KR20110138082A - Composition for immunopotentiating comprising the extract of herbal formula, ojeok-san - Google Patents
Composition for immunopotentiating comprising the extract of herbal formula, ojeok-san Download PDFInfo
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- KR20110138082A KR20110138082A KR1020100058204A KR20100058204A KR20110138082A KR 20110138082 A KR20110138082 A KR 20110138082A KR 1020100058204 A KR1020100058204 A KR 1020100058204A KR 20100058204 A KR20100058204 A KR 20100058204A KR 20110138082 A KR20110138082 A KR 20110138082A
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Abstract
Description
본 발명은 오적산 추출물을 유효성분으로 함유하는 면역 증강용 조성물, 이를 함유하는 면역 증강용 식품 조성물 및 면역기능 저하로 인한 질환의 예방 또는 치료용 조성물에 관한 것이다.
The present invention relates to a composition for enhancing immunity, comprising a potic acid extract as an active ingredient, a food composition for enhancing immunity, and a composition for preventing or treating diseases caused by a decrease in immune function.
오적산은 1107년 전후 진사문(陳師文) 등이 수정 발간한 태평혜민화제국방 상한문(太平惠民和劑局方 傷寒門)에 처음으로 수록된 처방으로서, 창출(蒼朮), 마황(麻黃), 진피(陳皮), 후박(厚朴), 길경(桔梗), 지각(枳殼), 당귀(當歸), 건강(乾薑), 백작약(白芍藥), 백복령(白茯笭), 천궁(川芎), 백지(白芷), 반하(半夏), 계피(桂皮), 감초(甘草), 생강(生薑), 총백(蔥白)으로 구성되어 있으며, 치감상풍한(治感傷風寒), 두신통(頭身痛), 사지역내흉복작통(四肢逆冷胸腹作統) 구사(嘔瀉) 또는 상냉생식(傷冷生食)에 적응되는 처방이다. 한국에서는 1990년부터 전통의학 한약처방 56개를 국가 보험에서 급여를 하고 있으며, 지난 20년간 이들 56개 한약 처방 중에서 오적산은 투약일수나 약제비 순위에서 1위를 차지하는 한약 처방이다. 2008년 건강보험에서도 국민건강보험 급여 한약제제 56종 중 오적산은 전체 투약일수(Days of medication)의 19%, 전체 약제비(medical expenses)의 33%를 차지하는 다빈도로 사용하는 중요한 한약 처방으로 조사되었다(National Health Insurance Corporation, 2008 National Health Insurance Statistical Yearbook, 2008 P.290). Ojeoksan was first published in the Taepyeong Hyeminje National Defense Sanhan Gate, first revised and published by Jinsamun around 1107. Dermis, Tak-bak, Gil-gyeong, Crust, Dang-gu, Health, Baek-jak-pyeon, Baek-bok-ryeong, Cheong-gung It consists of white paper, white, half cinnamon, cinnamon, licorice, ginger and baekbaek. It is a prescription that is adapted to the use of 身 사, intrathoracic abdominal pain (四 상 作 統) or normal cold regeneration (식 生 食). In Korea, 56 traditional Chinese herbal medicine prescriptions have been paid by national insurance since 1990. Of these 56 Chinese medicine prescriptions, Oeroksan ranks first in terms of number of medication days or drug costs. In 2008 health insurance, Oh Jeung-san among 56 national health insurance-beneficial herbal medicines was found to be an important prescription for Chinese medicine, which takes up 19% of the days of medication and 33% of medical expenses. National Health Insurance Corporation, 2008 National Health Insurance Statistical Yearbook, 2008 P.290).
오적산의 효능에 대해 발표된 것으로, 오적산의 간, 신장 등 독성에 대한 안전성, 소염, 진통, 해열, 고지혈증에 대한 치료 효과, 갱년기 불안장애 치료 등이 알려져 있으나, 면역 활성과 관련된 효능은 보고된 바가 없다.
It is published about the efficacy of Ojeok-san, which is known for its safety against liver and kidney toxicity, anti-inflammatory, analgesic, antipyretic, hyperlipidemia, and treatment of menopausal anxiety disorders. It has not been done.
이에 본 발명자들은 오적산 처방 추출물에 대한 면역활성과 관련된 효능을 연구하였다. 이를 위하여, 대식세포 활성화 효과(Friedl R, Moeslinger T, Kopp B, and Spieckermann PG. Stimulation of nitric oxide synthesis by the aqueous extract of Panax ginsena root in Raw 264.7 cells. British Journal of pharma- cology, 2001;134:1663-1670)와 비장세포의 증식 효과를 선택하였다.
In this regard, the present inventors studied the efficacy related to the immunological activity against the formulation of Ohjeksan prescription. To this end, macrophage activation effects (Friedl R, Moeslinger T, Kopp B, and Spieckermann PG.Stimulation of nitric oxide synthesis by the aqueous extract of Panax ginsena root in Raw 264.7 cells.British Journal of pharmacology, 2001; 134: 1663-1670) and the proliferation effect of splenocytes.
특히, NO의 생성 촉진 효과는 대식세포 활성화의 지표로 대식세포에서 생성되는 NO는 침입한 병원체를 없애고 기본적인 숙주 방어체계를 작동시키는 역할을 하는 것으로 알려져 있다(Farrell AJ, Blake DR. Nitric oxide. Annals of the Rheumatic Dieases. 1996;55:7-20). 특히 Th 세포의 분화단계 중 ThO 상태에서 Thl과 Th2로 나누어질 때, Th1로의 분화를 돕는 작용을 하는 것으로 알려져 있다. 또한 백혈구의 활성 중 암세포, 기생충 또는 박테리아를 살해하는 세포독성 활성은 NO를 유리시킴으로써 나타나는 것으로 보고되고 있다(Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB Journal. 1992;6: 3051-3064). 또한 기관지 염증에 NO가 T-cell과 내피세포간의 면역조절(immunomodulating)을 하며 (Bingisser RM, Holt PG Immunomodulating mechanism in the lower respiratory tract; nitric oxide mediated intraction between alveolar macrophage, epithelial cell, and T-cell, SWISS Medical Weekly. 2001;131;171-179) 강력한 혈소판 응집 억제작용이 있다는 것이 알려져 있다.
In particular, the NO production promoting effect is an indicator of macrophage activation. NO produced in macrophages is known to play a role in eliminating invading pathogens and acting as a basic host defense system (Farrell AJ, Blake DR. Nitric oxide. of the Rheumatic Dieases. 1996; 55: 7-20). In particular, when Thhl is divided into Thl and Th2 in the ThO differentiation stage, it is known to have a function of helping to differentiate to Th1. In addition, cytotoxic activity of killing cancer cells, parasites or bacteria among leukocyte activity has been reported to be released by the release of NO (Nathan C. Nitric oxide as a secretory product of mammalian cells.FASEB Journal. 1992; 6: 3051-3064) ). In addition, NO is immunomodulating between T-cell and endothelial cells (Bingisser RM, Holt PG Immunomodulating mechanism in the lower respiratory tract; nitric oxide mediated intraction between alveolar macrophage, epithelial cell, and T-cell, SWISS Medical Weekly. 2001; 131; 171-179) is known to have a potent platelet aggregation inhibitory effect.
이에 본 발명자들은 오적산 처방 추출물이 비장 세포의 증식을 촉진하며, 대식세포에서 NO의 생성을 유발하며, 생쥐에서 면역감작의 증진에 대해 우수한 효과를 나타내므로 오적산 처방 추출물이 면역 활성화 및 증강 효과가 있음을 확인함으로써 본 발명을 완성하였다.
In this regard, the present inventors suggest that the formulation of Ojeoksan prescription extract promotes the proliferation of splenocytes, induces the production of NO in macrophages, and shows an excellent effect on the enhancement of immune sensitization in mice. By confirming that the present invention was completed.
본 발명은 오적산 처방 추출물을 유효성분으로 함유하는 면역 증강용 조성물, 이를 함유하는 면역 증강용 식품 조성물 및 면역기능 저하로 인한 질환의 예방 또는 치료용 조성물을 제공하기 위한 것이다.
The present invention is to provide a composition for the enhancement of immunity, a composition for enhancing immunity, and a composition for the prevention or treatment of diseases caused by a decrease in immune function.
상기의 과제를 해결하기 위하여, 본 발명은 오적산 처방 추출물을 유효성분으로 함유하는 면역 증강용 조성물을 제공한다.
In order to solve the above problems, the present invention provides a composition for enhancing immunity containing a prescription formulation of Ohpisan as an active ingredient.
본 발명에서 사용되는 용어 "오적산"은, 창출(蒼朮, Atractylodis Rhizoma), 마황(麻黃, Ephedrae Herba), 진피(陳皮, Citri Unshii Pericarpium), 후박(厚朴, Magnoliae Cortex), 길경(桔梗, Platycodi Radix), 지각(只殼, Aurantii Fructus Immaturus), 당귀(當歸, Angelicae Gigantis Radix), 건강(乾薑, Zingiberis Rhizoma), 백작약(芍藥, Paeoniae Radix), 백복령(茯, Hoelen), 백지(白芷, Angelicae Dahuricae Radix), 천궁(川芎, Cnidii Rhizoma), 반하(半夏, Pinelliae Tuber), 계피(桂皮, Cinnamon Bark), 감초(甘草, Glycyrrhizae Radix et Rhizoma), 생강(生薑, Zingiberis Rhizoma Crudus) 및 총백(蔥白, Allii Radix)으로 구성된 조성물을 의미한다. 상기 조성물은 "동의보감"에 따른 것이며, 오적산의 면역활성과 관련된 효능에 대해서는 보고된 바가 없다.
The term "ojeoksan" used in the present invention, Atractylodis Rhizoma, Ephedrae Herba, Dermis, Citri Unshii Pericarpium, Magnoliae Cortex, Gilkyung , Platycodi Radix, Aurantii Fructus Immaturus, Angelicae Gigantis Radix, Health (Zingiberis Rhizoma), Paeoniae Radix, Paekoniae Radix, Hoelen, White Paper ( White, Angelicae Dahuricae Radix, Cnidii Rhizoma, Pinelliae Tuber, Cinnamon Bark, Glycyrrhizae Radix et Rhizoma, Ginger (Zingiberis Rhizoma Crudus) And Allii Radix. The composition is according to "consensus" and has not been reported for the efficacy associated with the immunological activity of miscalculated acid.
본 발명에서 사용되는 용어 "추출물"은, 오적산으로부터 액체의 용매를 사용하여 분리된 특정 성분을 의미한다. 상기 용매로는 물, C1 내지 C4 알코올 또는 이들의 혼합용매를 사용할 수 있다. 유효성분을 효율적으로 추출하기 위하여 메탄올, 에탄올 또는 이들과 물의 혼합물로 추출하는 것이 바람직하다. 메탄올과 에탄올의 농도는 10 내지 90%가 바람직하며, 50 내지 70%인 것이 보다 바람직하나 이에 제한되는 것은 아니다. 상기 추출용매는 오적산 중량의 5 내지 20 배를 사용하는 것이 바람직하다.As used herein, the term "extract" means a specific component that is separated from a miscalculated acid using a solvent of the liquid. As the solvent, water, C 1 to C 4 alcohol or a mixed solvent thereof may be used. In order to efficiently extract the active ingredient, it is preferable to extract with methanol, ethanol or a mixture of these and water. The concentration of methanol and ethanol is preferably 10 to 90%, more preferably 50 to 70%, but is not limited thereto. The extraction solvent is preferably used 5 to 20 times the weight of the cumulative acid.
추출방법은 열수 추출, 냉침 추출, 환류 냉각 추출, 초음파 추출 및 증기 추출로 이루어진 군으로부터 선택된 어느 하나를 사용할 수 있으며, 추출 후 추출물을 동결건조하여 추출물을 수득할 수 있다. 추출용매로 물을 사용할 경우에는, 열수추출인 것이 바람직하며 물의 온도는 90 내지 120℃, 추출시간은 1 내지 3 시간이 바람직하다.
The extraction method may be any one selected from the group consisting of hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction and steam extraction, the extract may be obtained by lyophilizing the extract after extraction. When water is used as the extraction solvent, hot water extraction is preferred, the temperature of the water is preferably 90 to 120 ℃, extraction time is 1 to 3 hours.
또한, 본 발명은 상기 면역 증강용 조성물을 유효성분으로 함유하는 면역기능 저하로 인한 질환의 예방 또는 치료용 약학적 조성물을 제공한다. The present invention also provides a pharmaceutical composition for the prevention or treatment of diseases caused by a decrease in immune function containing the composition for enhancing immune function as an active ingredient.
본 발명에서 사용되는 용어 "예방"은, 상기 오적산 추출물을 포함하는 조성물의 투여로 질환을 억제 또는 지연시키는 모든 행위를 의미한다. 또한, 본 발명에서 사용되는 용어 "치료"는, 상기 오적산 추출물을 포함하는 조성물의 투여로 질환의 증세가 호전되거나 완치되는 모든 행위를 의미한다. The term "prevention" as used in the present invention means any action that inhibits or delays the disease by administration of the composition comprising the above-obtained acid extract. In addition, the term "treatment" as used in the present invention means all the actions that improve or cure the symptoms of the disease by administration of the composition comprising the acid cumulative extract.
본 발명에서 사용되는 용어 "면역증강"은, 면역세포의 초기활성화 과정에서 비특이적으로 항원에 대해 면역반응을 촉진하는 기능, 또는 면역계의 세포의 활성을 증대시킴으로써 면역을 강화하는 기능을 의미한다. As used herein, the term "immune-enhanced" means a function that promotes an immune response to an antigen in the process of initial activation of immune cells, or enhances immunity by increasing the activity of cells of the immune system.
본 발명에서 면역기능 저하로 인한 질환은, 감기, 알러지 질환, 만성피로, 암으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다. 또한, 상기 알러지 질환의 예는 아토피, 천식, 알러지성 비염을 들 수 있다. In the present invention, the disease due to impaired immune function may be any one selected from the group consisting of colds, allergic diseases, chronic fatigue, and cancer. In addition, examples of the allergic disease include atopy, asthma, allergic rhinitis.
본 발명의 구체적인 실시예에서는, 오적산 추출물을 대식세포에 처리했을 때 농도의 조절으로 산화질소(NO) 생성이 증가함을 확인함으로써 오적산 추출물이 대식세포를 활성화시킨다는 것을 알 수 있었다(도 1). 또한, 비장세포 증식실험을 통하여 오적산 추출물이 농도의존적으로 비장세포의 증식을 유도하며(도 2), 배양액 내 IL-2 농도가 증가함을 확인하였다(도 3). 나아가, 마우스의 생체 내 실험(in vivo)에서, OVA를 항원으로 감작시킨 마우스에 오적산 추출물을 투여한 경우 비장세포가 증가하고(도 4), IgG1 및 IgM의 혈중농도가 증가함을 확인하였다(도 5). 따라서 본 발명의 오적산 추출물은 면역반응을 증강시키는데 우수한 효과가 있음을 알 수 있다. In a specific embodiment of the present invention, it was found that the nitric acid extract activates macrophages by confirming that the production of nitric oxide (NO) is increased by controlling the concentration when the ohmic acid extract is treated with macrophages (FIG. 1). ). In addition, it was confirmed through the spleen cell proliferation experiment that the erythroid extract induces the growth of splenocytes in a concentration-dependent manner (FIG. 2), and the IL-2 concentration in the culture medium was increased (FIG. 3). Furthermore, in vivo experiments, it was confirmed that the splenocytes were increased (Fig. 4), and the blood concentrations of IgG1 and IgM were increased when the OJ extract was administered to mice sensitized with OVA. (FIG. 5). Therefore, it can be seen that the ohjeok extract of the present invention has an excellent effect on enhancing the immune response.
본 발명의 조성물은 투여를 위하여, 상기 기재한 유효성분 이외에 약학적으로 허용 가능한 담체, 부형제 또는 희석제를 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀롤로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.
The composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described active ingredient for administration. The carriers, excipients and diluents 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용할 수 있다. 상세하게는, 제형화할 경우 통상 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제로는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하나, 이에 한정되는 것은 아니다. 이러한 고형제제는 상기 오적산 추출물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘 카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등을 첨가하여 조제될 수 있다. 비경구 투여를 위한 제제는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제 및 좌제를 포함한다. 비수성 용제 및 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. Specifically, when formulated, it may be prepared by using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrating agents, and surfactants which are commonly used. Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, and the like. Such solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like, with the oxalic acid extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. It may be prepared by adding various excipients such as humectants, sweeteners, fragrances, preservatives and the like in addition to liquid oral liquids or liquid paraffin for oral use. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를들어, 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. 상기 오적산 추출물의 일일 투여량은 바람직하게는 1 mg/kg 내지 500 mg/kg이며, 필요에 따라 일일 1회 내지 수회로 나누어 투여할 수 있다.The compositions of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the condition and weight of the patient, the extent of the disease, Depending on the drug form, route of administration, and time, it may be appropriately selected by those skilled in the art. The daily dosage of the oxalic acid extract is preferably 1 mg / kg to 500 mg / kg, and may be administered once to several times daily if necessary.
또한, 본 발명은 상기 면역 증강용 조성물을 유효성분으로 함유하는 조성물의 치료학적 유효량을 개체에 투여하는 단계를 포함하는 면역기능 저하로 인한 질환의 예방 또는 치료하는 방법을 제공한다.
In addition, the present invention provides a method for preventing or treating a disease due to impaired immune function, comprising administering to a subject a therapeutically effective amount of a composition containing the composition for enhancing immune function as an active ingredient.
또한 본 발명은 상기 면역 증강용 조성물을 유효성분으로 함유하는 면역 증강용 식품 조성물을 제공한다. The present invention also provides an immune enhancing food composition containing the immune enhancing composition as an active ingredient.
상기 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 천연 과일쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 또한, 상기 식품 조성물은 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 껌류, 아이스크림류, 스프, 음료수, 차, 기능수, 드링크제, 알콜 음료 및 비타민 복합제 중 어느 하나의 형태일 수 있다.
The food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof, organic acids , Protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Others may contain pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. In addition, the food composition is any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soups, drinks, tea, functional water, drinks, alcoholic beverages and vitamin complexes Can be.
또한 상기 식품 조성물은 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물"로서의 적합여부는 다른 규정이 없는 한 식품의약품안정청에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.
In addition, the food composition may further include food additives, and the suitability as a "food additive" standards for the item according to the General Regulations of the Food Additives Code and General Test Law, etc., unless otherwise specified. And based on the criteria.
상기 "식품첨가물공전"에 수재된 품목으로 예를 들어, 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성품, 감색소, 감초추출물, 결정셀롤로오스, 고랭색소, 구아검 등의 천연첨가물, L-글루타민산나트륨제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합 제제류들을 들 수 있다.
Examples of the items listed in the "Food Additives Code" include, for example, chemical synthetic products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as navy, licorice extract, crystalline cellulose, high quenching pigment, and guar gum. And mixed preparations such as sodium L-glutamate, alkalescent additives, preservatives and tar dyes.
이 때, 식품 조성물을 제조하는 과정에서 음료를 포함한 식품에 첨가되는 본 발명에 따른 추출물은 필요에 따라 그 함량을 적절히 가감할 수 있으며, 바람직하게는 식품 100중량%에 1 내지 15 중량% 포함되도록 첨가하는 것이 바람직하다.
At this time, the extract according to the present invention is added to the food containing the beverage in the process of preparing the food composition, if necessary, can be appropriately added or subtracted, preferably 1 to 15% by weight in 100% by weight of food. It is preferable to add.
또한, 본 발명은 오적산을 추출용매로 추출하여 추출액을 제조하는 단계; 및 상기 추출액을 건조하는 단계를 포함하는 오적산 처방 추출물을 유효성분으로 함유하는 면역 증강용 조성물의 제조방법을 제공한다.
In addition, the present invention comprises the steps of preparing the extract by extracting the acid as an extract solvent; And it provides a method for producing an immune enhancing composition containing the Ohsiksan prescription extract comprising the step of drying the extract as an active ingredient.
상기 추출용매로는 물, C1 내지 C4 알코올 또는 이들의 혼합용매를 사용할 수 있다. 유효성분을 효율적으로 추출하기 위하여 메탄올, 에탄올 또는 이들과 물의 혼합물로 추출하는 것이 바람직하다. 메탄올과 에탄올의 농도는 10 내지 90%가 바람직하며, 50 내지 70%인 것이 보다 바람직하나 이에 제한되는 것은 아니다. 상기 추출용매는 오적산 중량의 5 내지 20 배를 사용하는 것이 바람직하다. 물 추출물의 경우 열수추출인 것이 바람직하며 90~120℃에서 1~3 시간 추출한 것이 가장 바람직하다.As the extraction solvent, water, C 1 to C 4 alcohol or a mixed solvent thereof may be used. In order to efficiently extract the active ingredient, it is preferable to extract with methanol, ethanol or a mixture of these and water. The concentration of methanol and ethanol is preferably 10 to 90%, more preferably 50 to 70%, but is not limited thereto. The extraction solvent is preferably used 5 to 20 times the weight of the cumulative acid. In the case of the water extract is preferably hot water extraction is most preferably extracted for 1 to 3 hours at 90 ~ 120 ℃.
추출방법은 열수 추출, 냉침 추출, 환류 냉각 추출, 초음파 추출 및 증기 추출로 이루어진 군으로부터 선택된 어느 하나를 사용할 수 있으며, 추출 후 추출물을 동결건조하여 추출물을 수득할 수 있다.
The extraction method may be any one selected from the group consisting of hot water extraction, cold extraction, reflux cooling extraction, ultrasonic extraction and steam extraction, the extract may be obtained by lyophilizing the extract after extraction.
본 발명의 오적산 처방 추출물은 생쥐의 비장세포의 생장을 증가시키고, 대식세포에서 NO의 생성을 증가시키고, 생쥐에서 면역 증진에 대해 우수한 효과를 나타내므로 면역기능의 강화에 효과적이므로, 면역기능 강화용 조성물, 면역기능 강화용 식품 조성물 및 면역기능 저하로 기인하는 질환의 예방 또는 치료용 약학적 조성물로 이용하는데 유용하다.
Ojeoksan prescription extract of the present invention increases the growth of splenocytes of mice, increases the production of NO in macrophages, and is effective in enhancing immune function because it shows an excellent effect on immune enhancement in mice, thereby enhancing immune function. It is useful for use as a pharmaceutical composition for preventing or treating a disease caused by a composition for use, a food composition for enhancing immune function and a decrease in immune function.
도 1은, 오적산 추출물의 대식세포에 대한 면역활성 검색결과로서, 대식세포가 활성화될 때 NO의 생성량을 측정한 결과이다.
도 2는, 오적산 추출물의 비장세포에 대한 면역활성 검색결과로서, 비장세포의 증식 유발 효과를 검색한 결과를 도 2에 나타내었다.
도 3은, 오적산 추출물의 비장세포에 대한 면역활성 검색결과로서, 비장세포의 증식효과 검색 후 남은 세포를 수집하여 배양액 내의 IL-2 level을 측정한 결과를 나타낸 것이다.
도 4는, 오적산 추출물의 생체 내에서의 면역 활성화 효과를 나타낸 것으로, OVA를 항원으로 감작시킨 생쥐에 약물을 투여 한 후 비장세포를 분리하여 각각의 투여군에서 분리한 비장세포에 mitogen으로 OVA, LPS, Con A를 처리하여 mitogen effect 결과를 나타낸 것이다.
도 5는, 오적산 추출물의 생체 내에서의 면역 활성화 효과를 나타낸 것으로, OVA를 항원으로 감작시킨 생쥐에 약물을 투여 한 후 혈중 OVA-specific IgG, OVA-specific IgG1, IgM level을 측정한 결과를 나타낸 것이다. FIG. 1 is a result of detecting the activity of macrophages of the Ojeok extract by measuring the amount of NO produced when macrophages are activated.
FIG. 2 shows the results of searching for proliferation-inducing effects of splenocytes as a result of immunological activity on splenocytes of the Ojic acid extract.
Figure 3, as a result of the immune activity search results for splenocytes of the Ohjeoksan extract, showing the results of measuring the IL-2 level in the culture by collecting the remaining cells after the proliferation effect of the splenocytes.
Figure 4 shows the in vivo immune activating effect of the extract of Ojeoksan extract, after administration of the drug to mice sensitized with OVA, splenocytes were isolated and splenocytes isolated from the respective administration groups as mitogen OVA, LPS and Con A were treated to show mitogen effect results.
5 shows the in vivo immune activation effect of the Ojeoksan extract, the results of measuring the blood OVA-specific IgG, OVA-specific IgG1, IgM levels after administration of the drug to mice sensitized with OVA antigen It is shown.
이하,본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 더욱 쉽게 이해하기 위하여 제공되는 것일 뿐,실시예에 의하여 본 발명의 내용이 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be presented to assist 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 examples.
실시예 1 : 오적산 처방 추출물의 제조Example 1 Preparation of a Ohmic Acid Prescription Extract
오적산의 구성은 '동의보감'을 참조하여, 창출(蒼朮, Atractylodis Rhizoma), 마황(麻黃, Ephedrae Herba), 진피(陳皮, Citri Unshii Pericarpium), 후박(厚朴, Magnoliae Cortex), 길경(桔梗, Platycodi Radix), 지각(只殼, Aurantii Fructus Immaturus), 당귀(當歸, Angelicae Gigantis Radix), 건강(乾薑, Zingiberis Rhizoma), 백작약(芍藥, Paeoniae Radix), 백복령(茯, Hoelen), 백지(白芷, Angelicae Dahuricae Radix), 천궁(川芎, Cnidii Rhizoma), 반하(半夏, Pinelliae Tuber), 계피(桂皮, Cinnamon Bark), 감초(甘草, Glycyrrhizae Radix et Rhizoma), 생강(生薑, Zingiberis Rhizoma Crudus), 총백(蔥白, Allii Radix)의 약재를, 각각 약 7.5 : 3.75 : 3.75 : 3 : 3 : 3 : 3 : 3 : 3 : 3 : 2.625 : 2.625 : 2.625 : 2.625 : 2.25 : 3.75 : 3.75의 질량비로 사용하였다.
For the composition of Ojeoksan, refer to 'Agreement' and create, Atractylodis Rhizoma, Ephedrae Herba, Citri Unshii Pericarpium, Magnoliae Cortex, Gilkyung , Platycodi Radix, Aurantii Fructus Immaturus, Angelicae Gigantis Radix, Health (Zingiberis Rhizoma), Paeoniae Radix, Paekoniae Radix, Hoelen, White Paper ( White, Angelicae Dahuricae Radix, Cnidii Rhizoma, Pinelliae Tuber, Cinnamon Bark, Glycyrrhizae Radix et Rhizoma, Ginger (Zingiberis Rhizoma Crudus) ), The mass ratio of medicinal herb (Allii Radix) is about 7.5: 3.75: 3.75: 3: 3: 3: 3: 3: 3: 3: 2.625: 2.625: 2.625: 2.625: 2.25: 3.75: 3.75 Used as.
진피, 길경, 당귀, 건강, 백작약, 백복령, 백지, 천궁, 생강은 (주)옴니허브(Yeongcheon, Korea)에서 구입하였으며, 창출, 마황, 후박, 지각, 반하, 감초는 HMAX(Chungbuk, Korea), 계피는 화림제약, 총백은 지역시장에서 각각 구입하였다.
Dermis, Gilgyeong, Dongguk, Health, Baekjak, Baekbokyeong, Baekji, Cheongung, and Ginger were purchased from Yeongcheon, Korea, and HMAX (Chungbuk, Korea) Cinnamon was purchased from Hwarim Pharmaceuticals and Chongbaek was purchased from local markets.
각 약재를 비율별로 혼합한 후 약재 무게 10배의 물로 100 내지 120분간 열수추출기(COSMOS-660, 경서기계산업, Korea)를 이용하여 추출하였다. 추출액을 여과한 후 대형회전농축기(EYELA N-11, Japan)로 1차 농축 후 동결건조기 (PVTFD100R, Ilshin Lab., Korea)를 사용하여 황갈색의 추출물 분말을 회수하였다. 오적산 추출물 분말의 수율은 약 21.0%였다.
Each medicinal herb was mixed by ratio and extracted with hot water extractor (COSMOS-660, Gyeongseo Machinery Industry, Korea) for 100 to 120 minutes with 10 times the weight of medicinal herbs. After extracting the filtrate was concentrated first with a large rotary concentrator (EYELA N-11, Japan) and then using a freeze dryer (PVTFD100R, Ilshin Lab., Korea) to recover the yellowish brown extract powder. The yield of the ohmic acid extract powder was about 21.0%.
실험예 1 : 대식세포에 대한 면역활성 검색Experimental Example 1 Search for Immune Activity Against Macrophages
1) 세포배양1) Cell Culture
쥐의 대식세포주(Murin macrophage cell line)인 Raw 264.7세포를 ATCC(American Type Culture Collection, U.S.A.)에서 분양받아 사용하였다. 배양배지로 10% 우태아혈청(FBS; Gibco BRL., Grand Island, NY, U.S.A.), 항생제로 페니실린-스트렙토마이신(penicillin-streptomycin) 혼합제(Gibco BRL., Grand Island, NY, U.S.A.)를 포함하는 Dulbecco's modified Eagle Medium (DMEM; Gibco BRL., Grand Island, NY, U.S.A.)을 사용하였으며 37℃, 5% CO2의 환경에서 배양하였다. 주 2 내지 3회 배양배지를 교환하였으며 세포가 활성화되지 않은 상태를 유지하도록 배양플라스크에 부착된 세포들은 스크랩퍼를 사용하여 분리하고 주 1회 계대배양 하였다.
Raw 264.7 cells, a Murin macrophage cell line, were distributed from ATCC (American Type Culture Collection, USA) and used. 10% Fetal Bovine Serum (FBS; Gibco BRL., Grand Island, NY, USA) as a culture medium, Penicillin-streptomycin mixture (Gibco BRL., Grand Island, NY, USA) as an antibiotic Dulbecco's modified Eagle Medium (DMEM; Gibco BRL., Grand Island, NY, USA) was used and incubated in an environment of 37 ° C., 5% CO 2 . The culture medium was exchanged 2-3 times a week and the cells attached to the culture flasks were separated using a scraper and passaged once a week to keep the cells inactive.
2) 대식세포의 세포독성 검색2) Cytotoxicity Screening of Macrophages
오적산 추출물이 대식세포주의 세포 독성에 미치는 효과를 검색하기 위하여 96-웰 플레이트에 RAW 264.7세포를 3×103 cells/well로 분주하고 약재 추출물을 PBS에 용해하여 농도별로 첨가하였다. 2일간 배양한 후 테트라졸륨 염인 CCK-8 kit (Cell Counting Kit-8, Dojindo Laboratories, Tokyo, Japan) 용액을 첨가하여 4시간 더 배양한 다음 ELISA reader(Benchmark Plus, BIORAD, U.S.A.)를 이용하여 450 nm에서 흡광도를 측정하였다. 대조군 웰의 흡광도에 대한 약물첨가 웰의 흡광도의 비를 %로 계산하였고, 여러 웰의 평균값을 사용하였다. 계산식은 다음과 같다. In order to detect the effect of Ojeok extract on the cytotoxicity of macrophage lines, RAW 264.7 cells were divided into 3 × 10 3 cells / well in 96-well plates, and medicinal extracts were dissolved in PBS and added to each concentration. After 2 days of incubation, the solution was added for 4 hours with a tetrazolium salt CCK-8 kit (Cell Counting Kit-8, Dojindo Laboratories, Tokyo, Japan) solution, and then incubated for 4 hours using an ELISA reader (Benchmark Plus, BIORAD, USA). Absorbance was measured at nm. The ratio of absorbance of drug addition wells to absorbance of control wells was calculated in% and the mean value of several wells was used. The calculation is as follows.
% = {(약물첨가 well의 Abs450nm 평균값) - (Blank well의 Abs450nm 평균값)}/{(Control well의 Abs450nm 평균값) - (Blank well의 Abs450nm 평균값)} × 100
% = {(Average 450nm average value of drug well)-(Abs 450nm average value of blank well)} / {(Abs 450nm average value of control well)-(Abs 450nm average value of blank well)} × 100
3) 산화질소(NO) 생성량 측정3) Measurement of NOx Production
대식세포가 활성화될 때 산화질소 합성효고(Nitric oxide synthase; NOS)의 활성화로 NO가 생성되므로 대식세포의 활성화 지표로 NO의 생성량을 측정하였다. RAW 264.7 세포를 2.5×105 cells/well로 48 웰에 분주하여 CO2 배양기내에서 24시간 배양하여 안정화 시킨 후 추출물을 농도별로 처리하였다. 양성 대조군으로 LPS를 농도별로 처리하였으며 18 시간 배양 후 상층액을 분리하고 NO 생성량을 측정하였다. NO 생성량은 Griess reagent를 이용하여 535 nm에서 흡광도를 측정 후 농도를 계산하였다.
When macrophages are activated, NO is produced by activation of nitric oxide synthase (NOS), and the amount of NO production was measured as an indicator of macrophage activation. RAW 264.7 cells were divided into 48 wells at 2.5 × 10 5 cells / well, stabilized by incubation in a CO 2 incubator for 24 hours, and then treated with extracts. As a positive control, LPS was treated by concentration. After 18 hours of incubation, the supernatant was separated and NO production was measured. The amount of NO produced was calculated by measuring the absorbance at 535 nm using the Griess reagent and the concentration was calculated.
4) 결과4) Results
오적산 추출물의 처리로 인한 RAW 264.7(murin macrophage cell line) 세포의 활성화 지표 중 하나인 NO 생성 효과를 검색하였다. 1차적으로 오적산 추출물을 0, 2, 10, 20, 100, 200, 1000 μg/ml로 처리하여 세포독성 여부를 확인한 결과 세포독성을 나타내지 않은 200 μg/ml를 최고 농도로 설정하였다. 이에 12.5, 25, 50, 100, 200 μg/ml 의 농도에 대해 NO 생성 효과를 검색하여 그 결과를 도 1에 나타내었다. 결과는 mean±S.E.M.으로 나타냈으며, ANOVA 및 Bonferroni multiple comparison analysis를 사용하였다. 정상군과 유발군의 비교시 유의성 있는 차이는 #; p<0.05, ##; p<0.01로 나타냈으며 유발군과 약물 처리군과의 비교시 *; p<0.05, **; p<0.01로 나타내었다.
The effect of NO production, one of the activation indices of RAW 264.7 (murin macrophage cell line) cells, was investigated. First, it was determined that the cytotoxicity was treated with 0, 2, 10, 20, 100, 200, 1000 μg / ml, and 200 μg / ml, which did not show cytotoxicity, was set to the highest concentration. The results of the NO production for the concentration of 12.5, 25, 50, 100, 200 μg / ml was searched and the results are shown in FIG. The results were expressed as mean ± SEM, and ANOVA and Bonferroni multiple comparison analysis were used. Significant differences between the normal and induced groups were #; p <0.05, ##; p <0.01, and when compared with the induction group and the drug treatment group *; p <0.05, **; p <0.01.
도 1에 나타난 바와 같이, 오적산 추출물은 50 μg/ml이상의 농도에서 농도의존적으로 NO 생성 효과를 나타냈으며(p<0.01), 200 μg/ml에서는 양성 대조군으로 사용한 LPS 0.25 μg/ml와 유사한 수준으로 NO 생성이 증가되었다.
As shown in FIG. 1, the Ojic acid extract showed a concentration-dependent NO production effect at a concentration of 50 μg / ml or more (p <0.01), and a level similar to LPS 0.25 μg / ml used as a positive control at 200 μg / ml. NO production was increased.
실험예 2 : 비장세포에 대한 면역활성 검색Experimental Example 2 Screening of Immune Activity Against Splenocytes
1) 비장세포의 배양1) Culture of Splenocytes
6주령의 수컷 ICR 마우스(한림실험동물, 충북 음성)를 경추탈골 후 비장을 적출하였다. 적출한 비장을 HBSS(Hank's Balanced Salt Solution, Gbico BRL.)로 세척하고 syringe을 이용하여 비장세포를 분리한 후 RBC lysing buffer(Sigma Chemical Co, MO, U.S.A.)로 적혈구를 제거한 후 10% FBS와 페니실린-스트렙토마이신을 함유하는 RPMI 1640 배지(Gbico BRL.)에 부유시켜 5% CO2, 37℃에서 배양하였다.
Six-week-old male ICR mice (Hallim experimental animals, Chungbuk-negative) were extracted after the cervical spine bone. The extracted spleens were washed with HBSS (Hank's Balanced Salt Solution, Gbico BRL.), Splenocytes were separated using a syringe, and red blood cells were removed with RBC lysing buffer (Sigma Chemical Co, MO, USA), followed by 10% FBS and penicillin. -Suspended in RPMI 1640 medium (Gbico BRL.) Containing streptomycin and incubated at 5% CO 2 , 37 ℃.
2) 비장세포의 세포 증식 효과2) Cell proliferation effect of splenocytes
약재 추출물이 비장 세포 증식에 미치는 효과를 검색하기 위하여 96-웰 플레이트에 분리 배양한 비장 세포를 5×105 cells/well로 분주하고 약재 추출물을 PBS에 용해하여 농도별로 첨가하였다. 약재의 세포증식 효과에 대한 비교물질로 Con A와 LPS를 사용하였다. 48시간 배양한 후 테트라졸륨 염인 CCK-8 kit (Cell Counting Kit-8, Dojindo Laboratories, Tokyo, Japan) 용액을 첨가하여 4시간 반응시킨 후 ELISA reader를 이용하여 450 nm에서 흡광도를 측정하였다. 대조군 웰의 흡광도에 대한 약물첨가 웰의 흡광도의 비를 %로 계산하였고, 여러 well의 평균값을 사용하였다. 계산식은 다음과 같다. To investigate the effect of medicinal extracts on splenocyte growth, splenocytes isolated and cultured in 96-well plates were divided into 5 × 10 5 cells / well, and medicinal extracts were dissolved in PBS and added to each concentration. Con A and LPS were used as a comparative material for the cell proliferation effect of medicinal herbs. After 48 hours of incubation, the solution was added for 4 hours by adding a solution of tetrazolium salt (CK-8 kit (Cell Counting Kit-8, Dojindo Laboratories, Tokyo, Japan)), and then absorbance was measured at 450 nm using an ELISA reader. The ratio of absorbance of drug addition wells to absorbance of control wells was calculated in% and the average value of the various wells was used. The calculation is as follows.
% = {(약물첨가 well의 Abs450nm 평균값) - (Blank well의 Abs450nm 평균값)}/{(Control well의 Abs450nm 평균값) - (Blank well의 Abs450nm 평균값)} × 100
% = {(Average 450nm average value of drug well)-(Abs 450nm average value of blank well)} / {(Abs 450nm average value of control well)-(Abs 450nm average value of blank well)} × 100
3) 결과3) results
생쥐 비장세포를 분리하여 약물처리 후 비장세포의 증식 유발 효과를 검색한 결과를 도 2에 나타내었다. 결과는 mean±S.E.M.으로 나타냈으며, ANOVA 및 Bonferroni multiple comparison analysis를 사용하였다. 정상군과 유발군의 비교시 유의성 있는 차이는 #; p<0.05, ##; p<0.01로 나타냈으며 유발군과 약물 처리군과의 비교시 *; p<0.05, **; p<0.01로 나타내었다.
Mouse splenocytes were isolated and the results of searching for proliferation-inducing effects of splenocytes after drug treatment are shown in FIG. 2. The results were expressed as mean ± SEM, and ANOVA and Bonferroni multiple comparison analysis were used. Significant differences between the normal and induced groups were #; p <0.05, ##; p <0.01, and when compared with the induction group and the drug treatment group *; p <0.05, **; p <0.01.
도 2에 나타난 바와 같이, 양성대조군으로 사용한 T-cell mitogen인 Con A의 1 및 2 μg/ml 처리시 대조군의 300% 이상 세포증식을 유발하였으며(P<0.01), LPS 처리시 0.1 및 1.0 μg/ml에서 농도의존적으로 세포증식이 유발되었다(각 p<0.05, p<0.01). 오적산 추출물은 농도의존적으로 세포증식이 유발되는 경향을 나타냈으며 50 및 100 μg/ml에서 각각 대조군의 123.2%(p<0.05) 및 136.8% (p<0.01)로 증식을 유발하였고 200 μg/ml의 고농도에서는 다시 감소되는 것으로 나타났다.
As shown in FIG. 2, when 1 and 2 μg / ml of the T-cell mitogen Con A used as a positive control group, more than 300% of the control group induced cell proliferation (P <0.01), and 0.1 and 1.0 μg when treated with LPS. Cell growth was induced in a concentration-dependent manner at / ml (p <0.05, p <0.01, respectively). Ojeoksan extract showed a tendency to induce cell proliferation in a concentration-dependent manner, and proliferated at 50 and 100 μg / ml to 123.2% (p <0.05) and 136.8% (p <0.01) of the control, respectively, and 200 μg / ml At high concentrations of, it was again reduced.
또한, 비장세포의 증식효과 검색 후 남은 세포를 수집하여 배양액 내의 IL-2 level을 측정한 결과를 도 3에 나타내었다. 도 3에 나타난 바와 같이, 오적산 추출물의 처리시 대조군의 IL-2 농도보다 약 2.5배 이상 증가하는 것으로 나타났다.
In addition, the results of measuring the IL-2 level in the culture by collecting the cells remaining after the proliferation effect of the splenocytes are shown in FIG. As shown in FIG. 3, the treatment of the Ojic acid extract increased about 2.5 times more than the IL-2 concentration of the control group.
실험예 3 : OVA 감작 동물 모델에 대한 면역활성 검색Experimental Example 3 Screening of Immune Activity for OVA Sensitized Animal Models
1) 실험 동물 및 투여1) experimental animals and administration
6주령의 수컷 C57BL/6 마우스에 난백알부민(Ovalbumine)+알루미늄(OVA+Al)을 항원으로 100 μg + 200 μg/100 ㎕의 용량을 시험 기간 동안 3회 복강 주사하여 감작시켰다 (Day 1, Day 8 및 Day 15). 약물 시료를 Day 1부터 3주간 1회/일, 1 g/kg으로 경구투여하고 대조군은 부형제인 증류수를 투여하였다. 3주간 투여 후 채혈하여 전혈구수 측정 및 남은 혈액을 원심분리하여 수집한 혈장으로 IgG, IgM을 정량(ELISA법)하였다. 채혈 후 비장을 적출하여 비장세포를 분리하였다.
Six-week-old male C57BL / 6 mice were sensitized by three intraperitoneal injections of 100 μg + 200 μg / 100 μl of Ovalbumine + aluminum (OVA + Al) as an antigen (Day 1, Day). 8 and Day 15). Drug samples were administered orally at 1 g / kg once a day for three weeks from Day 1, and the control group was administered with distilled water as an excipient. After 3 weeks of administration, the blood was collected and the IgG and IgM were quantified (ELISA method). After collecting blood, the spleen was extracted to separate splenocytes.
2) 비장세포의 분열유발 활성효과(mitogen effect)2) mitogen effect of splenocytes
적출한 비장에서 비장세포를 분리하고 각 투여군별로 비장세포를 96 well에 분주한 후 대조군, 비장세포 활성화 물질(Concanavalin A, LPS)을 처리하여 사용하여 비장세포의 세포증식효과를 CCK-8 kit를 사용하여 검색하였다. 각 투여군별로 여분의 비장세포는 유전자의 발현 변화를 검색하기 위하여 RNA를 분리하였다.
Splenocytes were isolated from the spleens and the splenocytes were dispensed into 96 wells for each administration group, and then treated with control and splenocyte activating substances (Concanavalin A, LPS). Search using. The extra splenocytes of each dose group isolated RNA to detect changes in gene expression.
생체 내에서의 면역 활성화 효과를 평가하기 위하여 OVA를 항원으로 감작시킨 생쥐에 약물을 투여 한 후 비장세포를 분리하여 각각의 투여군에서 분리한 비장세포에 mitogen으로 OVA, LPS, Con A를 처리하여 mitogen effect를 확인하였으며, 그 결과를 도 4에 나타내었다. 결과는 mean±S.E.M.으로 나타냈으며, ANOVA 및 Bonferroni multiple comparison analysis를 사용하였다. 정상군과 유발군의 비교시 유의성 있는 차이는 #; p<0.05, ##; p<0.01로 나타냈으며 유발군과 약물 처리군과의 비교시 *; p<0.05, **; p<0.01로 나타내었다.
In order to evaluate the effects of immune activation in vivo, the drug was administered to mice sensitized with OVA antigen, and then splenocytes were isolated and treated with OVA, LPS and Con A as mitogen to splenocytes isolated from each administration group. The effect was confirmed, and the result is shown in FIG. The results were expressed as mean ± SEM, and ANOVA and Bonferroni multiple comparison analysis were used. Significant differences between the normal and induced groups were #; p <0.05, ##; p <0.01, and when compared with the induction group and the drug treatment group *; p <0.05, **; p <0.01.
도 4에 나타난 바와 같이, 무처리 군(control)을 대조군으로 이에 대한 세포 증식 효과를 %로 나타내면, OVA를 감작시킨 군(OVA+Al)에서, 각각 mitogen으로 OVA를 처리한 군은 116.0±4.9% (P>0.05), LPS 처리군은 150.6±5.0% (P<0.01), ConA 처리군은 153.6±2.0% (P<0.01)로 LPS 또는 ConA의 처리로 세포 증식이 증가되었음을 확인할 수 있었다. 또한, OVA 감작 생쥐에 각각의 약물을 투여한 군에서 OVA+Al군과 비교하면 오적산 처리군은 OVA, LPS와 ConA에 대해서 유의성있는 증식효과 (mitogen effect)를 나타냄을 확인할 수 있었다.
As shown in Figure 4, when the control group (control) as a control to show the cell proliferation effect on this, in the OVA-sensitized group (OVA + Al), each group treated with OVA with mitogen 116.0 ± 4.9 % (P> 0.05), LPS-treated group was 150.6 ± 5.0% (P <0.01), ConA-treated group 153.6 ± 2.0% (P <0.01) was confirmed that the increase in cell proliferation by treatment with LPS or ConA. In addition, compared to the OVA + Al group in the group to which each drug was administered to the OVA sensitized mice, the OJ-treated group showed a significant proliferative effect on the OVA, LPS and ConA.
3) IgG 및 IgM의 정량3) Quantification of IgG and IgM
수집한 혈장 내 IgG 및 IgM의 양은 각각의 측정 kit(Bethyl laboratories Inc., U.S.A)를 사용하여 ELISA법으로 정량하였다.
The amount of IgG and IgM in the collected plasma was quantified by ELISA method using the respective measurement kit (Bethyl laboratories Inc., USA).
생체 내에서의 면역 활성화 효과를 평가하기 위하여 OVA를 항원으로 감작시킨 생쥐에 약물을 투여 한 후 혈중 OVA-specific IgG, OVA-specific IgG1, IgM level을 측정하였으며, 그 결과를 도 5에 나타내었다. 결과는 mean±S.E.M.으로 나타냈으며, ANOVA 및 Bonferroni multiple comparison analysis를 사용하였다. 정상군과 유발군의 비교시 유의성 있는 차이는 #; p<0.05, ##; p<0.01로 나타냈으며 유발군과 약물 처리군과의 비교시 *; p<0.05, **; p<0.01로 나타내었다.
In order to evaluate the effect of immune activation in vivo, after administration of the drug to mice sensitized with OVA antigen, blood OVA-specific IgG, OVA-specific IgG1, IgM levels were measured, and the results are shown in FIG. The results were expressed as mean ± SEM, and ANOVA and Bonferroni multiple comparison analysis were used. Significant differences between the normal and induced groups were #; p <0.05, ##; p <0.01, and when compared with the induction group and the drug treatment group *; p <0.05, **; p <0.01.
도 5에 나타난 바와 같이, OVA+Al 감작으로 인하여 OVA-특이적 IgG, OVA-특이적 IgG1의 혈중 농도가 상승하였으며 오적산의 투여로 OVA+Al군과 비교하여 각각 3.3, 3.4배 상승하였음을 확인할 수 있었다. 또한 IgM의 혈중 농도는 정상군과 OVA+Al 감작군에서 차이가 없었으나 오적산의 투여로 OVA+Al군과 비교하여 3.2배 상승하였음을 확인할 수 있었다.
As shown in FIG. 5, the concentration of OVA-specific IgG and OVA-specific IgG1 was increased due to sensitization of OVA + Al, and 3.3 and 3.4 times higher than that of the OVA + Al group, respectively. I could confirm it. In addition, the blood concentration of IgM was not different between the normal group and the OVA + Al sensitized group.
하기에 상기 약학 및 식품조성물의 제제예를 하기와 같이 예시하나, 이는 본 발명을 한정하고자 함이 아닌 단지 구체적으로 설명하기 위한 것이다.
Hereinafter, the pharmaceutical examples of the pharmaceutical and food compositions are exemplified as follows, but for the purpose of illustrating the present invention only, it is not intended to limit the present invention.
제제예 1 : 산제의 제조Formulation Example 1 Preparation of Powder
약전 제제 총칙 중 산제의 제조방법에 따라 1 포당 하기의 성분 함량으로 제조하였다. According to the preparation method of powder in the pharmacopeia formulation, it was prepared in the following component content per one packet.
오적산 추출물 400 mgOjeoksan Extract 400 mg
유당 100 mg
탈크 10 mg
상기 성분들을 혼합하고 기말포에 충진하여 산제를 제조하였다.
The powders were prepared by mixing the ingredients and filling the final foam.
제제예 2 : 정제의 제조Formulation Example 2 Preparation of Tablet
약전 제제 총칙 중 정제의 제조방법에 따라 1정 당 하기의 성분 함량으로 제조하였다. According to the preparation method of the tablet in the pharmacopeia formulation, it was prepared in the following component content per tablet.
오적산 추출물 400 mgOjeoksan Extract 400 mg
옥수수전분 100 mg
유당 100 mg
스테아린산 마그네슘 2 mg2 mg magnesium stearate
상기의 성분들을 혼합 후 직타법으로 정제를 제조하였다.
After mixing the above components, a tablet was prepared by a direct stroke method.
제제예 3 : 캅셀제의 제조Formulation Example 3 Preparation of Capsule
약전 제제 총칙 중 캅셀제의 제조방법에 따라 1 캅셀당 하기의 성분 함량으로 제조하였다. According to the preparation method of the capsules in the pharmacopeia formulation, it was prepared in the following component content per capsule.
오적산 추출물 400 mgOjeoksan Extract 400 mg
옥수수전분 100 mg
유당 100 mg
스테아린산 마그네슘 2 mg2 mg magnesium stearate
상기의 성분들을 혼합하여 분말을 제조한 후, 상기 분말을 통상의 캡슐제의 제조방법에 따라 경질 캡슐에 충전하여 캡슐제를 제조하였다.
After the powder was prepared by mixing the above components, the powder was filled in a hard capsule according to the conventional method for preparing a capsule to prepare a capsule.
제제예 4 : 주사제의 제조Formulation Example 4 Preparation of Injection
약전 제제 총칙 중 주사제의 제조방법에 따라 1 앰플당(2 ㎖) 하기의 성분 함량으로 제조한다.According to the preparation method of injectables in the pharmacopeia formulation, it is prepared in the following component contents per ampule (2 ml).
오적산 추출물 400 mgOjeoksan Extract 400 mg
주사용 멸균 증류수 적량Appropriate sterile distilled water for injection
pH 조절제 적량pH adjuster
통상의 주사제의 제조방법에 따라 1 앰플 당(2 ㎖) 상기의 성분 함량으로 제조하였다.
According to the conventional method for preparing an injection, the amount of the above ingredient was prepared per ampoule (2 ml).
제제예 5 : 액제의 제조Formulation Example 5 Preparation of Liquid
약전 제제 총칙 중 액제제의 제조방법에 따라 액제 100 ㎖당 하기의 성분 함량으로 제조한다.According to the method for preparing a liquid formulation in the Pharmacopoeia General Formulation, it is prepared in the following component content per 100 ml of the liquid formulation.
오적산 추출물 1 g1 g of red pepper acid extract
이성화당 10 g10 g of isomerized sugar
만니톨 5 g5 g of mannitol
정제수 적량Purified water
통상의 액체의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고, 레몬향을 적량 가한 다음 상기의 성분을 혼합하였다. 그 다음 정제수를 가하여 전체 100㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조하였다.
Each component was added to and dissolved in purified water according to the usual liquid preparation method, and lemon flavor was added appropriately, followed by mixing the above components. Then, purified water was added thereto to adjust the total volume to 100 ml, and the solution was prepared by sterilization by filling in a brown bottle.
제제예 6 : 건강 식품의 제조Formulation Example 6 Preparation of Health Food
오적산 추출물 1 g1 g of red pepper acid extract
비타민 혼합물 적량Vitamin mixture proper amount
비타민 A 아세테이트 70 ㎍70 μg of Vitamin A Acetate
비타민 E 1.0 ㎎Vitamin E 1.0 mg
비타민 B1 0.13 ㎎Vitamin B1 0.13 mg
비타민 B2 0.15 ㎎Vitamin B2 0.15 mg
비타민 B6 0.5 ㎎Vitamin B6 0.5 mg
비타민 B12 0.2 ㎍0.2 μg of vitamin B12
비타민 C 10 ㎎
비오틴 10 ㎍10 μg biotin
니코틴산아미드 1.7 ㎎Nicotinic Acid 1.7 mg
엽산 50 ㎍
판토텐산 칼슘 0.5 ㎎Calcium Pantothenate 0.5mg
무기질 혼합물 적량Mineral mixture
황산제1철 1.75 ㎎Ferrous Sulfate 1.75 mg
산화아연 0.82 ㎎Zinc Oxide 0.82 mg
탄산마그네슘 25.3 ㎎Magnesium carbonate 25.3 mg
제1인산칼륨 15 ㎎Potassium monophosphate 15 mg
제2인산칼슘 55 ㎎Dibasic calcium phosphate 55 mg
구연산칼륨 90 ㎎Potassium Citrate 90 mg
탄산칼슘 100 ㎎
염화마그네슘 24.8 ㎎Magnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.
The composition ratio of the above-mentioned vitamin and mineral mixture is mixed with a composition suitable for a health food in a preferred embodiment, but the compounding ratio may be arbitrarily modified. The granules may be prepared and used for preparing a health food composition according to a conventional method.
제제예 7 : 건강 음료의 제조Formulation Example 7 Preparation of Healthy Drink
오적산 추출물 1 g1 g of red pepper acid extract
구연산 1000 ㎎
올리고당 100 g100 g oligosaccharides
매실농축액 2 gPlum concentrate 2 g
타우린 1 g1 g of taurine
정제수를 가하여 전체 900 ㎖Add 900 ml of purified water
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간 동안 85℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2ℓ 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용할 수 있다. 상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated and then stored in the present invention It can be used for the manufacture of healthy beverage composition. Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.
Claims (11)
Immunity-enhancing composition containing the formulation of Ohjeoksan prescription as an active ingredient.
The immune system of claim 1, wherein the Ohjeoksan is composed of ephedra, ephedra, dermis, hindu, gilkyung, crust, donkey, health, count, baekbokyeong, white paper, cheonggung, hwangja, cinnamon, licorice, ginger, and baekbaek. Composition.
The composition of claim 1, wherein the formulation of Ohpisan prescription extract is prepared by extracting Ohjisan with water, C 1 to C 4 alcohol, or a mixed solvent thereof.
According to claim 3, wherein the formulation of the five-acid prescription extract is immune enhancing composition, characterized in that prepared by extracting with methanol or ethanol.
A pharmaceutical composition for the prevention or treatment of diseases caused by reduced immune function, comprising the composition according to any one of claims 1 to 4 as an active ingredient.
An immune enhancing food composition comprising the composition according to any one of claims 1 to 4 as an active ingredient.
The pharmaceutical composition according to claim 5, wherein the disease caused by lowered immune function is any one selected from the group consisting of a cold, an allergic disease, chronic fatigue, and cancer.
The pharmaceutical composition according to claim 7, wherein the allergic disease is any one selected from the group consisting of atopy, asthma and allergic rhinitis.
상기 추출액을 건조하는 단계를 포함하는 오적산 처방 추출물을 유효성분으로 함유하는 면역 증강용 조성물의 제조방법.
Preparing a extract by extracting a misoic acid with an extraction solvent; And
Method of producing an immune enhancing composition containing the Ohsiksan prescription extract comprising the step of drying the extract as an active ingredient.
The method of claim 9, wherein the extraction solvent is water, C 1 to C 4 alcohols, or a mixed solvent thereof.
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