WO2013151281A1 - Composition containing mori cortex radicis extract as active ingredient for preventing or treating depression - Google Patents

Composition containing mori cortex radicis extract as active ingredient for preventing or treating depression Download PDF

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Publication number
WO2013151281A1
WO2013151281A1 PCT/KR2013/002657 KR2013002657W WO2013151281A1 WO 2013151281 A1 WO2013151281 A1 WO 2013151281A1 KR 2013002657 W KR2013002657 W KR 2013002657W WO 2013151281 A1 WO2013151281 A1 WO 2013151281A1
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Prior art keywords
extract
composition
stress
active ingredient
present
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PCT/KR2013/002657
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French (fr)
Korean (ko)
Inventor
한대석
이창호
김영언
김인호
장여정
허송욱
이미숙
조승목
김윤태
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한국식품연구원
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    • 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)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A61K36/605—Morus (mulberry)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • a composition for the prevention or treatment of depression comprising the extract of ifo / "/ Cortex Radicis as an active ingredient
  • the present invention was achieved by the task No. E0121401 under the support of the Ministry of Knowledge Economy, the research management specialized organization of the project is the Korea Food Research Institute, the research project name is “Korea Food Research Institute major projects”, the research title is “Mental Health Promotion Food Research Development project (Stress relief material development and industrialization technology research) ", The lead organization is Korea Food Research Institute.
  • the research period is 2012.01.01 ⁇ 2012.12.31.
  • the present invention relates to a composition for the prevention or treatment of depression, including the extract of baekryepi as an active ingredient.
  • the World Health Organization has identified mental health as' individuals can recognize their abilities, cope with the daily stresses of life, work productively, 'Good state to contribute to the community'. Stress here refers to changes in organisms caused by internal and external stimuli that break the balance of the living body. Selye, a pioneer of stress research, defined stress as a nonspecific reaction of the body to demand, and is one of the typical stress reactions from the adrenal cortex. The secretion of glucocorticoids was regarded as an important factor in the establishment of the biostress response (Selye 1993, 1958, 1976).
  • HPA axis hypothalamus-pituitary-adrenal gland
  • the hypothalamus secretes corticosteroid-releasing factors, which stimulate the anterior pituitary gland to release corticosteroids, and the released corticosteroids release glucocorticoids (Chrousos and Kino 2007).
  • Corticoids affect the body's organs, immune system, and central nervous system, causing problems with organ damage, weakened immune function, and decreased memory (Lahmame A., 1997; Majzoub, J., 2006; Chrousos GP & Gold PW. , 1992).
  • flavonoids such as cholin, stilbene, benzofuran, oxydihydromorusin, etc.Amilin, betelic acid, and cytostine It contains a variety of functional ingredients, including sitosterol and umbell iferon. It has been shown that antipyretic, antispasmodic, antiallergic, antidiabetic and anti-inflammatory effects have been shown in recent years, as well as the effects of protecting the liver and hair growth on hair loss, including whitening, antioxidant, diuretic and neuroinflammatory reactions. Known (Hikino et al., 1985; Kim et al., 1999 ,; Kim et al., 1995).
  • the present inventors made an intensive study to develop a substance that can effectively prevent or treat depression and to be safe for humans, especially plant-derived substances, and as a result, it has been found that the baekbaekpi, which has been conventionally used as a herbal medicine, is very effective in preventing or treating depression. By this, the present invention was completed.
  • an object of the present invention is to provide a composition for preventing or treating depression.
  • Another object of the present invention is to provide a food composition for improving or alleviating depression. Another object of the present invention is to provide a method for preventing, ameliorating or treating depression. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
  • the present invention provides a composition for the prevention or treatment of depression comprising an extract of Cortex Radicis as an active ingredient.
  • the present inventors made an intensive study to develop a substance that can effectively prevent or treat depression and to be safe for humans, especially plant-derived substances, and as a result, it has been found that the baekbaekpi, which has been conventionally used as a herbal medicine, is very effective in preventing or treating depression. It was.
  • composition of the present invention works very effectively in the prevention or treatment of depression.
  • the effects of the present invention are exerted by inhibiting the nuclear translocat ion of the glucocorticoid receptor (GR).
  • GR glucocorticoid receptor
  • Glucocorticoids bind to glucocorticoid receptors in cell substrates, which are activated by ligand binding. After the hormone binds to the corresponding receptor, the newly formed receptor-ligand complex binds to the glucocort icoid response element at the site where it translocates into the nucleus of the cell and elevates the target gene during gene expression regulation.
  • Cortisol is a steroid hormone secreted by the adrenal cortex and is also called glucocorticoid because it affects glucose metabolism. Cortisol breaks down proteins into glucose and affects the brain, causing behavioral reactions. The hypothalamus nucleus regulates the release of cortisol. Neurons in the nucleus contain a peptide called CR cortiotropin-releasing factor. Secrete, which in turn stimulates the anterior pituitary gland to release corticosteroids. This hormone is passed through the blood to the adrenal glands to release cortisol. As stress continues, cortisol continues to be released, blood pressure rises, muscle tissue is damaged, steroid diabetes, infertility, growth inhibition, etc., and immune function decreases.
  • the composition of the present invention inhibits the activation of the glucocorticoid receptor.
  • Glucocorticoid receptors are activated by binding of ligands.
  • the composition of the present invention inhibits the activation of the glucocorticoid receptor in a concentration dependent manner.
  • the composition of the present invention exhibits antistress and antidepressant effects by inhibiting the movement of activated glucocorticoid receptors into the nucleus.
  • treatment of the epidermis extract inhibits intranuclear migration of the glucocorticoid receptor induced by cortisol stimulation.
  • the intranuclear mobility of glucocorticoid receptors increased only by 2.76 ⁇ 0.07 times when treated with cortisol alone, whereas the intranuclear mobility of glucocorticoid receptors reacted by cortisol was increased by 1.96 ⁇ 0.29 times when treated with lettuce extract.
  • mobility in the nucleus was suppressed more than that in the case of cortisol treatment alone (FIG. 6).
  • the baekbaekpi used as an active ingredient in the composition of the present invention refers to the root of the mulberry plant which is a deciduous tree belonging to the mulberry (Moraceae).
  • various extracting solvents may be used.
  • a polar solvent or a nonpolar solvent can be used.
  • Suitable polar solvents include (i) water, (ii) alcohols (preferably methanol, ethanol, propane, butanol, normal-propanol, iso-propanol, normal-butanol 1-pentane, 2-butoxyethane Or ethylene glycol), (iii) acetic acid, (iv) dimethylformamide (DMF0) and (v) DMSOCdimethyl sulfoxide (v).
  • alcohols preferably methanol, ethanol, propane, butanol, normal-propanol, iso-propanol, normal-butanol 1-pentane, 2-butoxyethane Or ethylene glycol
  • acetic acid acetic acid
  • DMF0 dimethylformamide
  • DMSOCdimethyl sulfoxide v
  • Suitable as nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane pentane, hexane, 2,2,4-trimethylpentane, decane, cyclonucleic acid, cyclopentane-tan, diisobutalene, ⁇ Pentene : ⁇ chlorobutane, 1 chloropentane, 0- Xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane annealed diethyl Amines, ethers, carbon tetrachloride and THF.
  • the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) the lower alcohol and water A mixed solvent with (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) nucleic acid and (j) diethyl ether Include.
  • the extract of the present invention is obtained by treating water, ethane, or a combination thereof on lettuce skin.
  • the extract of baekbaekpi of the present invention is obtained by drying and grinding baekbaekpi, powdered, and then treated and homogenized ethane. :
  • Homogenization can be carried out through various methods known in the art. Preferably it is homogenized using a sonicator and a sonic dismembrator.
  • the term 'extract' has a meaning commonly used as a crude extract in the art as described above, but also broadly includes a fraction additionally extracting the extract.
  • the extract of baekbaekpi is not only obtained by using the above-described extraction solvent, but also includes the one obtained by additionally applying a purification process.
  • the fraction obtained through the purification method is also included in the extract of the lettuce skin.
  • sodium carbonate is added to the extract of Morus alba to completely dissolve the insoluble material.
  • the sodium carbonate is added to dissolve insoluble substances due to alcohol extraction, and then dried and powdered to obtain 100%. Make a state of acceptability.
  • the lettuce extract used in the present invention may be prepared in powder form by an additional process such as distillation under reduced pressure and freeze drying or spray drying.
  • the term 'comprising as an active ingredient' means to include an amount sufficient to achieve the efficacy or activity of the following extract of the lettuce extract.
  • the present invention is a composition extracted from baekbaekpi, a natural plant material, so there is no side effect to the human body, even if the dose is excessive, the quantitative upper limit contained in the composition of the baekbaekpi extract can be carried out by those skilled in the art within the appropriate range.
  • the composition of the present invention reduces the expression of c-Fos.
  • the early-expression gene, c-Fos is a neuroanatomical marker that tracks neuronal and brain activity associated with stress factors such as pain. It is activated in the form of cells.
  • the term "reduction (of c-Fos expression)" means that the amount of c ⁇ Fos is measurably reduced compared to a subject (control) not administered the composition of the present invention, preferably 30 It means reduced by more than%, more preferably reduced by more than 20%, even more preferably reduced by more than 10%.
  • the present invention provides a food composition for improving or alleviating depression, which comprises an extract of lettuce extract as an active ingredient.
  • an active ingredient as well as the extract of baekpipi, as well as ingredients commonly added in the manufacture of food, for example, protein, carbohydrates, fats, nutrients seasonings and flavors Includes the first.
  • Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and xyls, sorbitols, and erythrates.
  • sugars such as polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and xyls, sorbitols, and erythrates.
  • natural flavorings [tautin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring crabs (saccharin, aspartame) Etc.) can be used.
  • the invention provides a method of preventing, ameliorating, alleviating or treating depression comprising administering to a subject a composition comprising a pharmaceutically effective amount of an extract of Cortex Radicis .
  • the present invention provides a composition for the prevention or treatment of depression, including as an extract of the extract epithelium.
  • composition of the present invention exhibits an antistress effect by inhibiting intracellular nuclei migration of glucocorticoid receptors, thereby inhibiting stress-induced signaling processes.
  • composition of the present invention has an efficacy as an antagonist to the glucocorticoid receptor, like a conventional antidepressant, and can exhibit antidepressant efficacy.
  • FIG. 1 is a diagram showing a schematic diagram of a biostress sensor and a control sensor.
  • Figure 2 is a diagram showing a manufacturing schematic diagram of the biostress sensor.
  • 3 is a diagram showing a sonotrack system that can monitor the ultrasound of the specific area of the rat.
  • Figure 4 is an image of the activity of the biostress sensor for stress hormone (Cortisol) and antistress hormone (RU486) in HeLa cells.
  • 5 is a biostress sensor of lettuce extract in HeLa cells This is a picture of the activity inhibitory effect by concentration.
  • Figure 6 is an illustration showing the effect of the extract of lettuce on the mobility in the nucleus of GR.
  • FIG. 7 is a diagram showing the effect of the extract of Morus alba on GR activity in the skin of animals.
  • FIG. 8 is a diagram showing the effect of the extract of baekhyeolpi on the generation of ultrasound in the 22-kHz band.
  • 9 is a diagram showing the effect of the extract extracts on 50-kHz ultrasonic generation.
  • FIG. 10 is a diagram showing the effect of E. coli extract on c-Fos gene protein expression in hippocampal CA3 region of brain (A: normal control group, B: control group, C: stress + RU486 group, D: stress + MCR200 group) .
  • FIG. 11 is a diagram showing the effect of lettuce extract on c-Fos gene protein expression in the hypothalamic chamber nucleus of the brain (A: normal control, B: control, C: stress + RU486 group, D: stress + MCR200 group) to be.
  • the ultrasonic crusher (Fisher Scientific, ModellOO) was placed at an intensity of 7 and was treated four times for 20 minutes. After extraction, the filter was filtered through a filter paper (0.45 um, Millipore membrane filter, USA), and distilled water (30% of the total amount of the filtrate) was added to remove alcohol by using a rotary concentrator (Buchi Labor technik AG, Switzerland). When it was in the state was dried and lyophilized (IlshinBioBase, Korea) to powder.
  • glucocorticoid response element GRE, 5'-AGAGGATCTGTACAGGATGTTCTAGAT-3 '
  • pWPXUAddgene Biosensor carrier
  • Biosensor carriers were packaged, psPAX2 (Addgene, USA), and envelope vectors, pMD2G (Addgene, USA), 100 ug, 3.75 g, and 1,25, respectively, using SuperFect Trans feet ion Reagent (Qiagen, USA). Inserted into 293T cells grown in mm culture vessel (Fig. 2, Procedure 1).
  • Each culture solution was cultured for 48 and 72 hours, centrifuged at 7,000 rpm for 30 minutes, and the supernatant was passed through a 0.45 ⁇ syringe filter (millipore, USA) to produce a stress sensor (FIG. 2 ,).
  • Process 2 The produced luminescent expression stress sensor was infected with human-derived HeLa cells in which the glucocorticoid receptor (GR) is present (FIG. 2, processes 3 and 4).
  • GR glucocorticoid receptor
  • Biostress sensors GRE and control sensors (AGRE), which recognize the activity of glucocorticoid receptors (GR), were produced at the gene level (FIG. 1), which produced sensors using 293T cells.
  • the active effect of the biostress sensor was confirmed through the expression of a light emitting protein (luciferase) in HeLa cells (Fig. 2). Intracellular Antistress Mechanism Analysis of Extracts from Epidermis
  • ethanol extracts on the stress on the stress was compared with ethanol extracts from HeLa cells expressing glucocorticoid receptors. After incubating 1 ⁇ 10 4 cells in 96-well and passing the biosensor for 24 hours, the cells were incubated at 37 ° C. for 12 hours, and pretreated with epithelial extract dissolved in DMS0 for 3 hours, followed by 500 nM of glucocorticoids. Induction of activator / After 36 hours, biosensor activity was imaged and quantified using IVIS200.
  • anti-stress hormone (RU486, Sigma, USA), an antagonist of glucocorticoids, was treated with 500 nM of glucocorticoids after 3 hours of treatment with biosensor-inserted HeLa cells to confirm the inhibitory effect of luminescent protein expression (FIG. 4B). Fluorescence imaging
  • Fluorescently labeled GR (GR-EGFP) was inserted into C0S-1 cells that did not express GR itself via transient transfection method (Effect ine, Qiagen). Confocal microscopy was used to determine whether green fluorescence was expressed through cell culture for 24 hours, followed by treatment with extracts from the epidermis. After 3 hours, 100 nM of stress hormone (Cortisol) was treated for 30 minutes. Fluorescent cells were fixed with 4% paraformaldehyde and nuclear stained using DraQ, followed by confocal microscopy to obtain nuclear transfer images of fluorescently labeled GR, using an LSM image analyzer (Zeiss, Germany). Whole cell The degree of migration in the nucleus was evaluated by analyzing the ratio of the fluorescence intensity to the fluorescence intensity in the nucleus. In vivo assay
  • mice used in the experiment for the analysis of anti-stress efficacy were 8-week-old Wistar male rats, which were distributed from a central experimental animal (Korea), and had a weekly dip period in the nursery. Eight animals per group were categorized as randomized com lete block design and two were bred per cage. The groups classified in the experiment were normal control group (ND), stress control group (distilled water group), antistress group (stress + RU486; RU486 10 mg / kg body weight) ⁇ stress + epithelium 100 group (stress + MCR100; body weight 1 kg) The group was divided into 100 mg of sugar extract and 100 mg of stress + 200 extract (stress + MCR200; 200 mg extract per 1 kg of body weight).
  • the experiment was divided into 50 mg of the extract administration group, stress + epidermis 100 administration group (stress + MCR100; 100 mg of the extract extract per 100 kg body weight, 100 mg of stress + epidermis extract), 200 groups of stress + epidermis extract (stress + MCR200; 200 mg of extract extract per 1 kg body weight)
  • stress + MCR100 100 mg of the extract extract per 100 kg body weight, 100 mg of stress + epidermis extract
  • stress + MCR200 200 groups of stress + epidermis extract
  • the temperature and humidity of the breeding room were adjusted to 22 ⁇ 0.5 ° C and 55 ⁇ 5%, respectively, and the contrast period was adjusted to 12 hours. All animal-related care was controlled by the Korea Food Research Institute's Animal Experiment Ethics Committee (IACUC). Acknowledgment and supervision: Assessment of biostress sensor activity in skin
  • biosensor In order to detect the activity of biosensor in vivo, it is injected into skin by subcutaneous injection method stably for 15 days. 'Induce the biosensor to be inserted into the skin cells. After Biosensor Injection
  • the epidermis extract was administered once a day for 5 days.
  • the biostress sensor was activated after 15 minutes by giving 2 hours of confinement stress and intraperitoneal injection of D-luciferin (75 mg / kg).
  • a biological imaging device IVIS-200, Xenogen
  • the reaction caused by stress was confirmed in vivo by quantifying the image activity by the R0I value.
  • Ultrasonic zone analysis was performed to depress 8-week-old male Wistar rats for a week. After dissolution, it was orally administered for 14 days according to the experimental method. For 2 hours, confinement stress was imparted by using a cage and changed according to the emotional state of rats by using sonotrack (Metris, Netherlands) 4-channel ultrasonic sensors (microphones). The ultrasound zone was analyzed for 20 minutes while monitoring ( Figure 3). Sonotracks can detect up to 15-125 kHz, and ultrasound varies with age, environment, and emotional state. Generally, a 50-kHz vocalization occurs when the mouse is in a relaxed state, and a 22-kHz vocalization under fear or stress conditions. The anti-stress effect of the intake of lettuce extract was known based on the ultrasound zone that rats emit. Brain extraction
  • Formalin (Sigma, USA) was perfused through the heart with 300-400 mL at a flow rate of 25 mL per minute.
  • the brain is then removed, post-fixed with the same fixative for 24 hours, and then placed in PBS (phosphate buffered saline, 0.01 M, pH 7.4) containing 25% sucrose until the brain precipitates for 36-40 hours at 4 ° C. It was left.
  • Precipitated brain was rapidly shaken by cryo-spray and then micro-tome (Leica, Germany) was used for c-Fos gene expression study by cutting the hippocampus and hypothalamus into 30 Lim thickness of brain tissue. . c-Fos protein immunohistochemical staining
  • PBST PBS + 0.1% Triton x-100
  • a biostress sensor (GRE) or a control sensor ( ⁇ ? ⁇ ) was inserted into HeLa cells, respectively.
  • the post-stress hormone Cortisol
  • the amount of luciferase expression was measured using an IVIS-200 device to image and quantify the activity of the biostress sensor.
  • the control sensor there was no change in the expression of luciferase even in the treatment of hormones, whereas in the biostress sensor, luciferase expression was increased in proportion to the concentration of the stress hormone, thereby increasing the stress activity (FIG. 4). ).
  • the activity of the biostress sensor occurs in three major steps: first, the glucocorticoid ligand binds to the inactivating receptor (GR) in the cytoplasm (ligand binding), and second, the receptor activated by hormone binding is the nucleus. Translocation and third is the transduction of the target gene transcription by binding the receptor to glucocorticoid reaction factor (GRE).
  • GRE glucocorticoid reaction factor
  • GR a reporter using the green fluorescence protein (GFP)
  • GFP green fluorescence protein
  • the method was injected subcutaneously into the skin to induce the biostress sensor to be stably inserted into keratinocytes for 20 days.
  • a total of three groups were divided to explore the stress activity of the biostress sensor in vivo.
  • 1 negative control group PBS only was injected once daily for 5 days by the same method as drug injection.
  • 3 stress + MCR100 group Inject the baekpipi extract per 100 kg body weight to 100 mg dose once a day 5 days before, 5 days, 5 days 2 days to induce stress through confinement.
  • the physiological response to stress is regulated by the hypothalamus-ituitary-adrenal gland (HPA axis).
  • HPA axis functions to regulate nerves, endocrine, and immune functions for homeostasis during stress response.
  • the hypothalamus secretes corticotropin releasing factor (CRF), which stimulates the anterior pituitary gland to adrenal gland.
  • CRF corticotropin releasing factor
  • Cortical stimulating hormone is released and the released adrenal cortical stimulating hormone is secreted in the adrenal cortex to release the corticosterone, which affects the body's organs, immune system, central nervous system, etc. cause problems.
  • PVN paraventricular nucleus
  • c-Fos protein is well known to be expressed in various areas of the brain by acute harmful stimulation or stress.
  • Several related studies have observed an increase in c-Fos expression in brain stem, thalamus, hypothalamus, and hippocampus due to noxious stimulation or stress.
  • c-Fos expression is known to reflect behavioral and physiological reactions caused by stress. come.
  • c-Fos is Expressed within 20 minutes by stimulation such as stress and lasting for more than 24 hours, it is called an early-early gene and is widely known as a marker for measuring changes in metabolic activity in cells (Drgunow M et al. , 1989).
  • the present invention was intended to examine the anti-stress efficacy of the extracts of the lettuce extract by observing c-Fos protein expressed in the hippocampus, chamber nucleus, etc. after giving rapid confinement stress to the rat.

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Abstract

The present invention provides a composition containing Mori Cortex Radicis extract as an active ingredient for preventing or treating depression. The present invention exhibits an anti-stress effect by suppressing the nuclear translocation of glucocorticoid receptors, thereby impeding a stress-induced signal transmission process, and, similar to existing anti-depressives, can have an anti-depressive effect from an antagonist effect thereof for glucocorticoid receptors. Additionally, the present invention provides a food composition containing Mori Cortex Radicis extract as an active ingredient for improving or mitigating depression.

Description

【명세서】  【Specification】
【발명의 명칭] [Name of invention]
상백피 (ifo/"/ Cortex Radicis) 추출물을 유효성분으로 포함하는 우울증의 예방 또는 치료용 조성물  A composition for the prevention or treatment of depression comprising the extract of ifo / "/ Cortex Radicis as an active ingredient
【기술분야】 Technical Field
본 발명은 대한민국 지식경제부의 지원 하에서 과제번호 E0121401에 의해 이투어진 것으로서， 상기 과제의 연구관리전문기관은 한국식품연구원, 연구사업명은 "한국식품연구원 주요사업" , 연구과제명은 "정신건강 증진식품 연구개발사업 (스트레스 해소 소재 개발 및 산업화 기술 연구)" ， 주관기관은 한국식품연구원， 연구기간은 2012.01.01 ~ 2012.12.31이다.  The present invention was achieved by the task No. E0121401 under the support of the Ministry of Knowledge Economy, the research management specialized organization of the project is the Korea Food Research Institute, the research project name is "Korea Food Research Institute major projects", the research title is "Mental Health Promotion Food Research Development project (Stress relief material development and industrialization technology research) ", The lead organization is Korea Food Research Institute. The research period is 2012.01.01 ~ 2012.12.31.
본 특허출원은 2012년 04월 02일에 대한민국 특허청에 제출된 대한민국 특허출원 제 10-2012-0033853호에 대하여 우선권을 주장하며, 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다.  This patent application claims priority to Korean Patent Application No. 10-2012-0033853 filed with the Korean Intellectual Property Office on April 02, 2012, the disclosure of which is incorporated herein by reference.
본 발명은 상백피 추출물을 유효성분으로 포함하는 우울증의 예방 또는 치료용 조성물에 관한 것이다.  The present invention relates to a composition for the prevention or treatment of depression, including the extract of baekryepi as an active ingredient.
【배경기술】 Background Art
현대인은 사회 및 일상생활을 통해 끊임없이 스트레스를 경험하며 살아가고 있다. 즉, 사회가 급격히 발전하고 다변화되면서 요구되는 역할들이 증가함에 따라 까다로운 대인관계를 비롯한 불규칙적인 식생활, 부족한 수면 등의 지속적인 스트레스 상황에 노출될 수밖에 없으며， 개인의 적웅능력보다 강도가 크거나 장기간 지속되는 스트레스는 각종 정신적, 신체적 질환을 유발시키는 대표적인 요인으로 인식되고 있다. 따라서 최근에는 건강연구의 관심분야도 사망문제보다는 어떻게 정신적으로 안녕한 삶을 살 것인가로 시선이 이동되고 있으며 정신건강에 대한 관심과 스트레스를 경감시켜 줄 수 있는 식품이나 관련 웰빙 의식에 대한 관심도 고조되고 있는 실정이다.  Modern man is constantly experiencing stress through social and daily life. In other words, as society develops rapidly and diversifies, the required roles increase, which inevitably exposes people to constant stress situations such as difficult interpersonal relationships, irregular eating habits, and insufficient sleep. Stress is recognized as a representative factor causing various mental and physical diseases. In recent years, the focus of health research has shifted attention to how to live mentally well rather than death, as well as food and related well-being consciousness that can alleviate stress and mental health. There is a situation.
세계보건기구는 정신건강을 '개인이 자신의 능력을 인식하고, 삶의 일상적 스트레스에 대처할 수 있고, 생산적으로 일을 할 수 있으며, 지역사회에 기여할 수 있는 안녕한 상태' 라고 정의하였다. 여기서 말하는 스트레스란 생체의 균형을 깨뜨리는 내.외적인 자극에 의해 일어나는 유기체 내의 변화를 말하는 것으로 스트레스 연구의 선구자인 Selye는 스트레스를 요구에 대한 생체의 비특이적 반웅이라 정의하였고, 전형적인 스트레스 반웅의 하나로 부신피질로부터 당질글루코코르티코이드 (glucocorticoid)의 분비를 생체 스트레스 반웅의 성립에 중요한 인자로 보았다 (Selye 1993, 1958， 1976). The World Health Organization has identified mental health as' individuals can recognize their abilities, cope with the daily stresses of life, work productively, 'Good state to contribute to the community'. Stress here refers to changes in organisms caused by internal and external stimuli that break the balance of the living body. Selye, a pioneer of stress research, defined stress as a nonspecific reaction of the body to demand, and is one of the typical stress reactions from the adrenal cortex. The secretion of glucocorticoids was regarded as an important factor in the establishment of the biostress response (Selye 1993, 1958, 1976).
일반적으로 우리 생체는 스트레스에 대항하여 여러 반응을 일으키며 이로 인해 내분비계, 자율신경계 등에도 변화가 일어나게 되는데, 지속적이거나 만성적인 스트레스는 우울， 불안， 분노 등의 심리적 변화를 수반하게 되는 정신 장애와 중추 신경계， 면역계 장애， 신경성 위궤양 등과 같은 소화기 장애, 협심증 등과 같은 심혈관 장애 등의 여러 질환들의 발병기전과도 밀접한 관련성이 있다 (Kim et al . , 1992； Glavin GB. , 1985； Khansari et al .， 1990). 여기서 대표적인 스트레스에 의한 생리학적인 반웅은 시상하부-뇌하수체 -부신피질축 (hypothalamus— pituitary-adrenal gland; HPA axis)에 의해 조절되며， 이 HPA축이 스트레스 반웅 시 신체의 항상성 유지를 위해 신경， 내분비, 면역작용을 조절하는 기능을 하게 된다. 아 과정에서 시상하부는 부신 피질 자극호르몬 방출인자를 분비하여 뇌하수체 전엽을 자극하여 부신피질자극호르몬을 방출하고 방출된 부신피질자극호르몬은 글루코코르티코이드를 방출하게 되며 (Chrousos and Kino 2007) , 방출된 글루코코르티코이드는 몸의 기관과 면역계， 중추신경계 등에 영향을 주어 내장기관 손상, 면역기능 약화， 기억력 감소 등에도 문제를 일으키게 된다 (Lahmame A. , 1997； Majzoub, J. , 2006； Chrousos GP & Gold PW.， 1992) .  In general, our living body responds to stress, which causes changes in the endocrine system and the autonomic nervous system, and persistent or chronic stress is associated with psychological disorders such as depression, anxiety, and anger. It is also closely related to the pathogenesis of various diseases such as digestive disorders such as nervous system, immune system disorders, and gastric ulcers and cardiovascular disorders such as angina pectoris (Kim et al., 1992; Glavin GB., 1985; Khansari et al., 1990 ). Here, the representative stress-physiological reactions are controlled by the hypothalamus-pituitary-adrenal gland (HPA axis), which are used to maintain the homeostasis of the body during stress reaction. It will function to regulate immune function. In the subprocess, the hypothalamus secretes corticosteroid-releasing factors, which stimulate the anterior pituitary gland to release corticosteroids, and the released corticosteroids release glucocorticoids (Chrousos and Kino 2007). Corticoids affect the body's organs, immune system, and central nervous system, causing problems with organ damage, weakened immune function, and decreased memory (Lahmame A., 1997; Majzoub, J., 2006; Chrousos GP & Gold PW. , 1992).
현재 임상에서는 약물치료와 장기적인 정신과적 치료를 병행해 스트레스 관련 불안장애, 우울증 등을 치료하고 있으며， 약물치료의 경우에는 주로 디아제팜, 로라제팜， 클로나제팜 등과 같은 벤조디아제핀 계통의 약물이 주로 사용되고 있다. 하지만 웰빙의식의 고조로 이러한 약물들의 부작용을 줄일 수 있는 천연물로부터 유래된 항스트레스 효능을 가지는 물질에 대한 관심이 높아지고 있으며, 관련 연구가 활발히 진행되고 있다. 상백피는 뽕나무과 (Moraceae)에 속한 낙엽교목인 뽕나무 속 식물의 근피로， 구와논 (loiwanon), 멀베로푸란 (mulberrofuran) , 알바푸란 (albafuran) , 모루시논 (morucinon) , 상겐논 (sanggennon) , 코린 (cholin), 스틸벤 (stilbene), 벤조푸란 (benzofuran) , 옥시디하이드로모루신 (oxydihydromorusin) 등과 같은 플라보노이드를 다량 함유하고 있으며, 이외에도 아미린 (amyrin), 베를린산 (betulinic acid), 시토스테를 (sitosterol), 엄벨리페론 (umbell iferon) 등의 다양한 기능성 성분들을 포함하고 있다. 예로부터 상백피는 해열, 항경련， 항알러지， 항당뇨, 항염증 작용과 더불어 최근에는 간 보호 효과 및 탈모증에 대한 모발 성장 효과를 비롯한 미백， 항산화， 이뇨촉진， 신경성 염증반웅 억제 효과 등이 있는 것으로 알려져 있다 (Hikino et al . , 1985； Kim et al . , 1999,； Kim et al., 1995) . 하지만 항스트레스 관련 상백피의 약리학적 연구는 신경세포 보호 효과 정도로만 세포수준에서 관찰한 정도이며， 실질적이고 체계적인 연구는 아직 많이 이루어지고 있지 못한 실정이다. 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다. Currently, the clinic treats stress-related anxiety disorders and depression by combining drug treatment and long-term psychiatric treatment. In the case of drug treatment, benzodiazepine-based drugs such as diazepam, lorazepam, and clonazepam are mainly used. However, there is a growing interest in substances that have anti-stress effects derived from natural products that can reduce the side effects of these drugs due to the increase in well-being consciousness. It is the root of the deciduous tree of the deciduous tree in the family Moraceae, loiwanon, mulberrofuran, albafuran, morucinon, sanggennon, and corin. It contains a large amount of flavonoids such as cholin, stilbene, benzofuran, oxydihydromorusin, etc.Amilin, betelic acid, and cytostine It contains a variety of functional ingredients, including sitosterol and umbell iferon. It has been shown that antipyretic, antispasmodic, antiallergic, antidiabetic and anti-inflammatory effects have been shown in recent years, as well as the effects of protecting the liver and hair growth on hair loss, including whitening, antioxidant, diuretic and neuroinflammatory reactions. Known (Hikino et al., 1985; Kim et al., 1999 ,; Kim et al., 1995). However, pharmacological studies of antistress-related epithelium have been observed at the cellular level only to the extent of neuroprotective effect, and there are not many practical and systematic studies yet. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
【발명의 내용】 [Content of invention]
【해결하려는 과제】  [Problem to solve]
본 발명자들은 우울증을 효과적으로 예방 또는 치료할 수 있으며 인체에 안전한 물질， 특히 식물 -유래 물질을 개발하고자 예의 연구 노력하였고， 그 결과 종래부터 한약재로 사용되고 있는 상백피가 우울증을 예방 또는 치료하는데 매우 유효하다는 것을 규명함으로써, 본 발명을 완성하였다.  The present inventors made an intensive study to develop a substance that can effectively prevent or treat depression and to be safe for humans, especially plant-derived substances, and as a result, it has been found that the baekbaekpi, which has been conventionally used as a herbal medicine, is very effective in preventing or treating depression. By this, the present invention was completed.
따라서 본 발명의 목적은 우울증의 예방 또는 치료용 조성물을 제공하는데 있다.  Accordingly, an object of the present invention is to provide a composition for preventing or treating depression.
본 발명의 다른 목적은 우울증의 개선 또는 완화용 식품 조성물을 —제공하는데 있다. 본 발명의 또 다른 목적은 우을증의 예방， 개선 또는 치료 방법을 제공하는 데 있다. 본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다. Another object of the present invention is to provide a food composition for improving or alleviating depression. Another object of the present invention is to provide a method for preventing, ameliorating or treating depression. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
【과제의 해결 수단】 [Measures of problem]
본 발명의 일 양태에 따르면， 본 발명은 상백피 ( or/ Cortex Radicis) 추출물을 유효성분으로 포함하는 우울증의 예방 또는 치료용 조성물을 제공한다. 본 발명자들은 우울증을 효과적으로 예방 또는 치료할 수 있으며 인체에 안전한 물질， 특히 식물 -유래 물질을 개발하고자 예의 연구 노력하였고， 그 결과 종래부터 한약재로 사용되고 있는 상백피가 우울증을 예방 또는 치료하는데 매우 유효하다는 것을 규명하였다.  According to one aspect of the invention, the present invention provides a composition for the prevention or treatment of depression comprising an extract of Cortex Radicis as an active ingredient. The present inventors made an intensive study to develop a substance that can effectively prevent or treat depression and to be safe for humans, especially plant-derived substances, and as a result, it has been found that the baekbaekpi, which has been conventionally used as a herbal medicine, is very effective in preventing or treating depression. It was.
본 발명의 조성물은 우울증의 예방 또는 치료에 매우 효과적으로 작용한다.  The composition of the present invention works very effectively in the prevention or treatment of depression.
본 발명의 효과들은 글루코코르티코이드 수용체 (glucocorticoid receptor, GR)의 핵 내 이동 (nuclear translocat ion)을 억제시킴으로써 발휘된다.  The effects of the present invention are exerted by inhibiting the nuclear translocat ion of the glucocorticoid receptor (GR).
글루코코르티코이드는 글루코코르티코이드 수용체와 세포기질에서 결합하는데， 수용체는 리간드 (ligand) 결합에 의해 활성화된다. 호르몬이 해당 수용체와 결합한 후 새롭게 형성된 수용체-리간드 복합체는 세포의 핵 내로 전이하고 유전자 표출 조절시 표적 유전자를 상승시키는 부위에서 글루코코르티코이드 반웅 요소 (glucocort icoid response element)와 결합한다.  Glucocorticoids bind to glucocorticoid receptors in cell substrates, which are activated by ligand binding. After the hormone binds to the corresponding receptor, the newly formed receptor-ligand complex binds to the glucocort icoid response element at the site where it translocates into the nucleus of the cell and elevates the target gene during gene expression regulation.
코르티졸은 부신피질에서 분비되는 스테로이드 호르몬으로 포도당 대사에 영향을 주기 때문에 글루코코티코이드라고도 하는데， 코르티졸은 단백질을 분해하여 포도당으로 전환시키고, 뇌에 영향을 주어 행동적 반웅을 일으킨다. 시상하부의 실방핵은 코르티졸의 분비를 조절하는데 실방핵의 뉴런은 CR cortiotropin-releasing factor)라는 펩타이드를 분비하고， 이것은 다시 뇌하수체 전엽을 자극하여 부신피질자극 호르몬을 분비시킨다. 이 호르몬은 혈액을 통해 부신으로 전달되어 코르티졸을 방출한다 . 스트레스가 지속되어 코르티졸이 계속 분비되면 혈압이 오르고 , 근조직이 손상되며， 스테로이드 당뇨병， 불임, 성장억제 등이 나타나며 면역기능도 저하된다. Cortisol is a steroid hormone secreted by the adrenal cortex and is also called glucocorticoid because it affects glucose metabolism. Cortisol breaks down proteins into glucose and affects the brain, causing behavioral reactions. The hypothalamus nucleus regulates the release of cortisol. Neurons in the nucleus contain a peptide called CR cortiotropin-releasing factor. Secrete, which in turn stimulates the anterior pituitary gland to release corticosteroids. This hormone is passed through the blood to the adrenal glands to release cortisol. As stress continues, cortisol continues to be released, blood pressure rises, muscle tissue is damaged, steroid diabetes, infertility, growth inhibition, etc., and immune function decreases.
본 발명의 바람직한 구현예에 따르면， 본 발명의 조성물은 글루코코르티코이드 수용체의 활성화를 억제한다. 글루코코르티코이드 수용체는 리간드의 결합에 의해 활성화된다. 본 발명의 조성물은 농도 의존적으로 글루코코르티코이드 수용체의 활성화를 억제한다.  According to a preferred embodiment of the present invention, the composition of the present invention inhibits the activation of the glucocorticoid receptor. Glucocorticoid receptors are activated by binding of ligands. The composition of the present invention inhibits the activation of the glucocorticoid receptor in a concentration dependent manner.
본 발명의 바람직한 구현예에 따르면， 본 발명의 조성물은 활성화된 글루코코르티코이드 수용체가 핵 내로 이동하는 것을 억제함으로써, 항스트레스 및 항우울증 효과를 나타낸다.  According to a preferred embodiment of the present invention, the composition of the present invention exhibits antistress and antidepressant effects by inhibiting the movement of activated glucocorticoid receptors into the nucleus.
하기의 실시예에서 입증된 바와 같이， 상백피 추출물을 처리하면 코르티졸 자극에 의해 유도되는 글루코코르티코이드 수용체의 핵 내 이동을 억제시킨다. 예를 들면, 코르티졸만 처리한 경우 글루코코르티코이드 수용체의 핵 내 이동성은 대조군에 비해 2.76±0.07배 증가한 반면, 상백피 추출물을 전처리한 경우 코르티졸에 반웅한 글루코코르티코이드 수용체의 핵 내 이동성은 1.96 ±0.29배 증가하여, 코르티졸만 처리한 경우보다 핵 내 이동성이 억제되었다 (도 6).  As demonstrated in the examples below, treatment of the epidermis extract inhibits intranuclear migration of the glucocorticoid receptor induced by cortisol stimulation. For example, the intranuclear mobility of glucocorticoid receptors increased only by 2.76 ± 0.07 times when treated with cortisol alone, whereas the intranuclear mobility of glucocorticoid receptors reacted by cortisol was increased by 1.96 ± 0.29 times when treated with lettuce extract. Thus, mobility in the nucleus was suppressed more than that in the case of cortisol treatment alone (FIG. 6).
본 발명의 조성물에서 유효성분으로 이용되는 상백피는 뽕나무과 (Moraceae)에 속한 낙엽교목인 뽕나무 속 식물의 근피를 의미한다. 본 발명의 조성물에서 이용되는 상백피 추출물을 상백피에 추출용매를 처리하여 수득하는 경우에는， 다양한 추출용매가 이용될 수 있다. 바람직하게는， 극성 용매 또는 비극성 용매를 이용할 수 있다. 극성 용매로서 적합한 것은, (i) 물， (ii) 알코을 (바람직하게는， 메탄올， 에탄올， 프로판을， 부탄올， 노말 -프로판올， 이소 -프로판올， 노말-부탄올 1-펜탄을， 2-부톡시에탄을 또는 에틸렌글리콜)， (iii) 아세트산, (iv) DMF0(dimethyl- formamide) 및 (v) DMSOCdimethyl sulfoxide)를 포함한다. 비극성 용매로서 적합한 것은， 아세톤, 아세토나이트릴, 에틸 아세테이트， 메틸 아세테이트， 플루오로알칸 펜탄， 헥산， 2,2，4-트리메틸펜탄, 데칸， 사이클로핵산， 사이클로펜—탄， 디이소부탈렌， ：卜펜텐 ：卜클로로부탄, 1 클로로펜탄, 0- 자일렌， 디이소프로필 에테르， 2-클로로프로판, 를루엔, 1-클로로프로판, 클로로벤젠， 벤젠， 디에틸 에테르， 디에틸 설파이드， 클로로포름， 디클로로메탄, 1,2-디클로로에탄ᅳ 어닐린 디에틸아민, 에테르， 사염화탄소 및 THF를 포함한다. The baekbaekpi used as an active ingredient in the composition of the present invention refers to the root of the mulberry plant which is a deciduous tree belonging to the mulberry (Moraceae). In the case of obtaining the extract of the lettuce extract used in the composition of the present invention by treating the extract with the extract, various extracting solvents may be used. Preferably, a polar solvent or a nonpolar solvent can be used. Suitable polar solvents include (i) water, (ii) alcohols (preferably methanol, ethanol, propane, butanol, normal-propanol, iso-propanol, normal-butanol 1-pentane, 2-butoxyethane Or ethylene glycol), (iii) acetic acid, (iv) dimethylformamide (DMF0) and (v) DMSOCdimethyl sulfoxide (v). Suitable as nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane pentane, hexane, 2,2,4-trimethylpentane, decane, cyclonucleic acid, cyclopentane-tan, diisobutalene, 卜Pentene ： 卜 chlorobutane, 1 chloropentane, 0- Xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane annealed diethyl Amines, ethers, carbon tetrachloride and THF.
보다 바람직하게는， 본 발명에서 이용되는 추출용매는 (a) 물， (b) 탄소수 1-4의 무수 또는 함수 저급 알코을 (메탄올， 에탄올， 프로판올, 부탄올 등), (c) 상기 저급 알코올과 물과의 흔합용매， (d) 아세톤, (e) 에틸 아세테이트， (f) 클로로포름， (g) 부틸아세테이트， (h) 1,3- 부틸렌글리콜， (i) 핵산 및 (j) 디에틸에테르를 포함한다. 가장 바람직하게는， 본 발명의 추출물은 물， 에탄을 또는 이의 조합을 상백피에 처리하여 수득한 것이다.  More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) the lower alcohol and water A mixed solvent with (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) nucleic acid and (j) diethyl ether Include. Most preferably, the extract of the present invention is obtained by treating water, ethane, or a combination thereof on lettuce skin.
본 발명의 바람직한 구현예에 따르면， 본 발명의 상백피 추출물은 상백피를 건조시켜 분쇄하여 분말화한 후, 에탄을을 처리하고 균질화하여 수득한다. : According to a preferred embodiment of the present invention, the extract of baekbaekpi of the present invention is obtained by drying and grinding baekbaekpi, powdered, and then treated and homogenized ethane. :
균질화는 당업계에 공지된 다양한 방법을 통하여 실시할 수 있다. 바람직하게는 초음파기 (sonicator) 및 소닉디스멤브레이터 (sonic dismembrator)를 이용하여 균질화한다.  Homogenization can be carried out through various methods known in the art. Preferably it is homogenized using a sonicator and a sonic dismembrator.
본 명세서에서 사용되는 용어 '추출물' 은 상술한 바와 같이 당업계에서 조추출물 (crude extract)로 통용되는 의미를 갖지만， 광의적으로는 추출물을 추가적으로 분획 (fractionation)한 분획물도 포함한다. 즉， 상백피 추출물은 상술한 추출용매를 이용하여 얻은 것뿐만 아니라， 여기에 정제과정을 추가적으로 적용하여 얻은 것도 포함한다. 예컨대， 상기 추출물을 일정한 분자량 컷 -오프 값을 갖는 한외 여과막을 통과시켜 얻은 분획， 다양한 크로마토그래피 (크기， 전하， 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등， 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 상백피 추출물에 포함되는 것이다.  As used herein, the term 'extract' has a meaning commonly used as a crude extract in the art as described above, but also broadly includes a fraction additionally extracting the extract. In other words, the extract of baekbaekpi is not only obtained by using the above-described extraction solvent, but also includes the one obtained by additionally applying a purification process. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the extract of the lettuce skin.
본 발명의 바람직한 구현예에 따르면， 상백피 추출물에 탄산나트륨을 첨가하여 불용해성 물질을 완전히 용해시킨다. 바람직하게는 상백피 추출물에서 알코을 성분을 제거시킨 후， 탄산나트륨을 첨가하여 알코올 추출로 인한 불용해성 물질을 용해시키고， 건조 후 분말화하여 100% 수용화가 가능한 상태를 만든다. According to a preferred embodiment of the present invention, sodium carbonate is added to the extract of Morus alba to completely dissolve the insoluble material. Preferably, after removing the alcohol component from the extract of Morus alba, the sodium carbonate is added to dissolve insoluble substances due to alcohol extraction, and then dried and powdered to obtain 100%. Make a state of acceptability.
본 발명에서 이용되는 상백피 추출물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조될 수 있다.  The lettuce extract used in the present invention may be prepared in powder form by an additional process such as distillation under reduced pressure and freeze drying or spray drying.
본 명세서에서 용어 '유효성분으로 포함하는' 이란 하기의 상백피 추출물의 효능 또는 활성을 달성하는 데 층분한 양을 포함하는 것을 의미한다. 본 발명은 천연식물재료인 상백피로부터 추출한 조성물로서 과량 투여하여도 인체에 부작용이 없으므로 상백피 추출물이 본 발명의 조성물에 포함된 양적 상한은 당업자가 적절한 범위 내에서 선택하여 실시할 수 있다. 본 발명의 바람직한 구현예에 따르면， 본 발명의 조성물은 c-Fos의 발현을 감소시킨다. 조기발현유전자 (^mediate-early genes)인 c-Fos는 통증 등의 스트레스인자들과 관련된 신경로와 뇌활동의 양상을 추적하는 신경해부학적 표지자로 이용되는데, 신경전달물질에 의해 수 분내에 다양한 형태의 세포에서 활성화된다.  As used herein, the term 'comprising as an active ingredient' means to include an amount sufficient to achieve the efficacy or activity of the following extract of the lettuce extract. The present invention is a composition extracted from baekbaekpi, a natural plant material, so there is no side effect to the human body, even if the dose is excessive, the quantitative upper limit contained in the composition of the baekbaekpi extract can be carried out by those skilled in the art within the appropriate range. According to a preferred embodiment of the present invention, the composition of the present invention reduces the expression of c-Fos. The early-expression gene, c-Fos, is a neuroanatomical marker that tracks neuronal and brain activity associated with stress factors such as pain. It is activated in the form of cells.
본 명세서에서 용어 "(c-Fos 발현의) 감소" 는 본 발명의 조성물을 투여하지 않은 대상체 (대조군)에 비해 cᅳ Fos의 양이 측정 가능할 정도로 유의하게 감소된 것을 의미하며， 바람직하게는 30% 이상 감소된 것을 의미하고， 보다 바람직하게는 20% 이상 감소된 것을 의미하며， 보다 더 바람직하게는 10% 이상 감소된 것을 의미한다. 본 발명의 다른 양태에 따르면， 본 발명은 상백피 추출물을 유효성분으로 포함하는 우을증의 개선 또는 완화용 식품 조성물을 제공한다. 본 발명의 조성물이 식품 조성물로 제조되는 경우， 유효성분으로서 상백피 추출물뿐 만 아니라， 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어， 단백질, 탄수화물, 지방, 영양소ᅳ 조미제 및 향미제를 포함한다. 상술한 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드， 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리를, 소르비를, 에리트리를 등의 당알콜이다. 향미제로서 천연 향미제 [타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미게 (사카린， 아스파르탐 등)를 사용할 수 있다. As used herein, the term "reduction (of c-Fos expression)" means that the amount of c ᅳ Fos is measurably reduced compared to a subject (control) not administered the composition of the present invention, preferably 30 It means reduced by more than%, more preferably reduced by more than 20%, even more preferably reduced by more than 10%. According to another aspect of the present invention, the present invention provides a food composition for improving or alleviating depression, which comprises an extract of lettuce extract as an active ingredient. When the composition of the present invention is prepared as a food composition, as an active ingredient, as well as the extract of baekpipi, as well as ingredients commonly added in the manufacture of food, for example, protein, carbohydrates, fats, nutrients seasonings and flavors Includes the first. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and xyls, sorbitols, and erythrates. As flavoring agents, natural flavorings [tautin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring crabs (saccharin, aspartame) Etc.) can be used.
예컨대, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 상백피 추출물 이외에 구연산， 액상과당， 설탕, 포도당, 초산， 사과산， 과즙， 두충 추출액， 대추 추출액， 감초 추출액 등을 추가로 포함시킬 수 있다. 본 발명의 또 다른 양태에 따르면, 본 발명은 상백피 Cortex Radicis) 추출물의 약제학적 유효량을 포함하는 조성물을 대상 (subject)에 투여하는 단계를 포함하는 우울증의 예방， 개선, 완화 또는 치료 방법을 제공한다.  For example, when the food composition of the present invention is prepared with a beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc. have. According to another aspect of the invention, the invention provides a method of preventing, ameliorating, alleviating or treating depression comprising administering to a subject a composition comprising a pharmaceutically effective amount of an extract of Cortex Radicis .
【발명의 효과】 【Effects of the Invention】
본 발명의 특징 및 이점을 요약하면 다음과 같다:  The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 상백피 추출물올 유효성분으로 포함하는 우울증의 예방 또는 치료용 조성물올 제공한다.  (a) The present invention provides a composition for the prevention or treatment of depression, including as an extract of the extract epithelium.
(b) 본 발명의 조성물은 글루코코르티코이드 수용체의 핵 내 이동을 억제함으로써， 스트레스로 인해 유도되는 신호전달 과정을 저해하여 항스트레스 효과를 나타낸다.  (b) The composition of the present invention exhibits an antistress effect by inhibiting intracellular nuclei migration of glucocorticoid receptors, thereby inhibiting stress-induced signaling processes.
(c) 본 발명의 조성물은종래의 항우울제와 같이 글루코코르티코이드 수용체에 대한 길항제로써 효능을 가지고 있어， 항우울증 효능을 발휘할 수 있다.  (c) The composition of the present invention has an efficacy as an antagonist to the glucocorticoid receptor, like a conventional antidepressant, and can exhibit antidepressant efficacy.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 바이오스트레스 센서 및 대조 센서의 모식도를 나타낸 그림이다.  1 is a diagram showing a schematic diagram of a biostress sensor and a control sensor.
도 2는 바이오 스트레스 센서의 제작 모식도를 나타낸 그림이다. 도 3는 쥐가 내는 특정 영역대의 초음파를 모니터링 할 수 있는 소노트랙 시스템을 나타낸 그림이다.  Figure 2 is a diagram showing a manufacturing schematic diagram of the biostress sensor. 3 is a diagram showing a sonotrack system that can monitor the ultrasound of the specific area of the rat.
도 4는 HeLa 세포에서 스트레스 호르몬 (Cortisol) 및 항스트레스 호르몬 (RU486)에 대한 바이오스트레스 센서의 활성을 영상화한 그림이다. 도 5는 HeLa 세포에서 상백피 추출물의 바이오스트레스 센서 활성억제 효과를 농도별로 영상화한 그림이다. Figure 4 is an image of the activity of the biostress sensor for stress hormone (Cortisol) and antistress hormone (RU486) in HeLa cells. 5 is a biostress sensor of lettuce extract in HeLa cells This is a picture of the activity inhibitory effect by concentration.
도 6은 상백피 추출물이 GR의 핵 내 이동성에 미치는 효과를 나타낸 그림이다.  Figure 6 is an illustration showing the effect of the extract of lettuce on the mobility in the nucleus of GR.
도 7은 상백피 추출물이 동물의 피부에서의 GR활성에 미치는 효과를 나타낸 그림이다.  7 is a diagram showing the effect of the extract of Morus alba on GR activity in the skin of animals.
도 8은 상백피 추출물이 22-kHz대 초음파 발생에 미치는 영향을 나타낸 그림이다.  8 is a diagram showing the effect of the extract of baekhyeolpi on the generation of ultrasound in the 22-kHz band.
도 9는 상백피 추출물이 50-kHz대 초음파 발생에 미치는 영향을 나타낸 그림이다.  9 is a diagram showing the effect of the extract extracts on 50-kHz ultrasonic generation.
도 10은 상백피 추출물이 뇌의 해마 CA3 부위에서의 c-Fos 유전자 단백질 발현에 미치는 효과 (A: 정상대조군， B: 대조군, C: 스트레스 + RU486군， D: 스트레스 + MCR200군)를 나타낸 그림이다.  FIG. 10 is a diagram showing the effect of E. coli extract on c-Fos gene protein expression in hippocampal CA3 region of brain (A: normal control group, B: control group, C: stress + RU486 group, D: stress + MCR200 group) .
도 11은 상백피 추출물이 뇌의 시상하부 실방핵에서의 c-Fos 유전자 단백질 발현에 미치는 효과 (A: 정상대조군, B: 대조군， C: 스트레스 + RU486군, D: 스트레스 + MCR200군)를 나타낸 그림이다.  FIG. 11 is a diagram showing the effect of lettuce extract on c-Fos gene protein expression in the hypothalamic chamber nucleus of the brain (A: normal control, B: control, C: stress + RU486 group, D: stress + MCR200 group) to be.
【발명을 실시하기 위한 구체적인 내용】 [Specific contents to carry out invention]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. 실시예  Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . EXAMPLE
실험재료 및 방법 Experimental Materials and Methods
상백피 추출물 제조 Lettuce extract
강원도 홍천에서 채취하여 건조시킨 상백피 (ifor/ ( r ay Radicis)를 경동시장 (주)갑당약초 (한국)로부터 구입하여 수세 후 50°C 열풍건조기에서 2일간 건조시켜 분쇄기 (Waring Commercial, 미국)를 사용하여 분말화 하였다. 분말 상태의 상백피 500 g에 95% 주정 (발효 에탄올) 5 L를 투입하여 10%(w/v) 농도가 되게 하여 50°C의 항온교반기 (Vision Scientific Co., 한국) 및 초음파 발생장치 (Branson 5510, 미국)를 번갈아 사용하며 48시간 동안 추출하였다. 상백피 활성 물질의 추출을 좀 더 용이하게 하기 위해 추출과정 중 초음파 파쇄기 (Fisher Scientific, ModellOO)를 강도 7로 두어 20분씩 총 4회 처리하였다. 추출 후 여과지 (0.45 um, Millipore membrane filter, 미국)로 여과한 후 여과액 총량의 30%에 해당되는 증류수를 첨가하여 회전농축기 (Buchi Labor technik AG, 스위스)로 주정 성분을 제거시키고， 증류수만 남은 상태가 되었을 때 동결건조기 (IlshinBioBase, 한국)로 건조시켜 분말화 하였다. After harvesting the dried Sangbaekpi (ifor / (r ay Radicis) from Hongcheon, Gangwon-do, Korea from Kapdong Herb Co., Ltd. (Korea), after washing with water, it is dried for 2 days in a 50 ° C hot air dryer and then crushed (Waring Commercial, USA). 5 g of 95% spirit (fermented ethanol) was added to 500 g of powdered baekbaekpi in 10% (w / v) incubator at 50 ° C (Vision Scientific Co., Korea) And alternating ultrasonic generators (Branson 5510, USA) Extraction for 48 hours. In order to make the extraction of the epithelium active material easier, the ultrasonic crusher (Fisher Scientific, ModellOO) was placed at an intensity of 7 and was treated four times for 20 minutes. After extraction, the filter was filtered through a filter paper (0.45 um, Millipore membrane filter, USA), and distilled water (30% of the total amount of the filtrate) was added to remove alcohol by using a rotary concentrator (Buchi Labor technik AG, Switzerland). When it was in the state was dried and lyophilized (IlshinBioBase, Korea) to powder.
동물실험을 위한 상백피 추출물 시료 준비에 있어서는 동일 방법으로 추출하여 주정 성분을 완전히 제거시키고, 증류수만 남은 상태가 되었을 때 예비실험을 통해 예측된 수율을 고려하예 전체 수율의 5-10% 함량의 식용 탄산나트륨을 첨가하여 주정추출로 인한 불용해성 물질까지 완전히 용해시킨 후, 동결건조기 (IlshinBioBase, 한국)로 건조시켜 분말화하여 100% 수용화 가능한 상태로 만들었다. 바이오스트레스 센서 제작 및 생산  In the preparation of baekbaekpi extract samples for animal experiments, the extracts were extracted in the same way to completely remove alcoholic ingredients. When only distilled water remained, considering the yield predicted through preliminary experiments, edible sodium carbonate containing 5-10% of the total yield was obtained. After dissolving completely insoluble matters due to alcohol extraction by the addition, it was dried with a lyophilizer (IlshinBioBase, Korea) and powdered to make 100% soluble. Biostress sensor manufacture and production
모체 백터， pWPXUAddgene, 미국)에 5회 반복서열을 갖는 글루코코르티코이드 반웅 인자 (Glucocorticoid response element: GRE, 5 '-AGAGGATCTGTACAGGATGTTCTAGAT-3' )와 이에 의해 발현이 조절되어지는 루시퍼라아제 유전자를 삽입하여 전달 백터 (바이오센서 전달체)를 제작하였다 (도 1). 바이오센서 전달체를 포장백터， psPAX2(Addgene, 미국), 그리고 엔벨로프 백터， pMD2G(Addgene, 미국)를 각각 5 ug, 3.75 g 그리고 1,25 을 SuperFect Trans feet ion Reagent (Qiagen, 미국)를 이용하여 100 mm 배양용기에서 키운 293T 세포에 삽입하였다 (도 2， 과정 1). 48， 72시간 동안 배양된 세포에의 배양액 각각을 얻어 7,000 rpm의 중력가속기에서 30 분간 원심분리 한 후 상등액을 0.45 μιιι 주사기용 필터 (millipore, 미국)로 통과시켜서 스트레스 센서를 생산하였다 (도 2， 과정 2). 생산된 발광 발현 스트레스 센서를 스트레스 호르몬 수용체 (glucocorticoid receptor, GR)가 존재하는 인간유래의 HeLa 세포에 각각 감염 (Infection)시켰다 (도 2， 과정 3 및 4). 그런 다음， 발광유전자가 스트레스 호르몬에 반웅하여 발광단백질 발현을 유도하는지를 확인하기 위해 센서에 감염된 세포에 스트레스 호르몬 (glucocorticoid)을 첨가한 후 30-40시간 경과 후 발광 단백질의 발현을 형광 영상장비인 IVIS200(Xenogen Ins, 미국)을 이용하여 영상화 및 정량화 하였다 (도 2, 과정 5 및 6). 이때 음성대조구로서 5X GRE가 제거된 전달 백터 (AGRE)를 사용하였다 (도 1). Transfer vector by inserting glucocorticoid response element (GRE, 5'-AGAGGATCTGTACAGGATGTTCTAGAT-3 ') with 5 repetitions into the parent vector, pWPXUAddgene, USA) (Biosensor carrier) was produced (FIG. 1). Biosensor carriers were packaged, psPAX2 (Addgene, USA), and envelope vectors, pMD2G (Addgene, USA), 100 ug, 3.75 g, and 1,25, respectively, using SuperFect Trans feet ion Reagent (Qiagen, USA). Inserted into 293T cells grown in mm culture vessel (Fig. 2, Procedure 1). Each culture solution was cultured for 48 and 72 hours, centrifuged at 7,000 rpm for 30 minutes, and the supernatant was passed through a 0.45 μιιι syringe filter (millipore, USA) to produce a stress sensor (FIG. 2 ，). Process 2). The produced luminescent expression stress sensor was infected with human-derived HeLa cells in which the glucocorticoid receptor (GR) is present (FIG. 2, processes 3 and 4). Then, after adding the stress hormone (glucocorticoid) to the cells infected with the sensor to check whether the luminescent gene reacts with the stress hormone to induce the expression of the luminescent protein, After 30-40 hours, the expression of luminescent proteins was imaged and quantified using IVIS200 (Xenogen Ins, USA), a fluorescent imaging device (FIG. 2, Processes 5 and 6). At this time, a transfer vector (AGRE) from which 5X GRE was removed was used as a negative control (FIG. 1).
글루코코르티코이드 수용체 (glucocorticoid receptor, GR)의 활성을 인식하는 바이오스트레스 센서 (GRE)와 대조센서 (AGRE)를 유전자 수준에서 제작하였으며 (도 1)， 이를 293T 세포를 이용하여 센서를 생산하였다. 또한， 바이오스트레스 센서의 활성효과는 HeLa 세포에서 발광단백질 (luciferase)의 발현을 통해 확인하였다 (도 2). 상백피 주정 추출물의세포 내 항스트레스 기전 분석  Biostress sensors (GRE) and control sensors (AGRE), which recognize the activity of glucocorticoid receptors (GR), were produced at the gene level (FIG. 1), which produced sensors using 293T cells. In addition, the active effect of the biostress sensor was confirmed through the expression of a light emitting protein (luciferase) in HeLa cells (Fig. 2). Intracellular Antistress Mechanism Analysis of Extracts from Epidermis
상백피 주정 추출물이 스트레스에 미치는 영향을 글루코코르티코이드 수용체가 발현되는 HeLa 세포에서 상백피 주정 추출물을 처리하여 그에 따른 바이오센서의 활성을 비교하였다. 96—웰에 1X104 세포를 배양하고 24시간 경과 후 바이오센서를 전달하여 12시간 37°C에서 배양하였고， DMS0에 녹인 상백피 추출물을 농도별로 3시간 전처리한 후 글루코코르티코이드를 500 nM 처리하여 수용체의 활성올 유도하였다/ 36시간 경과 후 IVIS200을 이용하여 바이오센서 활성을 영상화, 정량화 하였다. 또한 글루코코르티코이드의 길항제인 항스트레스 호르몬 (RU486, Sigma, 미국)을 바이오센서 삽입된 HeLa 세포에 처리 3 시간 경과 후 글루코코르티코이드를 500 nM 처리하여 발광단백질 발현 억제효과를 확인하였다 (도 4B). 형광 이미징 The effect of ethanol extracts on the stress on the stress was compared with ethanol extracts from HeLa cells expressing glucocorticoid receptors. After incubating 1 × 10 4 cells in 96-well and passing the biosensor for 24 hours, the cells were incubated at 37 ° C. for 12 hours, and pretreated with epithelial extract dissolved in DMS0 for 3 hours, followed by 500 nM of glucocorticoids. Induction of activator / After 36 hours, biosensor activity was imaged and quantified using IVIS200. In addition, anti-stress hormone (RU486, Sigma, USA), an antagonist of glucocorticoids, was treated with 500 nM of glucocorticoids after 3 hours of treatment with biosensor-inserted HeLa cells to confirm the inhibitory effect of luminescent protein expression (FIG. 4B). Fluorescence imaging
형광 표지된 GR(GR-EGFP)을 GR이 자체적으로 발현되지 않는 C0S-1 세포에 일시적 형질주입 방법 (Effect ine, Qiagen)을 통해 삽입하였다. 24시간 세포배양을 통해 녹색형광이 발현되었는지를 공초점 현미경을 이용하여 확인한 후 상백피 주정 추출물을 처리하고, 3시간 경과 후 100 nM의 스트레스 호르몬 (Cortisol)을 30분 동안 처리하였다. 형광발현 세포를 4% 파라포름알데하이드로 고정한 후 DraQ를 이용하여 핵 염색과정을 거쳐 공초점 현미경을 이용하여 형광표지 GR의 핵 내 이동 이미지를 획득하였으며， LSM 이미지 분석기 (Zeiss, 독일)를 이용하여 세포 전체의 형광 강도에서 핵 안의 형광 강도에 대한 비율을 분석함으로써 핵 내 이동 정도를 평가하였다. 동물실험 (In vivo assay) Fluorescently labeled GR (GR-EGFP) was inserted into C0S-1 cells that did not express GR itself via transient transfection method (Effect ine, Qiagen). Confocal microscopy was used to determine whether green fluorescence was expressed through cell culture for 24 hours, followed by treatment with extracts from the epidermis. After 3 hours, 100 nM of stress hormone (Cortisol) was treated for 30 minutes. Fluorescent cells were fixed with 4% paraformaldehyde and nuclear stained using DraQ, followed by confocal microscopy to obtain nuclear transfer images of fluorescently labeled GR, using an LSM image analyzer (Zeiss, Germany). Whole cell The degree of migration in the nucleus was evaluated by analyzing the ratio of the fluorescence intensity to the fluorescence intensity in the nucleus. In vivo assay
항스트레스 효능 분석올 위해 실험에 사용된 쥐는 8주령 Wistar 수컷 흰쥐로 (주)중앙실험동물 (한국)에서 분양받아 사육실에서 일주일간 적웅기간을 갖도록 한 후 실험에 사용하였다. 각 군당 8마리씩 난괴법 (randomized com lete block design)으로 분류하였으며, 한 케이지 당 두 마리씩 사육하였다. 실험에서 분류한 군으로는 정상대조군 (ND), 스트레스 대조군 (증류수 투여군)， 항스트레스제 투여군 (스트레스 + RU486; 체중 1kg 당 RU486 10 mg 투여군)ᅳ 스트레스 + 상백피 100투여군 (스트레스 + MCR100; 체중 1kg 당 상백피 추출물 100 mg 투여군)， 스트레스 + 상백피 200 투여군 (스트레스 + MCR200; 체중 1kg 당 상백피 추출물 200 mg 투여군)으로 분류하였으며 정상대조군을 제외한 모든 실험군은 매일 한차례 일정한 시간인 오전 9-10시 사이에 상백피 주정 추출물 시료를 농도별로 체중에 비례하여 정확하게 맞추어 , 증류수에 녹인 후 실험 방법에 따라 M일간 경구투여 하였으며 대조군도 같은 시간 증류수로 경구투여 하였다. 또한, 생체 내 형광영상법을 이용한 동물실험의 경우， 8주령의 Wistar 수컷 흰쥐를 1주일간 적웅시킨 후, 그룹 당 7마리로 나누어 5일간 스트레스 + 상백피 50투여군 (스트레스 + MCR50; 체중 1 kg 당 상백피 추출물 50 mg 투여군, 스트레스 + 상백피 100 투여군 (스트레스 + MCR100; 체중 1 kg 당 상백피 추출물 100 mg 투여군, 스트레스 + 상백피 200 투여군 (스트레스 + MCR200; 체중 1 kg 당 상백피 추출물 200 mg 투여군)으로 나누어 실험을 진행하였다. 사육실의 온도와 습도는 각각 22±0.5°C, 55±5%로 조절하였고 명암주기는 12시간으로 조절하였으며， 모든 실험동물과 관계된 사육관리는 한국식품연구원 동물실험 윤리위원회 (IACUC)의 승인과 관리감독 하에 이루어졌다. 피부에서의 바이오스트레스 센서 활성 평가 Mice used in the experiment for the analysis of anti-stress efficacy were 8-week-old Wistar male rats, which were distributed from a central experimental animal (Korea), and had a weekly dip period in the nursery. Eight animals per group were categorized as randomized com lete block design and two were bred per cage. The groups classified in the experiment were normal control group (ND), stress control group (distilled water group), antistress group (stress + RU486; RU486 10 mg / kg body weight) ᅳ stress + epithelium 100 group (stress + MCR100; body weight 1 kg) The group was divided into 100 mg of sugar extract and 100 mg of stress + 200 extract (stress + MCR200; 200 mg extract per 1 kg of body weight). All experimental groups except the normal control group had a daily time between 9 and 10 am The alcohol extract samples were accurately adjusted in proportion to the weight of each concentration, dissolved in distilled water and orally administered for M days according to the experimental method, and the control group was orally administered with distilled water at the same time. In addition, in the case of animal experiments using in vivo fluorescence imaging, 8-week-old Wistar male rats were depressed for 1 week, divided into 7 animals per group for 5 days of stress + M. bark 50 groups (stress + MCR50; M. bark per 1 kg body weight). The experiment was divided into 50 mg of the extract administration group, stress + epidermis 100 administration group (stress + MCR100; 100 mg of the extract extract per 100 kg body weight, 100 mg of stress + epidermis extract), 200 groups of stress + epidermis extract (stress + MCR200; 200 mg of extract extract per 1 kg body weight) The temperature and humidity of the breeding room were adjusted to 22 ± 0.5 ° C and 55 ± 5%, respectively, and the contrast period was adjusted to 12 hours. All animal-related care was controlled by the Korea Food Research Institute's Animal Experiment Ethics Committee (IACUC). Acknowledgment and supervision: Assessment of biostress sensor activity in skin
생체 내에서 바이오센서의 활성을 탐색하기 위해 피부에 바이오센서를 피하주사 하는 방법으로 주입하여 15일 동안 안정적으로 바이오센서가 피부세포에 삽입되도록'유도하였다. 바이오센서 주입 후 약In order to detect the activity of biosensor in vivo, it is injected into skin by subcutaneous injection method stably for 15 days. 'Induce the biosensor to be inserted into the skin cells. After Biosensor Injection
15-20일 경과 후 상백피 추출물을 일일 1회 5일간 투여하였으며， 마지막 5일째에 감금 스트레스를 2시간 부여하고 D-루시페린 (1 kg 당 75 mg)을 복강 주사하여 15분 경과 후 바이오스트레스 센서 활성을 생체영상장비 (IVIS-200, Xenogen)를 이용하여 모니터링 하였으며， 영상 활성을 R0I 값으로 정량화함으로써 스트레스에 의한 반웅성을 생체 내에서 확인하였다. 초음파 영역대 분석 After 15-20 days, the epidermis extract was administered once a day for 5 days. On the last 5 days, the biostress sensor was activated after 15 minutes by giving 2 hours of confinement stress and intraperitoneal injection of D-luciferin (75 mg / kg). Was monitored using a biological imaging device (IVIS-200, Xenogen), and the reaction caused by stress was confirmed in vivo by quantifying the image activity by the R0I value. Ultrasound Zone Analysis
초음파 영역대 분석은 8주령 Wistar 수컷 흰쥐를 일주일간 적웅시키고， 정상군을 제외한 모든 실험군은 매일 한차례 일정한 시간인 오전 9ᅳ 10시 사이에 상백피 주정 추출물을 농도별로 체중에 비례하여 정확하게 맞추어, 증류수에 녹인 후 실험 방법에 따라 14일간 경구투여 하였으며 2시간 동안 감금장치를 이용하여 감금스트레스를 부여하고 소노트랙 (Metris, 네덜란드) 4 채널의 초음파 감지 장치 (microphones)를 이용하여 쥐의 감정 상태에 따라 변화되는 초음파 영역대를 20분 동안 모니터링 하면서 분석하였다 (도 3). 소노트랙은 15-125 kHz까지 감지할 수 있으며， 초음파는 주령， 환경, 감정 상태에 따라 달라지는데 일반적으로 쥐는 편안한 상태일 때 50-kHz 발성을, 공포나 스트레스 상황에 처하면 22- kHz 발성을 내는 것으로 알려져 있어 이것을 기준으로 상백피 추출물 섭취에 따른 항스트레스 효과를 쥐가 방출하는 초음파 영역대를 통해 알아보고자 하였다. 뇌 적출  Ultrasonic zone analysis was performed to depress 8-week-old male Wistar rats for a week. After dissolution, it was orally administered for 14 days according to the experimental method. For 2 hours, confinement stress was imparted by using a cage and changed according to the emotional state of rats by using sonotrack (Metris, Netherlands) 4-channel ultrasonic sensors (microphones). The ultrasound zone was analyzed for 20 minutes while monitoring (Figure 3). Sonotracks can detect up to 15-125 kHz, and ultrasound varies with age, environment, and emotional state. Generally, a 50-kHz vocalization occurs when the mouse is in a relaxed state, and a 22-kHz vocalization under fear or stress conditions. The anti-stress effect of the intake of lettuce extract was known based on the ultrasound zone that rats emit. Brain extraction
모든 행동실험이 끝난 직후 실험동물올 소듬 펜토바르비탈 (80 mg/kg, i.p.)을 주사하여 마취시키고, 뇌 적출올 위해 생리식염수 150 mL을 분당 30 mL 유속으로 심장을 통해 관류시키고, 이어 10% 포르말린 (Sigma, 미국)을 고정액으로 분당 25 mL 유속으로 300-400 mL을 심장을 통해 관류시켰다. 그 다음 뇌를 꺼내어 동일 고정액으로 24시간 동안 후고정 시킨 후 25%수크로오스가 함유된 PBS(phosphate buffered saline, 0.01 M, pH 7.4)에 넣어 4°C에서 36-40시간 동안 뇌가 침전될 때까지 방치하였다. 침전된 뇌는 크라이오 -스프레이를 이용하여 급속히 넁동시킨 후 마이크로톰 (Leica, 독일)을 이용하여 뇌조직 중 해마, 시상하부 부위를 30 Li m의 두께로 절편하여 c-Fos 유전자 발현 연구에 사용하였다. c-Fos 단백질 면역조직화학염색 Immediately after the completion of all behavioral experiments, anesthetized by injecting experimental animal allergens pentobarbital (80 mg / kg, ip), and 150 mL of saline was perfused through the heart at a flow rate of 30 mL per minute for brain extraction. Formalin (Sigma, USA) was perfused through the heart with 300-400 mL at a flow rate of 25 mL per minute. The brain is then removed, post-fixed with the same fixative for 24 hours, and then placed in PBS (phosphate buffered saline, 0.01 M, pH 7.4) containing 25% sucrose until the brain precipitates for 36-40 hours at 4 ° C. It was left. Precipitated brain was rapidly shaken by cryo-spray and then micro-tome (Leica, Germany) was used for c-Fos gene expression study by cutting the hippocampus and hypothalamus into 30 Lim thickness of brain tissue. . c-Fos protein immunohistochemical staining
뇌조직은 PBST(PBS + 0.1% 트리톤 x-100) 용액에 3회 씻은 후 c-Fos 관련 1차 항체로는 토끼 다클론 IgG(Santa Cruz biotechnology, 미국)를 PBST를 이용하여 500배 희석하여 준비하였다. 뇌조직은 PBST를 이용하여 3회 씻고， 블로킹 혈청 (PBST 10 mL에 노말 고트 혈청을 3방울 떨어뜨림 )을 처리하여 실온에서 1시간 지속적으로 흔들어주면서 배양시킨 후, 1차 항체로 4°C에서 24시간 지속적으로 흔들어 주면서 배양하였다. 그 후 조직을 PBST로 다시 3회 씻은 후， 2차 항체 (노말 고트 혈청을 3방울 + PBST 10 mL에 항 -토끼 IgG 바이오티닐화 항체 1방울 첨가)에 옮겨 담아 상온에서 1시간 동안 지속적으로 흔들어 주면서 배양하였다. 조직을 PBST에 3회 세척한 후， 상온에서 30분 동안 백타스테인 ABC-키트를 사용하여 ABC 시약 (A 용액 4방울 + PBST 10 mL에 B 용액 4방을 첨가)에 옮겨 담아 지속적으로 흔들어 주면서 배양하였다. PBS로 3회 이상 행군 후， 착색제로서 c-Fos 발현은 DAB(diaminobenzidine) 용액 (DAB 기질 키트， Vector lavoratories, 미국)을 사용하여 발색시켰다. 모든 처리가 끝난 뇌조직을 PBS에 옮겨 담고 증류수로 세척하여 슬라이드에 고정하여 건조시킨 후， 마운팅 용액 (Thermo Scientific, 미국)을 한 방울 떨어뜨려 커버글라스를 덮고 완전히 건조한 후 광학현미경 (light microscope magnificat ion)으로 40 배， 100 배 확대하여 c-Fos 면역반웅 신경세포를 관찰하였다. 이미지는 AxioVision 3.0 이미징 시스템 (Carl Zeiss Inc. , 독일)으로 캡쳐하여 4X4 cm 크기의 4각 격자를 사용하여 해마 CA3 부위 및 시상하부의 실방핵에서의 c-f0S 면역반웅 신경세포의 수를 측정하였다. 실험결과 및 고찰 Brain tissue was washed three times in PBST (PBS + 0.1% Triton x-100) solution and prepared by diluting rabbit polyclonal IgG (Santa Cruz biotechnology, USA) with PBST 500 times as the primary antibody for c-Fos. It was. Brain tissues were washed three times with PBST, treated with blocking serum (3 drops of normal goth serum in 10 mL of PBST), incubated with shaking for 1 hour at room temperature, and then incubated at 4 ° C with primary antibody. Incubation was continued for 24 hours. Then wash the tissue three times again with PBST, transfer to secondary antibody (3 drops of normal goth serum + 1 drop of anti-rabbit IgG biotinylated antibody in 10 mL of PBST) and shake continuously for 1 hour at room temperature. Incubated while giving. The tissues were washed three times in PBST, and then transferred to ABC reagent (4 drops of A solution + 4 drops of B solution in 10 mL of PBST) using Baxtastein ABC-Kit for 30 minutes at room temperature. It was. After three or more times in PBS, c-Fos expression was developed using DAB (diaminobenzidine) solution (DAB substrate kit, Vector lavoratories, USA) as colorant. All processed brain tissues were transferred to PBS, washed with distilled water, fixed on slides, dried, and then covered with a drop of mounting solution (Thermo Scientific, USA), covered with a cover glass, and completely dried, followed by a light microscope magnificat ion. ), 40-fold, 100-fold magnification was observed for c-Fos immune response neurons. Images were captured with an AxioVision 3.0 imaging system (Carl Zeiss Inc., Germany) to measure the number of cf 0S immunoreaction neurons in the hippocampal CA3 site and hypothalamus chamber nucleus using a 4 × 4 cm square grid. Experimental Results and Discussion
세포 내 바이오스트레스 센서의 활성 측정 Activity measurement of intracellular biostress sensors
제작된 바이오스트레스 센서를 세포내에서 평가하기 위하여 HeLa 세포에 바이오스트레스 센서 (GRE) 혹은 대조센서 (Δ ?Ε)를 각각 삽입시킨 후 스트레스 호르몬 (Cortisol)을 처리하였을 때 루시퍼라아제 발현 양을 IVIS-200 장비를 이용하여 측정함으로써 바이오스트레스 센서의 활성을 영상화 및 정량화 하였다. 대조센서에서는 호르몬의 처리에도 루시퍼라아제의 발현 변화가 없는 반면에 바이오스트레스 센서의 경우는 스트레스 호르몬의 농도에 비례하여 루시퍼라아제의 발현이 증가되어 스트레스 활성 또한 증가하였음을 확인할 수 있었다 (도 4). 또한 GR에 길항작용을 갖는 RU486을 처리하였을 때는 호르몬에 의해 증가된 활성이 농도 의존적으로 감소하는 것을 확인하였다. 이러한 농도 의존적 영상 결과로부터 바이오스트레스 센서가 스트레스 호르몬에 의해 유도된 활성을 정량적으로 측정할 수 있는 적절한 센서임을 확인할 수 있었으며， 세포 수준에서 이 시스템의 적용은 향후 항스트레스성 식품소재 개발을 위한 스크리닝에 유용하게 이용될 수 있음을 시사한다. 그 후， 상백피 추출물에 대해 바이오스트레스 센서 활성억제 효과를 농도별로 측정하였다. 그 결과 상백피 추출물은 농도 의존적으로 바이오스트레스 센서의 활성을 억제시키는 것으로 나타났다. 상백피 추출물 10 g/mL의 낮은 농도에서도 78¾의 바이오스트레스 센서 활성 억제 효과를 보였다 (도 5). 상백피 추출물의 세포내 항스트레스성효능의기전 규명 In order to evaluate the manufactured biostress sensor intracellularly, a biostress sensor (GRE) or a control sensor (Δ? Ε) was inserted into HeLa cells, respectively. When the post-stress hormone (Cortisol) was treated, the amount of luciferase expression was measured using an IVIS-200 device to image and quantify the activity of the biostress sensor. In the control sensor, there was no change in the expression of luciferase even in the treatment of hormones, whereas in the biostress sensor, luciferase expression was increased in proportion to the concentration of the stress hormone, thereby increasing the stress activity (FIG. 4). ). In addition, it was confirmed that when RU486, which has an antagonistic action on GR, the activity increased by hormone decreases in a concentration dependent manner. These concentration-dependent imaging results confirm that the biostress sensor is an appropriate sensor to quantitatively measure the stress hormone-induced activity.The application of this system at the cellular level is useful for screening for future antistress food material development. It suggests that it can be usefully used. Thereafter, the effect of inhibiting biostress sensor activity was measured for each concentration. As a result, the extract was inhibited in the concentration-dependent biostress sensor activity. Even at a low concentration of 10 g / mL of the extract of lettuce extract showed a 78¾ biostress sensor inhibitory effect (Fig. 5). Investigation of the Mechanism of Intracellular Antistress Efficacy
바이오스트레스 센서의 활성은 크게 세 단계를 거쳐 일어나게 되는데 첫째는 글루코코르티코이드 리간드가 세포질에 존재하는 불활성화 수용체 (GR)와 결합하는 단계 (리간드 결합)이고， 둘째는 호르몬 결합에 의해 활성화된 수용체가 핵 속으로 들어가는 단계 (translocation) 그리고 셋째는 글루코코르티코이드 반웅 인자 (GRE)에 수용체가 결합함으로써 표적유전자 전사를 유도하는 단계 (transact ivat ion)이다. 상백피 추출물의 스트레스 센서 활성억제 효과의 기전을 규명하기 위해 센서 수용체인 GR의 핵 내 이동성을 측정하였다. 녹색형광 유전자 (Green fluorescence protein; GFP)를 리포터로 하는 GR(EGFP-GR)을 C0S-1 세포에서 발현시키고， 코르티졸 (100 nM) 혹은 2.5 g/mL 상백피 추출물 전처리 후 코르티졸을 처리하여 30분경과 후 핵이동 변화를 공초점 현미경으로 확인하였다. 그 결과 코르티졸 처리에 의해 GR의 핵내 이동성은 2.76±0.07배 증가한 반면, 코르티졸만 처리한 경우보다 상백피 추출물을 전처리한 경우 코르티졸에 반웅한 GR의 핵내 이동성은 1.96±0.29배의 억제효과를 나타냈다 (도 6). 이로써 상백피의 바이오스트레스 센서 활성억제 효과는 호르몬 결합 저해효과보다는 핵 속으로의 전달 억제에 기인함을 확인할 수 있었다. 상백피 추출물의 피부에서의 바이오스트레스 센서 활성 평가 The activity of the biostress sensor occurs in three major steps: first, the glucocorticoid ligand binds to the inactivating receptor (GR) in the cytoplasm (ligand binding), and second, the receptor activated by hormone binding is the nucleus. Translocation and third is the transduction of the target gene transcription by binding the receptor to glucocorticoid reaction factor (GRE). In order to elucidate the mechanism of the inhibitory effect of stress sensor activity on the extracts of the lettuce extract, the intranuclear mobility of GR, the sensor receptor, was measured. GR (EGFP-GR), a reporter using the green fluorescence protein (GFP), was expressed in C0S-1 cells and treated with cortisol after pretreatment with cortisol (100 nM) or 2.5 g / mL epidermis extract. Post nuclear shift was confirmed by confocal microscopy. As a result, the intranuclear mobility of GR was increased by 2.76 ± 0.07 times by cortisol treatment, whereas cortisol was pretreated with cortex extract than cortisol alone. Intranuclear mobility of the reacted GR showed an inhibitory effect of 1.96 ± 0.29 fold (FIG. 6). As a result, it was confirmed that the inhibitory effect of biostress sensor activity of baekbaekpi was due to the inhibition of transfer into the nucleus rather than the inhibitory effect of hormone binding. Evaluation of Biostress Sensor Activity of Skin Extracts
상백피 추출물이 생체 내 바이오스트레스 센서 활성에 미치는 영향을 조사하기 위해 피부에 센서를 피하주사 하는 방법으로 주입하여 20일 동안 안정적으로 바이오스트레스 센서가 각질세포에 삽입되도록 유도하였다. 총 3개의 그룹을 나누어 생체 내에서 바이오스트레스 센서의 스트레스에 대한 활성을 탐색하였다. ① 음성대조군: PBS 만을 약물주사와 등일한 방법으로 5일 동안 매일 1회 주입하였다. ② 대조군 (스트레스 + PBS): PBS를 약물주사와 동일한 방법으로 감금하기 5일 전부터 매일 1회, 5일 동안 주입하였으며， 5일째 2시간 동안의 감금 (Restraint)을 통해 스트레스를 유발하였다. ③ 스트레스 + MCR100군: 체중 1 kg 당 상백피 추출물을 100 mg 용량으로 감금하기 5일 전부터 매일 1회， 5일 동안 주입하였으며， 5일째 2시간 동안의 감금을 통해 스트레스를 유발하였다.  In order to investigate the effect of the extract of E. coli on biostress sensor activity in vivo, the method was injected subcutaneously into the skin to induce the biostress sensor to be stably inserted into keratinocytes for 20 days. A total of three groups were divided to explore the stress activity of the biostress sensor in vivo. ① negative control group: PBS only was injected once daily for 5 days by the same method as drug injection. ② Control group (stress + PBS): PBS was injected once a day for 5 days from 5 days before confinement in the same manner as drug injection, and stress was induced through restraint (Restraint) for 5 hours on the 5th day. ③ stress + MCR100 group: Inject the baekpipi extract per 100 kg body weight to 100 mg dose once a day 5 days before, 5 days, 5 days 2 days to induce stress through confinement.
각 그룹 별로 2시간 동안 감금 스트레스를 주고, 스트레스에 의한 바이오센서의 영상을 IVIS-200 장비를 이용하여 모니터링 하였으며， 영상 활성을 R0I 값으로 정량화함으로써 스트레스에 의한 반웅성을 생체 내에서 확인하였다. 그 결과 감금 스트레스만을 준 경우 (대조군)， 음성대조군보다 바이오스트레스 센서의 활성이 증가하는 것을 확인할 수 있었으며， 미리 상백피 추출물을 주입하고 감금 스트레스를 준 경우 (MCR100)는 바이오스트레스 센서의 활성 변화가 대조군인 스트레스만 준 군보다 떨어지는 것을 확인 하였다 (도 7). 이 결과는 상백피 추출물의 스트레스에 대한 GR의 활성 변화를 생체 내에서 관찰한 최초의 결과이며， 상백피 추출물이 항스트레스 효과를 가지고 있음을 시사한다. 초음파 영역분석을 통한상백피 추출물의 항스트레스 효과  Each group was given confinement stress for 2 hours, and the image of the biosensor due to stress was monitored using the IVIS-200 equipment, and the reaction by stress was confirmed in vivo by quantifying the image activity by the R0I value. As a result, only the restraint stress (control), the activity of the biostress sensor was increased compared to the negative control group, and injecting the extract of the epidermis and confinement stress in advance (MCR100), the change in the activity of the biostress sensor was the control group Phosphorus stress was confirmed to fall from the group given only (Fig. 7). This result was the first to observe the change in the activity of GR against stress of the extract of the lettuce extract, suggesting that the extract has anti-stress effect. Antistress Effect of Epidermis Extract by Ultrasonic Region Analysis
상백피 추출물을 14 일간 경구 투여시키고 2시간 동안 감금스트레스를 부여한 후 소노트렉을 이용하여 쥐의 초음파 영역대를 모니터링한 결과 스트레스를 부여하지 않은 정상군의 쥐에서는 스트레스를 받지 않는 긍정적인 상태에서 발생되는 초음파인 5으 kHz 발성 (vocalization)이 스트레스 상황인 부정적인 상태에서 발생되는 초음파인 22-kHz 발성보다 현저하게 많이 발생되는 것을 관찰할 수 있었다. 또한 RU486 감금스트레스 + 항우울제 투여)군에는 단지 증류수만 급여하며 감금스트레스를 부여한 대조군군에 비해 긍정적인 상태에서 발생되는 초음파가 15.3%에서 56.8%로 증가됨으로써 항우울제에 의한 빠른 스트레스 회복을 관찰할 수 있었다. MCR100 및 MCR200군 (감금스트레스 + 상백피 추출물 체중 1 kg 당 100 mg 또는 200 mg 투여군)에서는 초음파가 각각 25.6%, 35.5¾>로 항우울제만큼은 아니었지만， 50-kHz 발성이 증가하고, 상백피 추출물을 200 mg/kg 농도로 급여한 군에서는 부정적인 상태에서 발생되는 22— kHz 발성이 감소됨으로써 항스트레스 효과를 관찰할 수 있었다 (도 8， 9). 상백피 추출물이 면역조직화학염색법을 통한 시상하부 및 해마에서의 c- Fos유전자 단백질 발현에 미치는 영향 After 14 days of oral administration of lettuce extract and confinement stress for 2 hours, sonotrec was used to monitor the ultrasound area of the rats. It was observed that 5 kHz vocalization, an ultrasonic wave generated in a positive state that was not received, occurred more significantly than 22-kHz vocalization, an ultrasonic wave generated in a negative state, which is a stress situation. In addition, the RU486 confinement stress + antidepressant treatment) group was fed only distilled water and the ultrasound generated in a positive state increased from 15.3% to 56.8% compared to the control group to which confinement stress was applied. . In the MCR100 and MCR200 groups (100 mg or 200 mg per kg body weight of the restraint stress + epidermis extract), the ultrasound was 25.6% and 35.5¾>, respectively, which was not as antidepressant but increased 50-kHz vocalization and 200 mg of epidermis extract. In the group fed / kg concentration, anti-stress effect was observed by decreasing 22-kHz vocalization occurring in a negative state (FIGS. 8 and 9). Effect of Morus bark Extract on the Expression of c-Fos Gene in Hypothalamus and Hippocampus by Immunohistochemical Staining
스트레스에 의한 생리학적 반응은 시상하부-뇌하수체ᅳ 부신피질축 (hypothalamus - ituitary-adrenal gland; HPA axis)에 의해 조절된다. HPA축은 스트레스 반응 시 항상성을 위해 신경， 내분비， 면역작용을 조절하는 기능을 하고 있으며， 이 과정에서 시상하부는 부신피질 자극 호르몬 방출인자 (corticotropin releasing factor; CRF)를 분비하여 뇌하수체 전엽을 자극하여 부신 피질 자극 호르몬을 방출하고 방출된 부신 피질 자극 호르몬은 부신피질에 분비되어 코르티코스테론을 방출하게 되며， 이는 몸의 기관과 면역계, 중추신경계 등에 영향을 주어 문제를 일으키게 된다. 특히 실방핵 (paraventricular nucleus; PVN)은 스트레스에 반응하는 부위로 코르티코스테론 자극 호르몬을 분비하는 뉴런이 집중되어 있어 스트레스 반웅연구에 객관적인 지표가 되고 있다. 특히 c-Fos 단백질은 급성 유해 자극이나 스트레스 등에 의해 뇌의 여러 영역에서 발현되는 것으로 잘 알려져 있다. 여러 관련 연구에서 유해 자극이나 스트레스에 의해 뇌간， 시상， 시상하부， 해마 등에서 c-Fos 발현의 증가를 관찰하였고， 일반적으로 c-Fos 발현은 스트레스에 의한 행동학적, 생리학적 반웅이 반영되는 것으로 알려져 왔다. c-Fos는 스트레스와 같은 자극에 의하여 20 분 이내에 발현되기 시작하여 24시간 이상 지속됨으로써 일명 전초기 유전자 (immediate-early gene)라 하고， 세포내 대사 활동성의 변화를 측정하는 표지자로 널리 알려져 있다 (Drgunow M et al . , 1989) . 이에 착안하여 본 발명에서도 쥐에게 급격한 감금스트레스를 부여한 후 해마, 실방핵 등에서 발현되는 c-Fos 단백질을 관찰함으로써 상백피 추출물의 항스트레스 효능을 살펴보고자 하였다. The physiological response to stress is regulated by the hypothalamus-ituitary-adrenal gland (HPA axis). The HPA axis functions to regulate nerves, endocrine, and immune functions for homeostasis during stress response. In this process, the hypothalamus secretes corticotropin releasing factor (CRF), which stimulates the anterior pituitary gland to adrenal gland. Cortical stimulating hormone is released and the released adrenal cortical stimulating hormone is secreted in the adrenal cortex to release the corticosterone, which affects the body's organs, immune system, central nervous system, etc. cause problems. Particularly, paraventricular nucleus (PVN) is a stress-responsive site, and the concentration of neurons releasing corticosteroid stimulating hormone is an objective indicator for stress reaction research. In particular, c-Fos protein is well known to be expressed in various areas of the brain by acute harmful stimulation or stress. Several related studies have observed an increase in c-Fos expression in brain stem, thalamus, hypothalamus, and hippocampus due to noxious stimulation or stress. In general, c-Fos expression is known to reflect behavioral and physiological reactions caused by stress. come. c-Fos is Expressed within 20 minutes by stimulation such as stress and lasting for more than 24 hours, it is called an early-early gene and is widely known as a marker for measuring changes in metabolic activity in cells (Drgunow M et al. , 1989). With this in mind, the present invention was intended to examine the anti-stress efficacy of the extracts of the lettuce extract by observing c-Fos protein expressed in the hippocampus, chamber nucleus, etc. after giving rapid confinement stress to the rat.
실험 수행 결과， 정상대조군 (A)에 비해 감금스트레스만 부여한 대조군 (B)에서 c-Fos 발현 정도가 유의적으로 높게 나타났음을 확인할 수 있었으며, 상백피 추출물의 섭취한 군은 대조군에 비해 c-Fos의 발현 정도가 감소되었음을 확인하였다 (도 10， 11). 상백피 추출물은 유의적으로 스트레스에 대한 c-Fos 발현 정도를 감소시키는데 영향을 미치는 것으로 나타났다. 이러한 결과는 감금스트레스에 의해 스트레스관련 신경세포들이 활성화 되어 스트레스 반웅을 유발시킴으로써 c-Fos 발현이 증가되었고， 상백피 추출물의 항스트레스 작용으로 c-Fos 발현이 억제된 것으로 보이는 결과라 사료된다. 이상으로 본 발명의 특정한 부분을 상세히 기술하였는바， 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. 참고 문헌  As a result of the experiment, it was confirmed that c-Fos expression level was significantly higher in the control group (B) given only the restraint stress than the normal control group (A). It was confirmed that the expression level was reduced (FIGS. 10 and 11). It was found that the extract of E. coli significantly affected the level of c-Fos expression against stress. These results suggest that c-Fos expression is increased by inducing stress reaction by activating stress-related neurons by confinement stress, and c-Fos expression seems to be suppressed by antistress action of E. coli extract. The specific parts of the present invention have been described in detail, and it is apparent to those skilled in the art that these specific technologies are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof. references
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Claims

【특허청구범위】 [Patent Claims]
【청구항 1】 [Claim 1]
상백피 (#07 Cortex Radicis) 추출물을 유효성분으로 포함하는 우울증의 예방 또는 치료용 조성물.  # 07 Cortex Radicis (# 07 Cortex Radicis) extract containing the extract as an active ingredient for the prevention or treatment of depression.
[청구항 2】 [Claim 2 ]
제 1 항에 있어서， 상기 상백피 추출물은 유기용매 , 물 또는 이들의 흔합물로 추출한 것을 특징으로 하는 조성물.  The method of claim 1, wherein the extract is an extract, characterized in that extracted with an organic solvent, water or a mixture thereof.
【청구항 3】 [Claim 3]
제 2 항에 있어서， 상기 유기용매는 에탄올, 메탄을， 아세톤, 핵산, 에틸아세테이트 및 메틸렌클로라이드로 구성된 군으로부터 선택되는 것올 특징으로 하는 조성물,  The composition of claim 2, wherein the organic solvent is selected from the group consisting of ethanol, methane, acetone, nucleic acid, ethyl acetate and methylene chloride,
【청구항 4】 【Claim 4 】
제 1 항에 있어서， 상기 상백피 추출물은 초음파기를 이용하여 추출한 것을 특징으로 하는 조성물.  The method of claim 1, wherein the extract of the lettuce extract, characterized in that the extract using a sonicator.
【청구항 5】 [Claim 5]
제 1 항에 있어서, 상기 조성물은 상기 상백피 추출물에 탄산나트륨을 첨가하여 불용해성 물질을 용해시킨 것올 특징으로 하는 조성물.  The composition of claim 1, wherein the composition is obtained by dissolving an insoluble substance by adding sodium carbonate to the extract of Morus bark.
【청구항 6】 [Claim 6]
제 1 항에 있어서， 상기 조성물은 글루코코르티코이드 수용체 (glucocorticoid receptor)의 활성화 (act ivat ion)를 억제시키는 것을 특징으로 하는 조성물.  The composition of claim 1, wherein the composition inhibits act ivat ions of the glucocorticoid receptor.
【청구항 7】 [Claim 7]
제 1 항에 있어서， 상기 조성물은 글루코코르티코이드 수용체 (glucocorticoid receptor)의 핵 내 이동 (nuclear translocation)을 억제시키는 것을 특징으로 하는 조성물. The method of claim 1, wherein the composition is a glucocorticoid A composition characterized by inhibiting nuclear translocation of a glucocorticoid receptor.
【청구항 8】 [Claim 8]
제 1 항에 있어서， 상기 조성물은 c-Fos의 발현을 감소시키는 것을 특징으로 하는 조성물.  The composition of claim 1, wherein the composition reduces the expression of c-Fos.
【청구항 9】 [Claim 9]
상백피 ( for/ Cortex Radicis) 추출물올 유효성분으로 포함하는 우울증의 개선 또는 완화용 식품 조성물.  For / Cortex Radicis extracts for improving or alleviating depression comprising an active ingredient as an active ingredient.
【청구항 10】 [Claim 10]
상백피 ( o / Cortex Radicis) 추출물의 약제학적 유효량을 포함하는 조성물을 대상 (subject)에 투여하는 단계를 포함하는 우울증의 예방, 개선， 완화 또는치료 방법 . A method of preventing, ameliorating, alleviating or treating depression comprising administering to a subject a composition comprising a pharmaceutically effective amount of an o / cortex radicis extract.
【청구항 11】 [Claim 11]
제 10 항에 있어서， 상기 상백피 추출물은 유기용매, 물 또는 이들의 흔합물로 추출한 것을 특징으로 하는 방법.  The method of claim 10, wherein the extract of the lettuce extract is characterized in that extracted with an organic solvent, water or a mixture thereof.
【청구항 12】 [Claim 12]
제 11 항에 있어서， 상기 유기용매는 에탄올， 메탄올, 아세톤, 핵산， 에틸아세테이트 및 메틸렌클로라이드로 구성된 군으로부터 선택되는 것을 특징으로 하는 방법 .  The method of claim 11, wherein the organic solvent is selected from the group consisting of ethanol, methanol, acetone, nucleic acid, ethyl acetate and methylene chloride.
【청구항 13】 [Claim 13]
제 10 항에 있어서， 상기 상백피 추출물은 초음파기를 이용하여 추출한 것을 특징으로 하는 방법.  The method of claim 10, wherein the extract of the lettuce extract is characterized in that it was extracted by using an ultrasonic wave.
【청구항 14】 [Claim 14]
제 10 항에 있어서， 상기 조성물은 상기 상백피 추출물에 탄산나트륨을 첨가하여 불용해성 물질을 용해시킨 것을 특징으로 하는 방법. The method of claim 10, wherein the composition is Sodium carbonate was added to dissolve the insoluble material.
【청구항 15】 [Claim 15]
제 10 항에 있어서， 상기 조성물은 글루코코르티코이드 수용체 (glucocorticoid receptor)의 활성화 (activation)를 억제시키는 것을 특징으로 하는 방법 .  The method of claim 10, wherein the composition inhibits activation of glucocorticoid receptors.
【청구항 16】 [Claim 16]
제 10 항에 있어서, 상기 조성물은 글루코코르티코이드 수용체 (glucocorticoid receptor)의 핵 내 이동 (nuclear translocat ion)을 억제시키는 것을 특징으로 하는 방법.  The method of claim 10, wherein the composition inhibits nuclear translocat ion of the glucocorticoid receptor.
【청구항 17】 [Claim 17]
제 10 항에 있어서， 상기 조성물은 c-Fos의 발현을 감소시키는 것을 특징으로 하는 방법 .  The method of claim 10, wherein the composition reduces the expression of c-Fos.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050067131A (en) * 2005-06-21 2005-06-30 주식회사 에스티씨나라 Composition comprising the extract of morus alba l having monoamine oxidase-inhibiting activity
KR20100012735A (en) * 2008-07-29 2010-02-08 문인술 Kimchi, to which functional extract of bamboo is added and method of manufacturing the same
KR20100024897A (en) * 2008-08-26 2010-03-08 한국화학연구원 Pharmaceutical composition for appetite suppression or prevention and treatment of psychiatric disorder, containing the extract of mori folium, fraction thereof or compound isolated therefrom, having mch-1 receptor antagonistic effect, as an active ingredient

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KR20100012735A (en) * 2008-07-29 2010-02-08 문인술 Kimchi, to which functional extract of bamboo is added and method of manufacturing the same
KR20100024897A (en) * 2008-08-26 2010-03-08 한국화학연구원 Pharmaceutical composition for appetite suppression or prevention and treatment of psychiatric disorder, containing the extract of mori folium, fraction thereof or compound isolated therefrom, having mch-1 receptor antagonistic effect, as an active ingredient

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