KR102191165B1 - Anti-inflammatory composition using an extract of radish root, etc. - Google Patents
Anti-inflammatory composition using an extract of radish root, etc. Download PDFInfo
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- KR102191165B1 KR102191165B1 KR1020190009452A KR20190009452A KR102191165B1 KR 102191165 B1 KR102191165 B1 KR 102191165B1 KR 1020190009452 A KR1020190009452 A KR 1020190009452A KR 20190009452 A KR20190009452 A KR 20190009452A KR 102191165 B1 KR102191165 B1 KR 102191165B1
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Abstract
본 발명은 무우 뿌리 추출물 등을 이용한 항염증용 조성물을 개시한다. 구체적으로 본 발명은 무우(Raphanus sativus L.) 뿌리 추출물, 삼채(Allium hookeri) 전초 추출물, 오가피(Acanthopanax sessiliflorum) 줄기 추출물, 황칠나무(Dendropanax morbiferus)의 잎과 줄기 혼합물 추출물 및/또는 더덕(Codonopsis lanceolata) 뿌리 추출물의 혼합물이 세균 외막 독소인 LPS(lipopolysaccharide)로 염증반응이 유도된 마우스 대식세포를 이용한 실험과 오발부민(Ovalbumin)과 LPS와 더불어 미세먼지를 함께 사용하여 염증 반응을 유발한 동물모델을 이용한 실험에서 뚜렷한 항염증 활성을 가짐을 개시한다.The present invention discloses a composition for anti-inflammatory using radish root extract and the like. Specifically, the present invention is radish ( Raphanus sativus L.) root extract, three ( Allium hookeri ) outpost extract, Ogapi ( Acanthopanax sessiliflorum ) stem extract, Hwangchil tree ( Dendropanax morbiferus ) leaf and stem mixture extract and / or deodeok ( Codonopsis lanceolata ) An experiment using mouse macrophages in which an inflammatory reaction was induced with LPS (lipopolysaccharide), a bacterial outer membrane toxin, and an animal model that induced an inflammatory reaction by using Ovalbumin and LPS together with fine dust. It is disclosed that it has a pronounced anti-inflammatory activity in the experiment using.
Description
본 발명은 무우 뿌리 추출물 등을 이용한 항염증용 조성물에 관한 것이다.The present invention relates to a composition for anti-inflammatory using radish root extract and the like.
염증은 물리적인 외상, 유해한 화학물질, 박테리아, 곰팡이, 바이러스에 의한 감염이나 생체 내 대사산물 중의 자극성 물질에 의하여 야기되는 병리적 상태에 대응하여 나타나는 국소적인 생체의 방어 반응이다. 염증 반응에는 다양한 생화학적 현상이 관여하지만, 특히 대식세포(Macrophage)가 화학적 자극 등에 의하여 산화질소(NO)와 여러 염증성 사이토카인(TNF-α, IL-1β, IL-6)을 생성함으로써 염증반응에서 중요한 역할을 한다고 알려져 있다(Ito T., et al., Curr Drug Traget Inflamm Allergy, 2(3):257-265, 2003).Inflammation is a local protective response in the body in response to physical trauma, infection by harmful chemicals, bacteria, fungi, viruses, or pathological conditions caused by irritants in metabolites in the body. Although various biochemical phenomena are involved in the inflammatory response, in particular, macrophages produce nitric oxide (NO) and various inflammatory cytokines (TNF-α, IL-1β, IL-6) by chemical stimulation. It is known to play an important role in (Ito T., et al., Curr Drug Traget Inflamm Allergy, 2(3):257-265, 2003).
현재 항염증제로서 널리 사용되고 있는 비스테로이드성 소염제(non-steroidal anti-inflammatory drugs, NSAIDS)는 위장관 장애, 간장애, 신장애 등의 심각한 부작용을 야기한다고 알려져 있다(Rainsford KD., Subcell biochem., 42:3-27, 2007; Guruprasad P. Aithal.,Rheumatology., 7:139-150, 2011; Praveen P. N. Rao et al.,Pharmaceuticals., 3:1530-1549, 2010).Non-steroidal anti-inflammatory drugs (NSAIDS), which are currently widely used as anti-inflammatory drugs, are known to cause serious side effects such as gastrointestinal disorders, liver disorders, and renal disorders (Rainsford KD., Subcell biochem., 42:3). -27, 2007; Guruprasad P. Aithal., Rheumatology., 7:139-150, 2011; Praveen PN Rao et al., Pharmaceuticals., 3:1530-1549, 2010).
따라서 항염 활성을 가지면서 부작용이 적고 효과가 지속적인 새로운 약물의 개발이 여전히 필요하다고 할 수 있다. Therefore, it can be said that it is still necessary to develop a new drug that has anti-inflammatory activity, has few side effects, and is effective.
본 발명은 무우 뿌리 추출물 등이 항염증 활성을 가짐을 개시한다. The present invention discloses that radish root extract and the like have anti-inflammatory activity.
본 발명의 목적은 무우 뿌리 추출물 등을 이용한 항염증용 조성물을 제공하는 데 있다.An object of the present invention is to provide a composition for anti-inflammatory using radish root extract and the like.
본 발명의 다른 목적이나 구체적인 목적은 이하에서 제시될 것이다.Other or specific objects of the present invention will be presented below.
본 발명자들은, 아래의 실시예에서 확인되는 바와 같이, 무우(Raphanus sativus L.) 뿌리 추출물, 삼채(Allium hookeri) 전초 추출물, 오가피(Acanthopanax sessiliflorum) 줄기 추출물, 황칠나무(Dendropanax morbiferus)의 잎과 줄기 혼합물 추출물 및/또는 더덕(Codonopsis lanceolata) 뿌리 추출물의 혼합물이 세균 외막 독소인 LPS(lipopolysaccharide)로 염증반응이 유도된 마우스 대식세포를 이용한 실험과 오발부민(Ovalbumin)과 LPS와 더불어 미세먼지를 함께 사용하여 천식과 염증 반응을 유발한 동물모델을 이용한 실험에서 뚜렷한 항염증 활성과 항천식 활성을 가짐을 확인하였고, 나아가 이들 혼합물이 항높은 산화 활성을 가짐과 함께 간세포의 제2상 해독화 효소인 GST(glutathione-S-transeferase)를 뚜렷하게 활성화시킴을 확인하였다. The present inventors, as confirmed in the examples below, radish ( Raphanus sativus L.) root extract, three ( Allium hookeri ) outpost extract, Ogapi ( Acanthopanax sessiliflorum ) stem extract, hwangchil tree ( Dendropanax morbiferus ) leaves and stems The mixture extract and/or the mixture of the root extract of Codonopsis lanceolata were tested using mouse macrophages in which the inflammatory reaction was induced with the bacterial outer membrane toxin LPS (lipopolysaccharide), and fine dust was used together with Ovalbumin and LPS. Thus, in an experiment using an animal model that induced asthma and inflammatory reactions, it was confirmed that it has distinct anti-inflammatory and anti-asthmatic activities. Furthermore, these mixtures have high anti-oxidative activity and GST, an enzyme that detoxifies
전술한 바를 고려할 때, 본 발명은 일 측면에 있어서, 무우 뿌리 추출물, 삼채 전초 추출물, 오가피 줄기 추출물, 황칠나무의 잎과 줄기 혼합물의 추출물, 더덕 뿌리 추출물 및 이들 추출물의 2 이상의 혼합물을 유효성분으로 포함하는 항염증용 조성물로 파악할 수 있고, 다른 측면에 있어서는 무우 뿌리 추출물, 삼채 전초 추출물, 오가피 줄기 추출물, 황칠나무의 잎과 줄기 혼합물의 추출물, 더덕 뿌리 추출물 및 이들 추출물의 2 이상의 혼합물을 유효성분으로 포함하는 항산화용 조성물로 파악할 수 있다. 또 다른 측면에 있어서 무우 뿌리 추출물, 삼채 전초 추출물, 오가피 줄기 추출물, 황칠나무의 잎과 줄기 혼합물의 추출물, 더덕 뿌리 추출물 및 이들 추출물의 2 이상의 혼합물을 유효성분으로 포함하는 간 해독 기능 증진용 조성물로 파악할 수 있다.In consideration of the above, the present invention, in one aspect, radish root extract, three herb extract, Ogapi stem extract, extract of a mixture of leaves and stems of Hwangchil tree, extract of deodeok root, and a mixture of two or more of these extracts as an active ingredient. It can be understood as an anti-inflammatory composition containing, and in other aspects, radish root extract, three herb extract, Ogapi stem extract, extract of a mixture of leaves and stems of Hwangchil tree, deodeok root extract, and a mixture of two or more of these extracts are active ingredients. It can be identified as an antioxidant composition containing as. In another aspect, as a composition for enhancing liver detoxification function comprising radish root extract, three herb extract, ogapi stem extract, extract of leaf and stem mixture of Hwangchil tree, deodeok root extract, and mixtures of two or more of these extracts as active ingredients I can grasp it.
본 발명의 항염증용 조성물, 항산화용 조성물 및 간 해독 기능 증진용 조성물에서, 상기 유효성분은 무우 뿌리 추출물, 삼채 전초 추출물, 오가피 줄기 추출물 및 황칠나무의 잎과 줄기 혼합물의 추출물의 혼합물이 바람직하고 특히 아래의 실시예를 참조할 대 이들 혼합비가 30:30:10:30 중량비인 것이 바람직하다.In the composition for anti-inflammatory, antioxidant, and liver detoxification function enhancement of the present invention, the active ingredient is preferably a mixture of radish root extract, three herb extract, stem extract, and extract of a mixture of leaves and stems of Hwangchil tree, and In particular, it is preferable that these mixing ratios are 30:30:10:30 by weight, referring to the examples below.
또 본 발명의 항산화용 조성물에 있어서는 상기 유효성분은 무우 뿌리 추출물, 삼채 전초 추출물, 오가피 줄기 추출물 및 더덕 뿌리 추출물의 혼합물이 바람직하고, 특히 아래의 실시예를 참조할 때 이들의 혼합비가 50:20:10:20인 것이 바람직하다.In addition, in the antioxidant composition of the present invention, the active ingredient is preferably a mixture of radish root extract, three herb extract, Ogapi stem extract, and deodeok root extract, and in particular, when referring to the examples below, the mixture ratio thereof is 50:20. It is preferably :10:20.
본 명세서에서, "추출물"이란 추출 대상을 물, 탄소수 1 내지 4의 저급 알콜(메탄올, 에탄올, 부탄올 등), 메틸렌클로라이드, 에틸렌, 아세톤, 헥산, 에테르, 클로로포름, 에틸아세테이트, 부틸아세테이트, N,N-디메틸포름아미드(DMF), 디메틸설폭사이드(DMSO), 1,3-부틸렌글리콜, 프로필렌글리콜 또는 이들의 혼합 용매를 사용하여 침출하여 얻어진 추출물, 이산화탄소, 펜탄 등 초임계 추출 용매를 사용하여 얻어진 추출물 또는 그 추출물을 분획하여 얻어진 분획물을 의미하며, 추출 방법은 활성물질의 극성, 추출 정도, 보존 정도를 고려하여 냉침, 환류, 가온, 초음파 방사, 초임계 추출 등 임의의 방법을 적용할 수 있다. 분획된 추출물의 경우 추출물을 특정 용매에 현탁시킨 후 극성이 다른 용매와 혼합·정치시켜 얻은 분획물, 상기 조추출물을 실리카겔 등이 충진된 칼럼에 흡착시킨 후 소수성 용매, 친수성 용매 또는 이들의 혼합 용매를 이동상으로 하여 얻은 분획물을 포함하는 의미이다. 또한 상기 추출물의 의미에는 동결건조, 진공건조, 열풍건조, 분무건조 등의 방식으로 추출 용매가 제거된 농축된 액상의 추출물 또는 고형상의 추출물이 포함된다. In the present specification, the term "extract" refers to water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N, Extract obtained by leaching using N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof, using a supercritical extraction solvent such as carbon dioxide, pentane, etc. It means the obtained extract or the fraction obtained by fractionating the extract, and the extraction method can be applied any method such as cold sedimentation, reflux, warming, ultrasonic radiation, supercritical extraction, etc. in consideration of the polarity, extraction degree, and preservation degree of the active substance. have. In the case of a fractionated extract, a fraction obtained by suspending the extract in a specific solvent and then mixing and policing it with a solvent having a different polarity, and adsorbing the crude extract on a column filled with silica gel, etc., and then adding a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof. It is meant to include the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract from which the extraction solvent has been removed by a method such as freeze drying, vacuum drying, hot air drying, or spray drying.
또 본 명세서에서 "유효성분"이란 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다.In addition, in the present specification, the term "active ingredient" refers to an ingredient capable of exhibiting a desired activity alone or with a carrier that is not itself active.
또 본 명세서에서, "항염증"은 아래에서 정의되는 염증성 질환의 개선(증상의 경감), 치료, 그러한 질환의 발병 억제 또는 지연을 포함하는 의미이다.In addition, in the present specification, "anti-inflammatory" is meant to include improvement (reduction of symptoms), treatment, and inhibition or delay of the onset of inflammatory diseases as defined below.
또 본 명세서에서, 상기 "염증성 질환"이란 외부의 물리·화학적 자극 또는 박테리아, 곰팡이, 바이러스, 각종 알레르기 유발 물질 등 외부 감염원의 감염 또는 자가면역에 대한 국부적 또는 전신적 생체 방어 반응으로 특정되는 염증 반응이 일으키는 병리적 증상으로서 정의될 있다. 이러한 염증 반응은 각종 염증 매개 인자와 면역세포와 관련된 효소(예컨대 iNOS, COX-2 등) 활성화, 염증 매개 물질의 분비(예컨대, NO, TNF-α, IL-6 등의 분비), 체액 침윤, 세포 이동, 조직 파괴 등의 일련의 복합적인 생리적 반응을 수반하며, 홍반, 통증, 부종, 발열, 신체의 특정 기능의 저하 또는 상실 등의 증상에 의해 외적으로 나타난다. 상기 염증성 질환은 급성, 만성, 궤양성, 알레르기성 또는 괴사성을 띨 수 있으므로, 어떠한 질환이 상기와 같은 염증성 질환의 정의에 포함되는 한 그것이 급성이든지, 만성이든지, 궤양성이든지, 알레르기성이든지 또는 괴사성이든지를 불문한다. 구체적으로 상기 염증성 질환에는 천식, 알레르기성 및 비-알레르기성 비염, 만성 및 급성 비염, 만성 및 급성 위염 또는 장염, 궤양성 위염, 급성 및 만성 신장염, 급성 및 만성 간염, 만성 폐쇄성 폐질환, 폐섬유종, 과민성 대장 증후군, 염증성 통증, 편두통, 두통, 허리 통증, 섬유 근육통, 근막 질환, 바이러스 감염(예컨대, C형 감염), 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, 프로스타글라딘 E 과다 증후군, 아테롬성 동맥 경화증, 통풍, 관절염, 류머티스성 관절염, 강직성 척추염, 호지킨병, 췌장염, 결막염, 홍채염, 공막염, 포도막염, 피부염(아토피성 피부염 포함), 습진, 다발성 경화증 등이 포함될 것이다. In addition, in the present specification, the term "inflammatory disease" refers to an inflammatory reaction specified by an external physical or chemical stimulus or an infection of an external infectious agent such as bacteria, fungi, viruses, various allergens, or a local or systemic biological defense response against autoimmunity. It can be defined as the pathological symptom that causes. These inflammatory reactions include activation of various inflammatory mediators and enzymes related to immune cells (eg, iNOS, COX-2, etc.), secretion of inflammatory mediators (eg, secretion of NO, TNF-α, IL-6, etc.), body fluid infiltration, It involves a series of complex physiological reactions such as cell migration and tissue destruction, and is externally manifested by symptoms such as erythema, pain, swelling, fever, deterioration or loss of specific functions of the body. Since the inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so long as any disease is included in the definition of such an inflammatory disease, whether it is acute, chronic, ulcerative, allergic, or Regardless of whether it is necrotic or not. Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibroids. , Irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, myofascial disease, viral infection (such as type C infection), bacterial infection, fungal infection, burn, surgical or dental surgery wound, pro Hyperstagladin E syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. Will be included.
본 발명의 항염증용 조성물, 항산화용 조성물 및 간 해독 기능 증진용 조성물(이하 "본 발명의 조성물")은 그 유효성분을 용도, 제형 등에 따라 치료를 의도하는 염증성 질환의 개선 활성 등을 나타낼 수 있는 한 임의의 양(유효량)으로 포함할 수 있는데, 통상적인 유효량은 조성물 전체 중량을 기준으로 할 때 0.001 중량 % 내지 15 중량 % 범위 내에서 결정될 것이다. 여기서 "유효량"이란 그 적용 대상인 포유동물 바람직하게는 사람에게 의료 전문가 등의 제언에 의한 투여 기간 동안 본 발명의 조성물이 투여될 때, 염증성 질환 등의 개선, 치료, 또는 그러한 병리적 증상의 발병 억제·지연 등 의도한 의료적·약리학적 효과를 나타낼 수 있는 유효성분의 양을 말한다. 이러한 유효량은 당업자의 통상의 능력 범위 내에서 실험적으로 결정될 수 있다.The anti-inflammatory composition, antioxidant composition, and liver detoxification function enhancement composition of the present invention (hereinafter, "composition of the present invention") may exhibit the active ingredient for improving inflammatory diseases intended for treatment depending on the use, formulation, etc. It may be included in any amount (effective amount) as long as it is present, and a typical effective amount will be determined within the range of 0.001% to 15% by weight based on the total weight of the composition. Herein, the term "effective amount" refers to the improvement, treatment, or suppression of the onset of such pathological symptoms when the composition of the present invention is administered to a mammal, preferably a human, to which the composition of the present invention is administered during the administration period according to the advice of a medical professional, etc. It refers to the amount of active ingredient that can exhibit the intended medical and pharmacological effects such as delay. Such effective amounts can be determined empirically within the range of ordinary skill in the art.
삭제delete
본 발명의 조성물은 구체적인 양태에 있어서, 식품 조성물로서 파악할 수 있다.The composition of the present invention can be grasped as a food composition in a specific aspect.
본 발명의 식품 조성물은 어떠한 형태로도 제조될 수 있으며, 예컨대 차, 쥬스, 탄산음료, 이온음료 등의 음료류, 우유, 요구르트 등의 가공 유류, 껌류, 떡, 한과, 빵, 과자, 면 등의 식품류, 정제, 캡슐, 환, 과립, 액상, 분말, 편상, 페이스트상, 시럽, 겔, 젤리, 바 등의 건강기능식품 제제류 등으로 제조될 수 있다. The food composition of the present invention may be prepared in any form, such as beverages such as tea, juice, carbonated beverages, ion beverages, processed oils such as milk and yogurt, gums, rice cakes, Korean sweets, bread, snacks, and noodles. Foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrup, gels, jelly, can be prepared in health functional food preparations such as bars.
또 본 발명의 식품 조성물은 법률상·기능상의 구분에 있어서 제조·유통 시점의 시행 법규에 부합하는 한 임의의 제품 구분을 띨 수 있다. 예컨대 한국 「건강기능식품에관한법률」에 따른 건강기능식품이거나, 한국 「식품위생법」의 식품공전(식약처 고시 「식품의 기준 및 규격」)상 각 식품유형에 따른 과자류, 두류, 다류, 음료류, 특수용도식품 등일 수 있다.In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food according to the Korean 「Health Functional Food Act」, or confectionery, bean, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (``Food Standards and Standards'' notified by the Ministry of Food and Drug Safety). , Special use food, etc.
본 발명의 식품 조성물에는 그 유효성분 이외에 식품첨가물이 포함될 수 있다. 식품첨가물은 일반적으로 식품을 제조, 가공 또는 보존함에 있어 식품에 첨가되어 혼합되거나 침윤되는 물질로서 이해될 수 있는데, 식품과 함께 매일 그리고 장기간 섭취되므로 그 안전성이 보장되어야 한다. 식품의 제조·유통을 규율하는 각국 법률(한국에서는 「식품위생법」임)에 따른 식품첨가물공전에는 안전성이 보장된 식품첨가물이 성분 면에서 또는 기능 면에서 한정적으로 규정되어 있다. 한국 식품첨가물공전(식약처 고시 「식품첨가물 기준 및 규격」)에서는 식품첨가물이 성분 면에서 화학적 합성품, 천연 첨가물 및 혼합 제제류로 구분되어 규정되어 있는데, 이러한 식품첨가물은 기능 면에 있어서는 감미제, 풍미제, 보존제, 유화제, 산미료, 점증제 등으로 구분된다. The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. In the Food Additive Code (“Food Sanitation Act” in Korea) governing the manufacture and distribution of food, food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (“Food Additive Standards and Standards” notified by the Ministry of Food and Drug Safety), food additives are classified into chemical synthetic products, natural additives and mixed preparations in terms of ingredients.These food additives are sweeteners and flavors in terms of function. It is divided into agent, preservative, emulsifier, acidulant, thickener, etc.
감미제는 식품에 적당한 단맛을 부여하기 위하여 사용되는 것으로, 천연의 것이거나 합성된 것 모두 본 발명의 조성물에 사용할 수 있다. 바람직하게는 천연 감미제를 사용하는 경우인데, 천연 감미제로서는 옥수수 시럽 고형물, 꿀, 수크로오스, 프룩토오스, 락토오스, 말토오스 등의 당 감미제를 들 수 있다. Sweeteners are used to impart a suitable sweetness to food, and both natural and synthetic can be used in the composition of the present invention. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.
풍미제는 맛이나 향을 좋게 하기 위하여 사용될 수 있는데, 천연의 것과 합성된 것 모두 사용될 수 있다. 바람직하게는 천연의 것을 사용하는 경우이다. 천연의 것을 사용할 경우에 풍미 이외에 영양 강화의 목적도 병행할 수 있다. 천연 풍미제로서는 사과, 레몬, 감귤, 포도, 딸기, 복숭아 등에서 얻어진 것이거나 녹차잎, 둥굴레, 대잎, 계피, 국화 잎, 자스민 등에서 얻어진 것일 수 있다. 또 인삼(홍삼), 죽순, 알로에 베라, 은행 등에서 얻어진 것을 사용할 수 있다. 천연 풍미제는 액상의 농축액이나 고형상의 추출물일 수 있다. 경우에 따라서 합성 풍미제가 사용될 수 있는데, 합성 풍미제는 에스테르, 알콜, 알데하이드, 테르펜 등이 이용될 수 있다. Flavoring agents can be used to improve taste or flavor, and both natural and synthetic can be used. Preferably, it is the case of using a natural one. In the case of using natural ingredients, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, roundtails, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used, and esters, alcohols, aldehydes, terpenes, and the like may be used as synthetic flavoring agents.
보존제로서는 소듐 소르브산칼슘, 소르브산나트륨, 소르브산칼륨, 벤조산칼슘, 벤조산나트륨, 벤조산칼륨, EDTA(에틸렌디아민테트라아세트산) 등이 사용될 수 있고, 또 유화제로서는 아카시아검, 카르복시메틸셀룰로스, 잔탄검, 펙틴 등을 들 수 있으며, 산미료로서는 연산, 말산, 푸마르산, 아디프산, 인산, 글루콘산, 타르타르산, 아스코르브산, 아세트산, 인산 등이 사용될 수 있다. 산미료는 맛을 증진시키는 목적 이외에 미생물의 증식을 억제할 목적으로 식품 조성물이 적정 산도로 되도록 첨가될 수 있다.As a preservative, sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, Pectin, etc. may be mentioned, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like can be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste.
점증제로서는 현탁화 구현제, 침강제, 겔형성제, 팽화제 등이 사용될 수 있다.As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.
본 발명의 식품 조성물은 전술한 바의 식품첨가물 이외에, 기능성과 영양성을 보충, 보강할 목적으로 당업계에 공지되고 식품첨가물로서 안정성이 보장된 생리활성 물질이나 미네랄류를 포함할 수 있다.In addition to the food additives described above, the food composition of the present invention may include a physiologically active substance or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutritional properties and ensuring stability as a food additive.
그러한 생리활성 물질로서는 녹차 등에 포함된 카테킨류, 비타민 B1, 비타민 C, 비타민 E, 비타민 B12 등의 비타민류, 토코페롤, 디벤조일티아민 등을 들 수 있으며, 미네랄류로서는 구연산 칼슘 등의 칼슘 제제, 스테아린산마그네슘 등의 마그네슘 제제, 구연산철 등의 철 제제, 염화 크롬, 요오드칼륨, 셀레늄, 게르마늄, 바나듐, 아연 등을 들 수 있다. Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, etc., and minerals include calcium preparations such as calcium citrate, magnesium stearate. Magnesium preparations such as, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, and the like.
본 발명의 식품 조성물에는 전술한 바의 식품첨가물이 제품 유형에 따라 그 첨가 목적을 달성할 수 있는 적량으로 포함될 수 있다.In the food composition of the present invention, the food additive as described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.
본 발명의 식품 조성물에 포함될 수 있는 기타의 식품첨가물과 관련하여서는 각국 식품공전이나 식품첨가물 공전을 참조할 수 있다.With respect to other food additives that may be included in the food composition of the present invention, reference may be made to the food code of each country or the food additive code.
본 발명의 조성물은 다른 구체적인 양태에 있어서는 약제학적 조성물로 파악될 수 있다.The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.
본 발명의 약제학적 조성물은 유효성분 이외에 약제학적으로 허용되는 담체를 포함하여 당업계에 공지된 통상의 방법으로 투여 경로에 따라 경구용 제형 또는 비경구용 제형으로 제조될 수 있다. 여기서 투여 경로는 국소 경로, 경구 경로, 정맥 내 경로, 근육 내 경로, 및 점막 조직을 통한 직접 흡수를 포함하는 임의의 적절한 경로일 수 있으며, 두 가지 이상의 경로를 조합하여 사용할 수도 있다. 두 가지 이상 경로의 조합의 예는 투여 경로에 따른 두 가지 이상의 제형의 약물이 조합된 경우로서 예컨대 1차로 어느 한 약물은 정맥 내 경로로 투여하고 2차로 다른 약물은 국소 경로로 투여하는 경우이다. The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including a local route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, when one drug is firstly administered by an intravenous route and secondly, the other drug is administered by a local route.
약학적으로 허용되는 담체는 투여 경로나 제형에 따라 당업계에 주지되어 있으며, 구체적으로는 "대한민국약전"을 포함한 각국의 약전을 참조할 수 있다. Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopeias of each country, including the "Korean Pharmacopoeia."
본 발명의 약제학적 조성물이 경구용 제형으로 제조될 경우, 적합한 담체와 함께 당업계에 공지된 방법에 따라 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 현탁액, 웨이퍼 등의 제형으로 제조될 수 있다. 이때 적합한 담체의 예로서는 락토스, 글루코스, 슈크로스, 덱스트로스, 솔비톨, 만니톨, 자일리톨 등의 당류, 옥수수 전분, 감자 전분, 밀 전분 등의 전분류, 셀룰로오스, 메틸셀룰로오스, 에틸셀룰로오스, 나트륨 카르복시메틸셀룰로오스, 하이드록시프로필메틸셀룰로오스 등의 셀룰로오스류, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 마그네슘 스테아레이트, 광물유, 맥아, 젤라틴, 탈크, 폴리올, 식물성유, 에탄올, 그리세롤 등을 들 수 있다. 제제화활 경우 필요에 따라적절한 결합제, 윤활제, 붕해제, 착색제, 희석제 등을 포함시킬 수 있다. 적절한 결합제로서는 전분, 마그네슘 알루미늄 실리케이트, 전분페리스트, 젤라틴, 메틸셀룰로스, 소듐 카복시메틸셀룰로스, 폴리비닐피롤리돈, 글루코스, 옥수수 감미제, 소듐 알지네이트, 폴리에틸렌 글리콜, 왁스 등을 들 수 있고, 윤활제로서는 올레산나트륨, 스테아르산나트륨, 스테아르산마그네슘, 벤조산나트륨, 초산나트륨, 염화나트륨, 실리카, 탈쿰, 스테아르산, 그것의 마그네슘염과 칼슘염, 폴리데틸렌글리콜 등을 들 수 있으며, 붕해제로서는 전분, 메틸 셀룰로스, 아가(agar), 벤토나이트, 잔탄 검, 전분, 알긴산 또는 그것의 소듐 염 등을 들 수 있다. 또 희석제로서는 락토즈, 덱스트로즈, 수크로즈, 만니톨, 소비톨, 셀룰로스, 글라이신 등을 들 수 있다. When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier It can be prepared in such a formulation. Examples of suitable carriers at this time include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol, etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, colorants, and diluents may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferryst, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and oleic acid as a lubricant Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polydecylene glycol, etc., and as disintegrating agents starch, methyl cellulose , Agar, bentonite, xanthan gum, starch, alginic acid, or a sodium salt thereof. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. are mentioned as a diluent.
본 발명의 약제학적 조성물이 비경구용 제형으로 제조될 경우, 적합한 담체와 함께 당업계에 공지된 방법에 따라 주사제, 경피 투여제, 비강 흡입제 및 좌제의 형태로 제제화될 수 있다. 주사제로 제제화할 경우 적합한 담체로서는 수성 등장 용액 또는 현탁액을 사용할 수 있으며, 구체적으로는 트리에탄올 아민이 함유된 PBS(phosphate buffered saline)나 주사용 멸균수, 5% 덱스트로스 같은 등장 용액 등을 사용할 수 있다. 경피 투여제로 제제화할 경우 연고제, 크림제, 로션제, 겔제, 외용액제, 파스타제, 리니멘트제, 에어롤제 등의 형태로 제제화할 수 있다. 비강 흡입제의 경우 디클로로플루오로메탄, 트리클로로플루오로메탄, 디클로로테트라플루오로에탄, 이산화탄소 등의 적합한 추진제를 사용하여 에어로졸 스프레이 형태로 제제화할 수 있으며, 좌제로 제제화할 경우 그 담체로는 위텝솔(witepsol), 트윈(tween) 61, 폴리에틸렌글리콜류, 카카오지, 라우린지, 폴리옥시에틸렌 소르비탄 지방산 에스테르류, 폴리옥시에틸렌 스테아레이트류, 소르비탄 지방산 에스테르류 등을 사용할 수 있다.When the pharmaceutical composition of the present invention is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, triethanol amine-containing PBS (phosphate buffered saline), sterile water for injection, or an isotonic solution such as 5% dextrose may be used. . When formulated into a transdermal dosage form, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. Nasal inhalants can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide, and when formulated as a suppository, the carrier is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.
약제학적 조성물의 구체적인 제제화와 관련하여서는 당업계에 공지되어 있으며, 예컨대 문헌[Remington's Pharmaceutical Sciences(19th ed., 1995)] 등을 참조할 수 있다. 상기 문헌은 본 명세서의 일부로서 간주 된다.Regarding the specific formulation of the pharmaceutical composition is known in the art, for example, Remington's Pharmaceutical Sciences (19th ed., 1995) and the like can be referred to. These documents are considered as part of this specification.
본 발명의 약제학적 조성물의 바람직한 투여량은 환자의 상태, 체중, 성별, 연령, 환자의 중증도, 투여 경로에 따라 1일 0.001mg/kg ~ 10g/kg 범위, 바람직하게는 0.001mg/kg ~ 1g/kg 범위일 수 있다. 투여는 1일 1회 또는 수회로 나누어 이루어질 수 있다. 이러한 투여량은 어떠한 측면으로든 본 발명의 범위를 제한하는 것으로 해석되어서는 아니 된다. The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any aspect.
본 발명의 조성물 특히 항염증용 조성물과 항산화용 조성물은 또 다른 구체적인 양태에 있어서, 화장료 조성물로 파악할 수 있다. 본 발명의 항염증용 조성물이 화장품 조성물로 파악될 경우 그 용도는 염증성 피부 트러블 억제, 염증성 피부 자극 완화 등의 용도로 이해될 수 있다.The composition of the present invention, in particular, an anti-inflammatory composition and an anti-oxidation composition can be recognized as a cosmetic composition in another specific aspect. When the anti-inflammatory composition of the present invention is identified as a cosmetic composition, its use may be understood as a use for suppressing inflammatory skin problems and alleviating inflammatory skin irritation.
본 발명의 조성물이 화장료 조성물로 파악될 경우에도 그 화장료 조성물은 그 용도상, 법률상 임의의 제품 구분을 띨 수 있으며, 구체적으로 피부 트러블 개선, 아토피 피부염 개선 등의 용도를 가진 기능성 화장품, 비기능성 일반 화장품 등일 수 있다. 제품 형태에 있어서도 임의의 제품 형태를 띨 수 있는데, 구체적으로 용액, 현탁액, 유탁액, 페이스트, 젤, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션, 스프레이 등의 제품 형태를 띨 수 있다. 구체적인 제품 형태에 있어서는 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포옴, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형 등일 수 있다.Even if the composition of the present invention is identified as a cosmetic composition, the cosmetic composition can be classified as a product for its purpose and legally, and specifically, functional cosmetics with uses such as improving skin troubles and improving atopic dermatitis, non-functional It may be general cosmetics, etc. The product form can also take any product form, specifically solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, waxes. It can take the form of a product such as a foundation or spray. In a specific product form, it may be a flexible lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder formulation.
본 발명의 화장료 조성물은 그 유효성분 이외에 화장료 조성물에 통상적으로 이용되는 성분들, 예컨대, 안정화제, 용해화제, 계면활성제, 비타민, 색소 및 항료와 같은 통상적인 보조제, 및 담체를 포함할 수 있다. In addition to the active ingredient, the cosmetic composition of the present invention may include components commonly used in the cosmetic composition, such as stabilizers, solubilizers, surfactants, vitamins, conventional adjuvants such as pigments and perfumes, and carriers.
본 발명의 제형이 페이스트, 크림 또는 젤인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되는데, 구체적으로 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜, 소르비탄의 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, and the like may be used.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르, 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 등이 이용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and crystallites Castle cellulose, aluminum metahydroxide, bentonite, agar, and the like can be used.
본 발명의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.
본 발명의 화장료 조성물은 그 유효성분을 포함하는 것을 제외하고는 당업계에 통상적으로 행하여지는 화장료 조성물의 제조방법에 따라 제조할 수 있다.The cosmetic composition of the present invention can be prepared according to a method for preparing a cosmetic composition conventionally performed in the art, except for including the active ingredient.
전술한 바와 같이, 본 발명에 따르면 무우 뿌리 추출물 등을 이용한 항염증용 조성물을 제공할 수 있다.As described above, according to the present invention, it is possible to provide an anti-inflammatory composition using radish root extract or the like.
본 발명의 항염증용 조성물은 염증성 질환 개선 용도, 염증성 피부 자극의 완화 용도 등으로 식품, 화장품, 약품 등으로 제품화될 수 있다.The anti-inflammatory composition of the present invention may be commercialized into foods, cosmetics, medicines, etc. for use in improving inflammatory diseases and alleviating inflammatory skin irritation.
도 1은 마우스 유래 대식세포인 RAW264.7에서 세포독성 살펴본 결과이다.
도 2 및 도 3은 마우스 유래 대식세포인 RAW264.7에서 염증 인자(NO 및 TNF-α)의 생성 정도를 측정한 결과이다.
도 4 및 도 5는 염증 동물모델 실험에서 염증성 사이토카인 등의 생성 정도를 측정한 결과이다.
도 6 및 도 7은 항산화 활성 실험 결과이다.
도 8은 GST 활성 증진 실험 결과이다.1 is a result of examining cytotoxicity in RAW264.7, which is a mouse-derived macrophage.
2 and 3 are results of measuring the production of inflammatory factors (NO and TNF-α) in RAW264.7, which is a mouse-derived macrophage.
4 and 5 are results of measuring the degree of production of inflammatory cytokines, etc. in an inflammatory animal model experiment.
6 and 7 are results of antioxidant activity experiments.
8 is a result of an experiment for enhancing GST activity.
이하 본 발명을 실시예를 참조하여 설명한다. 그러나 본 발명의 범위가 이러한 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described with reference to examples. However, the scope of the present invention is not limited to these examples.
<실시예> 항염 활성 실험, 항산화 활성 실험, 간 해독 기능 증진 실험 <Example> Anti-inflammatory activity test, antioxidant activity test, liver detoxification function improvement test
1. 재료 및 방법1. Materials and methods
1.1 재료1.1 material
추출 대상은 무우(Raphanus sativus L.) 뿌리, 삼채(Allium hookeri) 전초, 오가피(Acanthopanax sessiliflorum) 줄기, 황칠나무(Dendropanax morbiferus)의 잎과 줄기 혼합물, 더덕(Codonopsis lanceolata) 뿌리를 사용하였다.Extraction targets were radish ( Raphanus sativus L.) roots, Allium hookeri outpost, Ogapi ( Acanthopanax sessiliflorum ) stem, Hwangchil tree ( Dendropanax morbiferus ) leaf and stem mixture, and deodeok ( Codonopsis lanceolata ) roots were used.
추출물은, 분말로 제조한 각 추출 대상 1kg에 물 10L를 가하고 100℃에서 5시간동안 환류추출기를 이용하여 열수추출하고, 열수추출물은 Whatman No.2로 여과한 다음, 여과액을 감압농축하고 동결건조하여 분말상으로 얻었다.For the extract, 10L of water was added to 1 kg of each extraction target prepared as a powder, and hot water was extracted using a reflux extractor at 100°C for 5 hours. The hot water extract was filtered with Whatman No.2, and the filtrate was concentrated under reduced pressure and frozen. It was dried and obtained in powder form.
이렇게 얻어진 각 추출물을 아래의 표와 같은 중량비로 혼합하여 실험에 사용하였다.Each extract thus obtained was mixed in a weight ratio as shown in the table below and used in the experiment.
1.1 항염 활성 세포 실험1.1 Anti-inflammatory activity cell test
(1) 세포배양(1) Cell culture
마우스 유래 대식세포인 RAW264.7 세포는 10% FBS와 1% 페니실린-스트렙토마이신을 함유된 DMEM(Dulbecco’s modified Eagle’s medium) 배지에 배양하였다. 5% 이산화탄소가 존재하에서 37℃에서 배양하였다.RAW264.7 cells derived from mouse macrophages were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS and 1% penicillin-streptomycin. It was incubated at 37°C in the presence of 5% carbon dioxide.
(2) 세포독성평가(2) Cytotoxicity evaluation
RAW 264.7 세포를 1×105 cells/well의 농도로 24시간 배양한 후, 다양한 농도의 시료를 처리하였다. 2시간 후, lipopolysaccharide(LPS, 1 mg/mL)의 농도로 24시간 자극한 후, 세포독성을 측정하였다. 세포 독성은 배지에 10배 희석한 CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)를 100 mL를 분주한 후, 1시간 동안 반응시켜, 490 nm에서 흡광도를 측정하였다.RAW 264.7 cells were cultured at a concentration of 1×10 5 cells/well for 24 hours, and then samples of various concentrations were treated. After 2 hours, after stimulation with a concentration of lipopolysaccharide (LPS, 1 mg/mL) for 24 hours, cytotoxicity was measured. For cytotoxicity, 100 mL of CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) diluted 10-fold in the medium was dispensed and reacted for 1 hour, and the absorbance was measured at 490 nm.
(3) NO 생성량 측정(3) Measurement of NO generation
배양액 내의 NO질소는 Griess분석법을 이용하여 측정하였다. 배양 상층액과 동량의 Griess 시약 (5% phosphoric acid와 1% salfanilamide의 혼합용액)을 첨가하여, 실온에서 20분간 반응시킨 후, 540 nm에서 흡광도를 측정하였다.NO nitrogen in the culture medium was measured using the Griess assay. Griess reagent (mixed solution of 5% phosphoric acid and 1% salfanilamide) in the same amount as the culture supernatant was added, reacted at room temperature for 20 minutes, and absorbance was measured at 540 nm.
(4) 사이토카인 생성량 측정(4) Measurement of cytokine production
RAW 264.7 세포를 24 well에서 2.5×105 cells/well의 농도로 분주하여, 24시간 배양하였다. 배양 상층액 제거 후, 다양한 농도의 시료를 처리하였다. 2시간 후, LPS로 24시간 자극하여, 배양 상층액 내의 TNF (Tumor necrosis factor)-a 생성량을 ELISA (Enzyme-linked immunosorbent assay) 법으로 측정하였다. RAW 264.7 cells were dispensed in 24 wells at a concentration of 2.5×10 5 cells/well and cultured for 24 hours. After removal of the culture supernatant, samples of various concentrations were treated. After 2 hours, by stimulation with LPS for 24 hours, the amount of TNF (Tumor necrosis factor)-a produced in the culture supernatant was measured by ELISA (Enzyme-linked immunosorbent assay) method.
1.2 항염 활성 동물실험1.2 Anti-inflammatory activity animal test
(1) 실험동물(1) Experimental animals
동물실험은 Sprague Dawley계의 백서 mouse의 체중 160-190 g정도를 무게별로 그룹으로 나누고 동신대학교 한의대의 동물사육실(습도 40-60%, 온도 24-26 ℃)에서 실시하였다. 먹이 급여는 고형사료(Pellet, GMO, Korea)를 자유롭게 먹도록 하였고 물을 충분히 공급하였다. 4일 이상 안정을 취하게 한 후 다시 군을 분리하였다. Animal experiments were carried out in the animal breeding room (humidity 40-60%, temperature 24-26 ℃) of the Sprague Dawley type white paper mice, divided into groups by weight of 160-190 g. For feeding, solid feed (Pellet, GMO, Korea) was freely eaten and water was supplied sufficiently. After being stabilized for more than 4 days, the groups were separated again.
(2) 시험 전처리 과정(2) Test pretreatment process
실험 mouse는 과민성 호흡 관련 천식 증가 및 염증 반응 유도를 위해 Ovalbumin(OVA, 1 mg/kg)+Lipopolysaccharide(LP, 5 mg/kg)를 주사용 생리식염수로 희석한 후 5일 간격으로 2회 복강투여 하여 전신을 감작시켰다. 이때 OVA는 10 mg/kg aluminum potassium sulfate(Sigma-Aldrich Co.)를 인산완충액(PBS) 용해한 후 혼합하여 사용하였다. 복강투여후 5일간 휴식을 취하게 하였다(Jang et al., 2015). Experimental mouse increased irritable respiratory-related asthma and inflammatory response For induction, Ovalbumin (OVA, 1 mg/kg) + Lipopolysaccharide (LP, 5 mg/kg) was diluted with physiological saline for injection, and then intraperitoneally administered twice every 5 days to sensitize the whole body. At this time, OVA was used after dissolving 10 mg/kg aluminum potassium sulfate (Sigma-Aldrich Co.) in a phosphate buffer (PBS). After intraperitoneal administration, they were given a break for 5 days (Jang et al., 2015).
또한 대기 오염 물질인 미세먼지 노출시험은 복강투여 후 6일째 되는 날에 2% 미세먼지(Standard Reperance Matter, NIST, USA)+3% ovalbumin+5% Lipopolysaccharide를 생리식염수에 녹여 밀폐용기에서 연무기(이노테크, 한국)를 사용하여 1일(4시간 간격으로 1회 15분간 투여) 총 4회 60분간 노출시켰다. 그리고 복강투여 후 7일, 9일, 11일 총 4회 시료를 연무하여 처리하였다. In addition, on the 6th day after intraperitoneal administration, 2% fine dust (Standard Reperance Matter, NIST, USA) + 3% ovalbumin + 5% Lipopolysaccharide is dissolved in physiological saline, and a nebulizer (inno. Tech, Korea) was used for a total of 4 exposures for 60 minutes per day (once administered at 4 hour intervals for 15 minutes). And after intraperitoneal administration, the samples were treated by misting a total of 4 times on the 7th, 9th, and 11th days.
(3) 시험 처리구(3) Test treatment section
실험 처리구는 아무 것도 처리하지 않는 무처리구(normal), 미세먼지+OVA+LPS(control), 미세먼지+OVA+LPS+dexamethasone(DEXA, 양성대조구, 3 mg/kg), 미세먼지+OVA+LPS+시료100 mg/㎏(HS100), 미세먼지+OVA+LPS시료200 mg/㎏(HS200), 미세먼지+OVA+LPS시료400 mg/㎏(HS400) 총 6처리구로 하였고 반복수는 5마리(n=5)로 처리하였다. 여기서 시료는 SSA4를 사용하였다.Experimental treatments were untreated (normal), fine dust + OVA + LPS (control), fine dust + OVA + LPS + dexamethasone (DEXA, positive control, 3 mg/kg), fine dust + OVA + LPS +
(4) Cytokine 변화 분석 - ELISA(4) Cytokine change analysis-ELISA
염증성 사이토카인(TNF-α, IL-1β, IL-6, IFN-γ)의 protein level에서의 발현량을 확인하였다. 마우스에서 채혈된 혈액을 원심분리기(Mega21, Hanil, Korea)에서 3,000 rpm, 30 min간 원심분리한 혈청을 사용하여 측정하였으며, sandwich ELISA mouse kit(R&D system, Mckinley place NE, USA)를 이용하였다. The expression level of inflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ) at the protein level was confirmed. Blood collected from mice was measured using serum centrifuged at 3,000 rpm for 30 min in a centrifuge (Mega21, Hanil, Korea), and a sandwich ELISA mouse kit (R&D system, Mckinley place NE, USA) was used.
(5) Cytokine 변화 분석: RT-PCR(5) Cytokine change analysis: RT-PCR
대기오염에 의한 염증과 관련된 물질의 변화를 측정하기 위하여, 폐 조직으로부터 extraction kit를 이용하여 total RNA를 추출하고 PCR 방법을 통해서 mRNA의 변화를 관찰하였다. 분리된 total RNA 5μg과 2.5 μl Oligo (dT), DEPC-treated water를, RT premix(Bioneer, Korea)에 넣어 Mastercycler gradient(Eppendorf, Germany)를 이용하여 50 μl cDNA를 합성하여 아래의 프라이머를 이용하여 PCR(GeneAmp PCR system 9700, Applied Biosystems, USA) 을 수행하였다. In order to measure changes in substances related to inflammation caused by air pollution, total RNA was extracted from lung tissue using an extraction kit, and changes in mRNA were observed through PCR. 5 μg of the separated total RNA, 2.5 μl Oligo (dT), and DEPC-treated water were added to RT premix (Bioneer, Korea), and 50 μl cDNA was synthesized using Mastercycler gradient (Eppendorf, Germany). PCR (GeneAmp PCR system 9700, Applied Biosystems, USA) was performed.
IL-1β: (F) 5'-ATGGCAACTGTTCCTGAACTCAACT-3', (R) 5'-CAGGACAGGTATAGATTCTTTCCTTT-3'IL-1β: (F) 5'-ATGGCAACTGTTCCTGAACTCAACT-3', (R) 5'-CAGGACAGGTATAGATTCTTTCCTTT-3'
IL-6 : (F) 5'-TTGCCTTCTTGGGACTGATG-3'(F), (R) 5'-CAGAATTGCCATTGCACAACT-3' IL-6: (F) 5'-TTGCCTTCTTGGGACTGATG-3'(F), (R) 5'-CAGAATTGCCATTGCACAACT-3'
IFN-γ : (F) 5’-AATGAACGCTACACACTGCA-3', (R) 5’-TGAAGAAGGTAGTAATCAGG-3' IFN-γ: (F) 5'-AATGAACGCTACACACTGCA-3', (R) 5'-TGAAGAAGGTAGTAATCAGG-3'
TNF-α : (F) 5’-CCACATCTCCCTCCAGAAAA-3', (R) 5’-AGGGTCTGGGCCATAGAACT-3'TNF-α: (F) 5'-CCACATCTCCCTCCAGAAAA-3', (R) 5'-AGGGTCTGGGCCATAGAACT-3'
1.3 항산화 활성 실험1.3 Antioxidant activity test
(1) DPPH radical 소거능(1) DPPH radical scavenging ability
여러 농도의 시료를 메탄올 용매로 용해하여, 900 μL의 DPPH 용액(100 μM)과 각 시료 100 μL를 혼합하여 교반한다. 이 혼합 시료를 암소에서 30분간 반응시킨 후 517 nm에서 흡광도를 측정하였다. DPPH 라디칼 소거 활성을, 3회 반복실험을 통해 시료 무처리군 대비 백분율로 결과를 산출하였다. 표준시료는 gillic acid를 사용하였다.Samples of various concentrations are dissolved in a methanol solvent, and 900 μL of DPPH solution (100 μM) and 100 μL of each sample are mixed and stirred. The mixed sample was reacted in the dark for 30 minutes, and then absorbance was measured at 517 nm. DPPH radical scavenging activity was calculated as a percentage compared to the sample untreated group through three replicates. The standard sample was gillic acid.
(2) ABTS cation radical 소거능 측정(2) ABTS cation radical scavenging ability measurement
7 mM ABTS[2,2-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt] 용액과 2.45 mM potassium persulfate를 최종농도로 혼합하여 실온에서 하루 동안 방치한 다음 ABTS radical 생성을 유도하였다. ABTs radical은 732 nm에서 흡광도 값이 0.7 nm가 되도록 하기 위해 희석액으로 PBS(phosphate buffer saline, pH 7.4) buffer를 활용하였다. 96 well plate에 희석한 ABTS 용액 200 μL와 시료 100 μL(MeOH에 녹여)을 농도별로 희석한고 1분 후 microplate reader에 넣고 흡광도 732 nm에서 측정하였다. ABTS 라디칼 소거 활성을, 3회 반복실험을 통해 시료 무처리군 대비 백분율로 결과를 산출하였다. 표준시료는 gillic acid를 사용하였다.7 mM ABTS[2,2-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt] solution and 2.45 mM potassium persulfate were mixed at a final concentration and left at room temperature for one day, and then ABTS radical formation was induced. . For the ABTs radical, a PBS (phosphate buffer saline, pH 7.4) buffer was used as a diluent to make the absorbance value 0.7 nm at 732 nm. 200 μL of the ABTS solution and 100 μL of the sample (dissolved in MeOH) diluted in a 96 well plate were diluted by concentration and placed in a microplate reader after 1 minute, and the absorbance was measured at 732 nm. ABTS radical scavenging activity was calculated as a percentage of the sample untreated group through three replicates. The standard sample was gillic acid.
1.4 간 해독 활성(GST) 실험 1.4 Liver detoxification activity (GST) experiment
(1) 세포배양(1) Cell culture
본 실험에 사용한 세포주로는 계대 보존 중인 마우스 유래의 간암 세포주인 Hepa1c1c7(murine hepatoma cell line) 세포를 10% FBS(fetal bovine serum)와 1% antibiotics(penicillin/streptomycin)를 첨가한 MEM 배지를 이용하여 5% CO2가 존재하는 37"C 배양기에서 l주일에 2-3회 계대배양하였다.As the cell line used in this experiment, hepa1c1c7 (murine hepatoma cell line) cells, which are mouse-derived liver cancer cell lines under passage preservation, were used in MEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin). It was subcultured 2-3 times per week in a 37"C incubator in the presence of 5% CO 2 .
(2) 세포내 GST 활성(2) intracellular GST activity
세포내 GST 활성은 배양한 세포를 0.4% trypan blue 염색법으로 세포수를 측정한 후 1 x 104 cells/well의 농도로 96-well microtiter plate에 200 μL씩 분주후 24 hr동안 배양하였다. 배지를 제거하고 새로운 MEM배지에 200 μL씩 첨가 및 시료를 첨가한 후 48시간 배양하였다. 다시 배지액을 제거하고 PBS로 4회 세척한 후 세포를 용해시켰다. 용해된 세포를 2% triton X-100액에 50 μL를 첨가하고 10분간 혼합시켰다. GST는 0.1 M potassium phosphate buffer에 녹인 2.5 mM GSH와 l mM CDNB(1-chloro-2,4-dinitrobenzene) 반응액 100 μL를 첨가하고 1분간 혼합시킨 다음 흡광도 405 nm에서 5분간 측정하였다. GST활성은 slop/min/mg protein으로 계산하여 시료 무처리군인 대조군의 GST 활성에 대한 시료를 처리한 GST 활성의 비율로 표현하였다.Intracellular GST activity was measured by measuring the number of cells in cultured cells by 0.4% trypan blue staining, and then 1 x 10 4 After dispensing 200 μL into a 96-well microtiter plate at the concentration of cells/well, the cells were cultured for 24 hr. The medium was removed, 200 μL each was added to a new MEM medium, and samples were added, followed by incubation for 48 hours. The medium was removed again, washed 4 times with PBS, and then the cells were lysed. The lysed cells were added 50 μL to 2% triton X-100 solution and mixed for 10 minutes. For GST, 2.5 mM GSH dissolved in 0.1 M potassium phosphate buffer and 100 μL of l mM CDNB (1-chloro-2, 4-dinitrobenzene) reaction solution were added, mixed for 1 minute, and absorbance was measured at 405 nm for 5 minutes. GST activity was calculated as slop/min/mg protein and expressed as the ratio of GST activity treated with the sample to the GST activity of the control group, which was not treated with the sample.
통계처리Statistical processing
모든 측정값은 Excel statistic program(Microsoft, USA)을 이용하여 평균치와 표준오차(mean±standard error)로 표시하였고, 각 실험군 간의 통계학적 분석은 Window용 SPSS(SPSS, USA)를 사용하여 비모수적 방법으로 Mann-Whitney U test를 시행하였다. 각 실험군은 대조군에 비하여 α=0.05 수준(P<0.05)과 α=0.01 수준(P<0.01)에서 유의성을 검정하였다. All measured values were expressed as mean values and standard errors (mean±standard error) using an Excel statistic program (Microsoft, USA), and statistical analysis between each experimental group was performed using a non-parametric method using SPSS for Windows (SPSS, USA). As a result, the Mann-Whitney U test was performed. Each experimental group was tested for significance at the α=0.05 level (P<0.05) and the α=0.01 level (P<0.01) compared to the control group.
2. 실험 결과2. Experiment result
2.1 항염증 세포실험2.1 Anti-inflammatory cell test
세포독성 실험 결과를 도 1에 나타내었고, NO 생성 및 TNF-α 생성 정도를 도 2 및 도 3에 나타내었다. 혼합물 시료는 모두 세포독성을 보이지 않았으며, 또 농도 의존적으로 NO 생성 및 TNF-α 생성을 억제하였다. 특히 D(SSA4) 시료가 활성이 높았다(A: SSA1, D:SSA4). 동물실험에서는 이러한 이유로 D 시료만을 사용하였다. The cytotoxicity test results are shown in Fig. 1, and the levels of NO production and TNF-α production are shown in Figs. 2 and 3. All of the mixture samples did not show cytotoxicity, and inhibited NO production and TNF-α production in a concentration-dependent manner. In particular, the D(SSA4) sample showed high activity (A: SSA1, D:SSA4). In animal experiments, only D sample was used for this reason.
2.2 항염 활성 동물실험2.2 Anti-inflammatory activity animal test
ELISA 측정 결과를 도 4에, RT-PCR 측정 결과를 도 5에 나타내었다. ELISA 측정 결과 및 RT-PCR 측정 결과 모두에서, 시료는(SSA4, HS)는 투여 용량에 비례하여 염증성 사이토카인 등의 생성을 억제하였다. The ELISA measurement results are shown in Fig. 4 and the RT-PCR measurement results are shown in Fig. 5. In both the ELISA measurement results and the RT-PCR measurement results, the samples (SSA4, HS) inhibited the production of inflammatory cytokines and the like in proportion to the dose administered.
2.3 항산화 활성2.3 antioxidant activity
DPPH 라디칼 소거 활성과 ABTS 라디칼 소거 활성을 도 6 및 도 7에 나타내었다. 도 6 및 도 7을 참조하여 보면, SSA1 시료가 가장 활성이 높음을 알 수 있다.DPPH radical scavenging activity and ABTS radical scavenging activity are shown in FIGS. 6 and 7. 6 and 7, it can be seen that the SSA1 sample has the highest activity.
2.2 간 해독 증진 활성 실험2.2 Liver detoxification enhancement activity experiment
간 해독 증진 활성 실험 결과를 도 8에 나타내었다. 간 해독 증진 활성에 있어서는 SSA4가 가장 높은 활성을 보였다.Figure 8 shows the results of the liver detoxification enhancing activity. SSA4 showed the highest activity in promoting liver detoxification.
Claims (8)
상기 무 뿌리 추출물, 삼채 전초 추출물, 오가피 줄기 추출물 및 황칠나무의 잎과 줄기 혼합물의 추출물의 혼합물이 30:30:10:30 중량비(오가피 줄기 추출물이 10 중량임)의 혼합물인,
항염증용 식품 조성물.
Including a mixture of radish root extract, three herb extract, Ogapi stem extract, and extract of a mixture of leaves and stems of Hwangchil tree as an active ingredient,
The mixture of the radish root extract, the extract of three herbs, the extract of the stem extract of Hwangchil, and the extract of the leaf and stem mixture of Hwangchil tree is a mixture of 30:30:10:30 weight ratio (the extract of the stem extract is 10 weight),
Food composition for anti-inflammatory.
상기 추출물은 물, 에탄올 또는 이들의 혼합용매 추출물인 것을 특징으로 하는 조성물.
The method of claim 1,
The extract is a composition, characterized in that water, ethanol or a mixed solvent extract thereof.
상기 무 뿌리 추출물, 삼채 전초 추출물, 오가피 줄기 추출물 및 황칠나무의 잎과 줄기 혼합물의 추출물의 혼합물이 30:30:10:30 중량비(오가피 줄기 추출물이 10 중량임)의 혼합물인,
항염증용 화장품 조성물.
Including a mixture of radish root extract, three herb extract, Ogapi stem extract, and extract of a mixture of leaves and stems of Hwangchil tree as an active ingredient,
The mixture of the radish root extract, the extract of three herbs, the extract of the stem extract of Hwangchil, and the extract of the leaf and stem mixture of Hwangchil tree is a mixture of 30:30:10:30 weight ratio (the extract of the stem extract is 10 weight),
Cosmetic composition for anti-inflammatory.
상기 추출물은 물, 에탄올 또는 이들의 혼합용매 추출물인 것을 특징으로 하는 조성물.
The method of claim 3,
The extract is a composition, characterized in that water, ethanol or a mixed solvent extract thereof.
상기 무 뿌리 추출물, 삼채 전초 추출물, 오가피 줄기 추출물 및 황칠나무의 잎과 줄기 혼합물의 추출물의 혼합물이 30:30:10:30 중량비(오가피 줄기 추출물이 10 중량임)의 혼합물인,
간 해독 기능 증진용 식품 조성물.
Including a mixture of radish root extract, three herb extract, Ogapi stem extract, and extract of a mixture of leaves and stems of Hwangchil tree as an active ingredient,
The mixture of the radish root extract, the extract of three herbs, the extract of the stem extract of Hwangchil, and the extract of the leaf and stem mixture of Hwangchil tree is a mixture of 30:30:10:30 weight ratio (the extract of the stem extract is 10 weight),
Food composition for improving liver detoxification function.
상기 추출물은 물, 에탄올 또는 이들의 혼합용매 추출물인 것을 특징으로 하는 조성물.The method of claim 5,
The extract is a composition, characterized in that water, ethanol or a mixed solvent extract thereof.
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