  1. 오수유 추출물을 유효성분으로 포함하는 난독성 (ovotoxicity) 예방 또는 치료용 약학적 조성물.Pharmaceutical composition for the prevention or treatment of ovotoxicity comprising sewage extract as an active ingredient.
  2. 제1항에 있어서,The method of claim 1,
    상기 난독성은 4-vinylcyclohexene diepoxide (VCD)에 의해 유도되는 것을 특징으로 하는, 약학적 조성물.The egg toxicity is characterized in that induced by 4-vinylcyclohexene diepoxide (VCD), pharmaceutical composition.
  3. 제1항에 있어서,The method of claim 1,
    상기 오수유 추출물은 난소 세포의 세포사멸 (apoptosis)을 억제하는 것을 특징으로 하는, 약학적 조성물.The sewage oil extract is characterized in that to inhibit the apoptosis (apoptosis) of ovarian cells, pharmaceutical compositions.
  4. 제1항에 있어서,The method of claim 1,
    상기 오수유 추출물은 PI3K/Akt 신호전달 경로를 활성화시키는 것을 특징으로 하는, 약학적 조성물.The sewage extract is characterized in that to activate the PI3K / Akt signaling pathway, pharmaceutical composition.
  5. 오수유 추출물을 유효성분으로 포함하는 조기난소부전 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating premature ovarian failure comprising sewage extract as an active ingredient.
  6. 오수유 추출물을 유효성분으로 포함하는 조기난소부전 개선용 건강기능식품 조성물.Health functional food composition for improving early ovarian failure comprising sesame oil extract as an active ingredient.
  7. 제5항의 조성물을 개체에 투여하는 단계를 포함하는 조기난소부전의 예방 또는 치료 방법.A method for preventing or treating premature ovarian failure comprising administering to the subject a composition of claim 5.
  8. 오수유 추출물의 조기난소부전 예방 또는 치료 용도.Use for the prevention or treatment of premature ovarian failure of sesame oil extract.
PCT/KR2016/011302 2015-12-02 2016-10-10 Composition for preventing and treating premature ovarian failure, containing evodia rutaecarpa bentham extract having protective activity against ovotoxicity WO2017095011A1 (en)

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KR1020160129640A KR101860639B1 (en) 2015-12-02 2016-10-07 Composition comprising extract of evodiae fructus having protective activity against ovotoxicity for preventing or treating premature menopause

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US11690888B2 (en) * 2017-06-30 2023-07-04 Dongguk University Wise Campus Industry-Academy Cooperation Foundation Composition for preventing or treating menopausal syndrome, containing medicinal herb extract as active ingredient

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KR20020037292A (en) * 2000-11-13 2002-05-18 권승룡 Composition of phytoestrogens for hormone replacement therapy including the treatment of menopausal women
JP2005298450A (en) * 2004-04-15 2005-10-27 Taisho Pharmaceut Co Ltd Prophylactic or therapeutic agent composition for menopausal syndrome
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KR20020037292A (en) * 2000-11-13 2002-05-18 권승룡 Composition of phytoestrogens for hormone replacement therapy including the treatment of menopausal women
JP2005298450A (en) * 2004-04-15 2005-10-27 Taisho Pharmaceut Co Ltd Prophylactic or therapeutic agent composition for menopausal syndrome
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11690888B2 (en) * 2017-06-30 2023-07-04 Dongguk University Wise Campus Industry-Academy Cooperation Foundation Composition for preventing or treating menopausal syndrome, containing medicinal herb extract as active ingredient

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