KR101263356B1 - Food composition for the oral purpose with anti-inflammatory activity - Google Patents
Food composition for the oral purpose with anti-inflammatory activity Download PDFInfo
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- KR101263356B1 KR101263356B1 KR1020110016057A KR20110016057A KR101263356B1 KR 101263356 B1 KR101263356 B1 KR 101263356B1 KR 1020110016057 A KR1020110016057 A KR 1020110016057A KR 20110016057 A KR20110016057 A KR 20110016057A KR 101263356 B1 KR101263356 B1 KR 101263356B1
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- inflammatory
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- mixed
- food composition
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Abstract
본 발명은 항염증용 식품 조성물에 관한 것으로, 특히 염증의 예방 및 증상개선에 효과가 있는 곡물 및 과실에서부터 유효활성 성분의 추출방법과 그 추출물을 함유하는 항염증용 식품 조성물에 관한 것이다.
이를 위한 본 발명의 항염증용 식품 조성물은, 상심자(오디), 서리태(검정콩) 및 양파 추출물을 각각 추출한 후, 이들을 적정의 비율로 혼합하여 적정량의 용량범위에서 항산화 효과, 염증성 부종 완화 효과, 급성 및 만성염증 억제효과, 염증유발 효소인 Lipoxygenase, Cyclooxygenase-2, iNOS(inducible Nitric oxide synthase) 및 Type I collagenase의 발현 억제효과, 염증유발인자인 TNF-a(tumor necrosis factor-alpha), IL-1b(interleukin 1-beta), IL-6(interleukin 6) 및 Prostaglandins의 생성억제효과 등이 우수한 소염진통제 및 관절염의 예방 및 증상개선에 효과가 있는 기능성 식품 조성물에 관한 것이다. The present invention relates to an anti-inflammatory food composition, and more particularly, to a method for extracting an active ingredient from grains and fruits effective for preventing inflammation and improving symptoms and an anti-inflammatory food composition containing the extract.
For the anti-inflammatory food composition of the present invention, after extracting the lettuce (Su), seotae (black soybean) and onion extract, respectively, and mixed them in an appropriate ratio of the antioxidant effect, inflammatory edema alleviation effect, Inhibitory effect of acute and chronic inflammation, inhibitory effects of inflammatory enzymes Lipoxygenase, Cyclooxygenase-2, iNOS (inducible Nitric oxide synthase) and Type I collagenase, TNF-a (tumor necrosis factor-alpha) and IL- The present invention relates to an anti-inflammatory analgesic agent having excellent anti-inflammatory effects such as 1b (interleukin 1-beta), IL-6 (interleukin 6), and prostaglandins, and a functional food composition effective in preventing and improving symptoms.
Description
본 발명은 항염증용 식품 조성물에 관한 것으로, 특히 양파와 상심자 및 서리태로부터 유효활성 성분의 추출 및 정제방법과 그 추출물을 함유하는 항염증용 식품 조성물에 관한 것이다.The present invention relates to an anti-inflammatory food composition, and more particularly, to a method for extracting and purifying an active ingredient from onions, lettuce and frost, and an anti-inflammatory food composition containing the extract.
본 발명은 양파 추출물과 상심자 추출물 및 서리태 추출물을 적정비로 혼합하여 진통효과, 염증유발인자(일산화질소, 프로스타그란딘) 생성 억제작용, 염증유발 사이토카인(TNF-α, IL-1β, IL-6) 생성 억제작용, 염증관련 효소(COX2, iNOS,Lipoxygenase, Type I Collagenase) 활성 저해작용, 염증관련 효소(iNOS, COX-2) 발현 억제작용, 항산화 작용(DPPH 소거작용, H2O2 소거작용, superoxide radical 소거작용) 등이 우수한 소염진통제, 관절염 예방 및 증상 개선제를 위한 항염증용 식품 조성물에 관한 것이다.The present invention is a mixture of onion extract, lettuce extract, and frost extract in an appropriate ratio analgesic effect, inhibitory effect on the production of inflammation-causing factors (nitrogen monoxide, prostaglandin), inflammation-induced cytokines (TNF-α, IL-1β, IL-6) Inhibits production, inhibits the activity of inflammation-related enzymes (COX2, iNOS, Lipoxygenase, Type I Collagenase), inhibits the expression of inflammation-related enzymes (iNOS, COX-2), antioxidants (DPPH scavenging, H2O2 scavenging, superoxide radical scavenging The present invention relates to an anti-inflammatory food composition for anti-inflammatory analgesics, arthritis prevention and symptom ameliorating agents having excellent effects.
일반적으로 양파(Allium cepa L.)는 양총이라고도 불리며 식품공전 원재료 분류에서 근채류로써 식용이 가능하다. 다른 근채류와 비교하여 탄수화물, 단백질, 비타민 및 무기질(칼륨, 철, 인, 나트륨 등)이 많이 함유되어 있고 갈락탄(galactan), 자일란(xylan), 메틸 펜토오즈(methyl pentose) 및 헤미셀룰로오즈(hemicellulose)도 많다. 매운 성분은 알릴 엔-프로필 디설파이드(allyl n-propyl disulfide), 알리신(allicin), 디하이드로젠 설파이드(dihydrogen sulfide) 등이 있는데, 이것은 열을 가하면 기화되거나 분해되어 설탕의 50배의 단맛을 내는 프로필메르캡탄(propylmercaptan)을 형성한다. 퀘르세틴(quercetin, C15H10O7)은 양파(적양파 혹은 흑양파 포함)의 껍질 부분에 많이 들어 있으며, 지방성분의 산패를 막아주며 고혈압의 예방에 효과가 있다고 알려져 있다. 잎에는 100g 중에 비타민A 5,000IU, 비타민C 45mg, 칼슘 80mg, 마그네슘 24mg, 칼륨 220mg이 들어 있다. 일반적으로 디하이드로젠 설파이드(dihydrogen sulfide), 메르캡탄(mercaptan), 알콜(alcohol)류, 디설파이드(disulfide)류(thiol methyldisulfide, allyldisulfide), 트리설파이드(trisulfide)류, 알데히드(aldehyde)류 등이 양파의 주요 향기성분으로 알려져 있다. [참고문헌: 1. 원색한국식물도감, ㈜ 교학사, pp67(1996) 2. 대한식물도감, 향문사, pp92(1979) 3. 원색한국기준식물도감, 아카데미서적, pp122(1996)]In general, onion (Allium cepa L.) is also called yanggun and can be eaten as a root vegetable in the classification of raw materials of food industry. Compared to other root vegetables, it contains more carbohydrates, proteins, vitamins and minerals (potassium, iron, phosphorus, sodium, etc.), galactan, xylan, methyl pentose and hemicellulose ) Spicy ingredients include allyl n-propyl disulfide, allicin, and dihydrogen sulfide, which are vaporized or decomposed when heated to give 50 times sweeter sugar. To form a mercaptan (propylmercaptan). Quercetin (C15H10O7) is found in the skin of onions (including red onions or black onions), and is known to prevent fat loss and prevent high blood pressure. The leaves contain vitamin I 5,000 IU, vitamin C 45 mg,
상심자(오디)는 상심, 상실, 오심, 흑심이라고도 불리며, 상지 또는 오디나무라고 불리는 뽕나무(Morus alba L.)의 열매로서, 식품공전에 주원료(열매, 잎, 어린가지)로 사용 가능한 동식물로 분류된다. 작은 열매가 모여서 하나의 덩어리로 이루어져 있으며 긴 원형이고 길이 1 ~ 2㎝, 지름 0.5 ~ 0.8㎝이다. 황자색, 자홍색 또는 암자색을 띠고 짧은 줄기가 있다. The mulberry is also the fruit of the mulberry tree (Morus alba L.), also called the lettuce, the mulberry, the nausea, and the black heart. It is an animal and plant that can be used as the main ingredient (fruit, leaf, sprig) in food festival. Are classified. Small fruits are gathered and consist of one lump, long round, 1 ~ 2㎝ long, 0.5 ~ 0.8㎝ in diameter. Yellow, magenta or dark purple with short stems.
또한, 본초강목, 소경, 진장기, 본조식감 등의 고전 문헌에 따르면 성질은 차고, 맛은 달며, 독이 없으며, 소갈증을 해소하고 오장을 편안하게 하고, 당뇨와 관절에 이롭게 하며, 혈기를 통하게 하고, 오래 먹으면 허기짐을 잊을 수 있다고 한다. 상심자의 성분으로는 Albafuran A, Albafuran B, Albafuran C, Albanol A, Albanol B, Alboctalol, alpha-Pinene(+,-), Artecanin, Astragalin, Bergapten, beta-Amyrin, beta-Amyrin acetate, 3-beta-Hydroxystigmast-5-en-7-one, beta-Resorcylaldehyde, beta-Sitosterol, Betulinic acid Mulberrofuran T, omega-Hydroxymoracin N, Oxydihydroresveratrol, Oxyresveratrol (E-form), Quercetin, Rutin, Resorcinol, Resveratrol, Sanggenon C, Sanggenon D, Sanggenon E, Sanggenon P, Scopoletin, Stigmasterol, Umbelliferone, Zeatin 등이다. [참고문헌: 1. 한국의 약용식물, ㈜ 교학사, pp73,(2000) 2. 약초의 성분과 이용, 일월서각, pp180(1976) 3. 한약(생약)규격집 주해서, 한국메디칼인덱스사, pp202(1998) 4. 식품원재료, 한국식품의약품안전청, 원재료명-뽕나무 검색결과]In addition, according to the classical literature, such as herbal wood, small diameter, jinjanggi, main breakfast, etc., the nature is cold, the taste is sweet, there is no poison, quenches thirst, relaxes the five intestines, benefits diabetes and joints, If you eat long, you can forget hunger. The components of the loser include Albafuran A, Albafuran B, Albafuran C, Albanol A, Albanol B, Alboctalol, alpha-Pinene (+,-), Artecanin, Astragalin, Bergapten, beta-Amyrin, beta-Amyrin acetate, 3-beta- Hydroxystigmast-5-en-7-one, beta-Resorcylaldehyde, beta-Sitosterol, Betulinic acid Mulberrofuran T, omega-Hydroxymoracin N, Oxydihydroresveratrol, Oxyresveratrol (E-form), Quercetin, Rutin, Resorcinol, Resveratrol, Sanggen Con Sang Don Con , Sanggenon E, Sanggenon P, Scopoletin, Stigmasterol, Umbelliferone and Zeatin. [Reference: 1. Medicinal Plants of Korea, Kyohaksa Co., Ltd., pp73, (2000) 2. Ingredients and Utilization of Herbs, Ilwol Seokgak, pp180 (1976) 3. Collection of Herbal Medicines (Medicinal Herbs) Standards, Korea Medical Index, pp202 (1998) 4. Food Ingredients, Korea Food and Drug Administration, Ingredients-Mulberry Search Results]
한편, 서리태(Glycine max (L.) Merr.)는 흑두 또는 검정콩(Black beans)이라고 불리며 식품공전 원재료 분류에 두류로 분류된다. 100g당 영양가가 에너지 378kcal, 수분 11.7g, 단백질 34.3g, 지질 18.1g, 당질 26.5g, 섬유 4.0g, 회분 5.4g, 칼슘 224mg, 인 629mg, 철 7.8mg, 나트륨 5mg, 칼륨 1,539mg, 비타민 B1 0.34mg, 비타민B2 0.22mg, 나이아신 1.9mg이며, 불포화지방산(리놀레산), 비타민E , 칼륨 등이 풍부하고, 생리활성물질에는 Amino acid(aspartic acid, glutamic acid), Carotene, Vitamin(B1, B2, B12), Isoflavone, Daidzin, Genistin, Choline, Organic acid(folic acid, pantothenic acid), Lecithin, Saponins(soyasapogenol A, B, C, D, E의 aglycon) 등이 있다. On the other hand, Glycine max (L.) Merr. Is called black bean or black beans and is classified as bean in the food ingredient classification. Nutritional value per 100 g: energy 378 kcal, water 11.7 g, protein 34.3 g, lipid 18.1 g, sugar 26.5 g, fiber 4.0 g, ash 5.4 g, calcium 224 mg, phosphorus 629 mg, iron 7.8 mg,
또한, 종피에는 delphinidin-3-o-β-D-glucoside, cyanidin-3-o-β-D-glucoside, petuinidin-3-o-β-D-gluc-oside 등이 함유되어 있다. 고전 문헌에 의하면 서리태에는 기관지를 강하게 하고 내장의 점막을 튼튼하게 하는 작용이 있어 예로부터 기침의 묘약으로 이용되어 왔다. 서리태를 검은 설탕이나 벌꿀로 단맛을 내서 먹으면 기침 뿐만 아니라 기관지나 천식에도 효과적이다. 서리태를 끓인 물은 각기병·위궤양·신장병에도 좋다. 서리태는 해독효과가 있어 한방에서는 서리태와 팥을 볶아 가루로 만들어 독을 제거하는데 사용할 정도이다. [참고문헌: 1. 콩나물콩 품종별 생육특성 및 일반성분 조성, 콩연구회 18차 연구발표회 요지, pp4 ~ 7(1992) 2. 콩 고단백계통 종실 성분함량의 지역변이, 한육지, 25(3): pp157 ~ 162(1993) 3. 우리나라 재래종 대두의 단백질 및 지방함량에 관한 연구, 한육지, 7: pp40 ~ 44(1975) 4. 식품원재료, 한국식품의약품안전청, 원재료명-서리태 검색결과]The epidermis also contains delphinidin-3-o-β-D-glucoside, cyanidin-3-o-β-D-glucoside, and petuinidin-3-o-β-D-gluc-oside. According to the classical literature, Suritae has been used as a cough potion since ancient times because it has an effect of strengthening the bronchus and strengthening the mucous membrane of the intestine. Eating frosted sweet sugar with black sugar or honey is effective for bronchial or asthma as well as coughing. Boiling frosted water is good for each bottle, stomach ulcer, kidney disease. Seoritae has a detoxifying effect, so in Oriental medicine, it is roasted and dried to make poisonous powder. [References: 1. Growth Characteristics and Composition of General Components of Soybean Sprouts and Soybean Varieties, Journal of the Soybean Research Association, 18th Annual Meeting, pp4 ~ 7 (1992) 2. Regional Variation of Seed Contents of Soybean High Protein System : pp157 ~ 162 (1993) 3. A Study on the Protein and Fat Content of Korean Soybeans, Han Yuji, 7: pp40 ~ 44 (1975) 4. Food Ingredients, Korea Food and Drug Administration, Raw Materials Name-Suritae Search Results]
통증은 통각수용기에서 신경섬유로 향하는 구심성 신경로를 통하여 대뇌피질 및 계통영역을 자극하는 기전이 느끼는 불쾌한 감각이다. 통증 기간에 따라 급성 통증, 아급성 통증, 재발성 통증, 지속성인 암통증, 만성통증, 만성통증 증후군이 있다. 통증과 관련된 물질로는 아라키돈산이 사이클로옥시제나제(cyclooxygenese) 및 리포옥시제나제(lipooxigenase) 효소에 의해 생성되는 프로스타그란딘(prostaglandin), 루코트리엔(leukotriene), 트롬복산(thromboxane)과 세로토닌(serotonin), 카테콜아민(catecholamine), 프로톤(protons), 히스타민(histamine), 포타슘 이온 등이 있다. Pain is an unpleasant sensation that the mechanism of stimulating the cerebral cortex and the systemic region through the afferent neuropathy from pain receptors to nerve fibers. Depending on the duration of pain, there are acute pain, subacute pain, recurrent pain, persistent cancer pain, chronic pain and chronic pain syndrome. Pain-related substances include prostaglandin, leukotriene, thromboxane and serotonin, in which arachidonic acid is produced by the cyclooxygenese and lipooxigenase enzymes. ), Catecholamines, protons, histamine, potassium ions, and the like.
또한, 통증치료에는 물리치료(냉치료, 열치료, 경피적 전기 신경자극 치료, 운동치료), 약물치료(진통제, 비마약성 진통제, 비스테로이드성 항염증제, 항우울제), 주사치료(주로 근육통증부위에 국소적으로 주사)가 있지만 각각의 치료방법은 대개 부작용을 가지고 있기 때문에 현실적으로 적절하고 완벽한 치료를 기대하기 어렵다.In addition, pain therapy includes physical therapy (cold therapy, heat therapy, percutaneous electrical neurostimulation therapy, exercise therapy), drug therapy (painkillers, non-narcotic analgesics, nonsteroidal anti-inflammatory drugs, antidepressants), injection therapy (mainly local to the muscle pain area). Injections, but each treatment usually has side effects, making it difficult to expect adequate and complete treatment.
염증의 현재까지 알려진 기전은 세포손상으로 인한 히스타민 및 키닌 등의 방출로 혈관확장, 모세혈관 투과성 증가 및 염증부위로 대식세포의 집결이 일어나고, 이에 따라서 감염부위의 혈류량 증가, 부종, 면역세포 및 항체 이동, 통증, 발열 등의 현상이 일어난다. To date, the mechanism of inflammation is the release of histamine and kinin due to cell damage, resulting in vasodilation, increased capillary permeability, and aggregation of macrophages into inflammatory sites, resulting in increased blood flow, swelling, immune cells and antibodies at the site of infection. Movement, pain, and fever occur.
또한, 염증을 유발하는 인자에는 펩티드성 인자 이외에 프로스타그란딘, 루코트리엔, 혈소판활성인자와 같은 지질성 인자, 염증인자 합성효소, NO(nitric oxide)와 같은 자유라디칼, 세포접착분자, 면역계, 응고인자 등이 관여한다. 현재 염증의 치료제에는 이부프로펜과 같은 합성의약품, 항히스타민제, 스테로이드, 코티손, 면역억제제, 면역 항진제 등이 사용되고 있으나 치료효과가 일시적이거나 단순 증상완화, 과민반응, 면역체계 악화 등의 부작용이 많이 있고 염증의 근본적인 치료가 어렵다.In addition to the peptide-inducing factor, inflammation-causing factors include prostaglandins, leukotrienes, lipid factors such as platelet activators, inflammatory synthase, free radicals such as NO (nitric oxide), cell adhesion molecules, immune system, and coagulation. Factors are involved. Currently, synthetic drugs such as ibuprofen, antihistamines, steroids, cortisones, immunosuppressants, and immunosuppressive drugs are used, but there are many side effects such as temporary or simple symptom relief, hypersensitivity reactions, and immune system deterioration. Fundamental treatment is difficult.
퇴행성 관절염(degenerative arthritis)이란 골관절염(osteoarthritis, degenerative joint disease)이라고도 하며, 외상, 질병 및 기형으로 인한 경우도 있으나 환자의 대다수는 주로 관절을 보호하는 연골의 퇴행성 손상으로 인해 염증과 통증을 유발하는 경우이다. Degenerative arthritis, also called osteoarthritis (degenerative joint disease), is sometimes caused by trauma, disease, or malformations, but the majority of patients cause inflammation and pain mainly due to degenerative damage to cartilage that protects the joints. to be.
또한, 일반적으로 노화가 퇴행성 관절염의 발생을 증가시키기는 하지만 성별, 유전적 요소, 비만정도, 생활습관 등도 연관되어 있다. 퇴행성 관절염의 치료는 중증인 경우 관절경을 이용한 유리체와 활액막을 제거, 활막 절제술, 소파관절 성형술, 다발성 천공술, 인공관절 치환술과 같은 수술적 치료가 이루어 지며 중증이 아닌 경우 약물요법이 주로 이루어 진다. In addition, although aging generally increases the incidence of degenerative arthritis, sex, genetic factors, obesity, and lifestyle are also associated. In severe cases, degenerative arthritis is treated by arthroscopic removal of vitreous and synovial membranes, synovial resection, curettage, multiple perforation, arthroplasty and pharmacotherapy.
류마티스 관절염의 명확한 원인은 아직 밝혀지지 않았지만 자가면역(autoimmunity) 현상이 주요 기전으로 알려져 있다. 활막(synovium)의 염증으로 시작하여 주위의 연골과 뼈로 염증이 확산되어 관절(주로 초기에는 손가락, 손목, 발가락 관절이, 병이 진행함에 따라 팔꿈치, 어깨, 발목, 무릎 관절)의 변형이 초래될 뿐만 아니라 피하 결절, 혈관염, 폐섬유화증, 빈혈, 피부 궤양 등의 합병증도 발생한다. 여러 가지 약제들이 이 병의 치료에 사용되고 있으나 아직까지 류마티스 관절염을 완치하는 약제는 개발되어 있지 않다.The exact cause of rheumatoid arthritis is not known yet, but autoimmunity is known as a major mechanism. Inflammation of the synoviium begins to spread to surrounding cartilage and bones, leading to deformation of the joints (primarily the fingers, wrists, toes, and joints of the elbows, shoulders, ankles, and knees as the disease progresses). In addition, complications such as subcutaneous nodules, vasculitis, pulmonary fibrosis, anemia and skin ulcers occur. Various drugs are used to treat the disease, but no drug has been developed to cure rheumatoid arthritis.
관절염에 사용되는 약물 중에서 합성의약품에는 아스피린, 아세트아미노펜과 같은 소염 진통제를 비롯해서 쎄레브렉스, 바이옥스와 같은 NSAIDs(non-steroidal anti-inflammatory drugs)계열 제제, TNF 차단제(tumor necrosis factor 차단제- 류마티스 관절염의 경우 사용) 등이 사용되고 있으나 질병의 근원적인 치료가 불가능하고 소화기계 및 혈액응고기전의 심각한 부작용이 보고되고 있어서 사용에 주의를 요한다. Among the drugs used for arthritis, synthetic drugs include anti-inflammatory analgesics such as aspirin and acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs) such as cerebrex and viox, and tumor necrosis factor blockers-rheumatoid arthritis. ), But it is impossible to cure the disease, and serious side effects of the digestive system and blood coagulation have been reported.
또한, 천연물 의약품에는 현재 조인스정, 아드로다캡슐, 이모튼정, 세이미정, 아트렌정, 글루코사민, 황산 콘드로이친과 같은 제품이 사용되고 있으나 그 효과가 미미하거나 그 효과를 입증하기 위한 엄격한 임상연구를 거친 제품이 드물다. 실제로 국내에서 관절염 치료를 위해 글루코사민을 복용하는 것이 사회적으로 유행처럼 되었으나 미국 FDA에서 대규모 환자들을 대상으로 치료효과를 규명하기 위한 연구를 수행한 결과, 전혀 효과가 없는 것으로 밝혀졌다.In addition, products such as joins tablets, adroda capsules, imoton tablets, seimi tablets, atrene tablets, glucosamine, and chondroitin sulfate are currently used in natural medicines, but the products are insignificant or have undergone rigorous clinical research to prove their effectiveness. rare. Indeed, taking glucosamine for the treatment of arthritis in Korea has become a social epidemic, but a study conducted by the US FDA to investigate the therapeutic effect of a large number of patients showed no effect at all.
본 발명은 상기한 종래의 문제점을 감안하여, 천연 식물추출물을 이용하여 염증성 질환, 통증성 질환 및 관절염 증상 개선제를 발명하여 기존의 다른 합성의약품과 비교하여 부작용의 염려가 없으며, 단순증상 완화 이상의 관절염의 근본적인 증상 개선 및 예방에 탁월한 효과를 갖는 항염증용 식품 조성물을 제공하는 것을 목적으로 한다. In view of the above-mentioned problems, the present invention invents an inflammatory disease, painful disease and arthritis symptom improving agent using natural plant extracts, so that there is no concern of side effects compared to other synthetic drugs, arthritis more than simple symptom relief. It is an object of the present invention to provide an anti-inflammatory food composition having an excellent effect on the improvement and prevention of fundamental symptoms.
또한, 현재까지 개발된 소염, 진통제의 대부분이 약효의 평가를 COX-2, Lipoxygenase, iNOS 같은 염증유발효소의 활성을 저해하는데 주력하였다면, 본 발명은 이러한 효소들의 활성을 저해함은 물론이고 생체 내에서의 생합성(발현)도 억제하는 천연 추출물을 발명함으로써 염증과 통증의 근원적인 증상 개선을 도모하는 항염증용 식품 조성물을 제공하는 것을 목적으로 한다.In addition, if most of the anti-inflammatory and analgesics developed so far focused on inhibiting the activity of proinflammatory enzymes such as COX-2, Lipoxygenase, iNOS, the present invention not only inhibits the activity of these enzymes, but also in vivo It is an object of the present invention to provide an anti-inflammatory food composition which aims at improving the root symptom of inflammation and pain by inventing a natural extract that also inhibits biosynthesis (expression).
또한, 본 발명은 Type I collagenase의 활성을 저해하고 항산화 작용을 함으로써 관절 연골세포의 파괴를 질환의 발생 후는 물론 발생 전에도 방지하는 물질을 개발함으로써 질환의 치료 및 질환발생의 고위험군에 있는 고령자들의 퇴행성 관절염의 발병을 예방하는 항염증용 식품 조성물을 제공하는데 또 다른 목적이 있다.In addition, the present invention by developing a substance that inhibits the activity of Type I collagenase and antioxidant activity to prevent the destruction of articular chondrocytes before and after the disease, degenerative of elderly people in the high-risk group of disease treatment and disease development Another object is to provide an anti-inflammatory food composition for preventing the development of arthritis.
상기 목적을 달성하기 위한 본 발명의 항염증성의 경구 투여용 기능성 식품 조성물은, 양파 추출물과 상심자 추출물 및 서리태 추출물의 혼합된 식물 조성물을 주성분으로 함유하여, 염증성 질환과 통증성 질환 및 관절염의 예방과 증상을 개선하는 것을 특징으로 한다.Functional food composition for anti-inflammatory oral administration of the present invention for achieving the above object contains a mixed plant composition of onion extract, lettuce extract and frost extract as a main component, prevention of inflammatory diseases and pain diseases and arthritis It is characterized by improving the symptoms.
상기 혼합된 식물 조성물은 상심자 추출물, 서리태 추출물 및 양파 추출물이 1 : (0.1 ~ 5) : (0.1 ~ 50)의 중량비로 각각 혼합되게 하는 것이 바람직하다.The mixed plant composition is preferably to be mixed in the weight ratio of 1: 1 (0.1 ~ 5): (0.1 ~ 50) of the lettuce extract, frost extract and onion extract.
상기 목적을 달성하기 위한 본 발명의 항염증성의 경구 투여용 기능성 식품 조성물은, 상심자 추출물과 서리태 추출물이 혼합된 식물 조성물을 주성분으로 함유하여, 염증성 질환과 통증성 질환 및 관절염의 예방과 증상을 개선하는 것을 특징으로 한다.Functional food composition for anti-inflammatory oral administration of the present invention for achieving the above object, containing the plant composition mixed with the extract of the loser and frost extract as a main component, to prevent and symptomatic symptoms of inflammatory diseases and pain diseases and arthritis It is characterized by improving.
상기 혼합된 식물 조성물은 상심자 추출물과 서리태 추출물이 1 : (0.1 ~ 5)의 중량비로 혼합되게 하는 것이 바람직하다.The mixed plant composition is preferably to be mixed in the weight ratio of 1: 1 (0.1 ~ 5) of the lettuce extract and frost extract.
상기 목적을 달성하기 위한 본 발명의 경구 투여용 기능성 식품 조성물은, 상심자 추출물과 양파 추출물이 혼합된 식물 조성물을 주성분으로 함유하여, 염증, 통증, 관절염의 예방 및 증상을 개선하는 것을 특징으로 한다.Functional food composition for oral administration of the present invention for achieving the above object, by containing the plant composition mixed with the lettuce extract and onion extract as a main component, it is characterized in that the prevention and symptoms of inflammation, pain, arthritis .
상기 혼합된 식물 조성물은 상심자 추출물 및 양파 추출물이 1 : (0.1 ~ 50)의 중량비로 혼합되도록 하는 것이 바람직하다.The mixed plant composition is preferably to be mixed in the weight ratio of the lettuce extract and onion extract 1: 1: (0.1 ~ 50).
또한, 상기 목적을 달성하기 위한 본 발명의 항염증성의 경구 투여용 기능성 식품 조성물은, 서리태 추출물과 양파 추출물이 혼합된 식물 조성물을 주성분으로 함유하여, 염증, 통증, 관절염의 예방 및 증상을 개선하는 것을 특징으로 한다.In addition, the functional food composition for anti-inflammatory oral administration of the present invention for achieving the above object contains a plant composition mixed with frost extract and onion extract as a main component, to prevent inflammation, pain, arthritis and improve symptoms It is characterized by.
상기 혼합된 식물 조성물은 서리태 추출물 및 양파 추출물이 1 : (0.1 ~ 50)의 중량비로 혼합되도록 하는 것이 바람직하다.The mixed plant composition is preferably such that the frost extract and onion extract is mixed in a weight ratio of 1: (0.1 ~ 50).
상기 항염증성의 기능성 식품 조성물이 투여되는 경구 투여용 기능성 식품에는 캡슐, 환, 과립, 분말, 캔디, 껌, 정제, 음료, 시럽 중 어느 하나가 되도록 하는 것이 바람직하다.Functional food for oral administration to which the anti-inflammatory functional food composition is administered is preferably one of capsules, pills, granules, powders, candy, gum, tablets, beverages, and syrups.
본 발명의 항염증성의 기능성 식품 조성물에 함유된 추출물 중 상심자 추출물을 제조하기 위한 제조방법은, 상심자의 분쇄 후 4 ~ 10배 중량비의 0 ~ 99% 에탄올 수용액으로 30 ~ 80℃에서 2 ~ 8시간, 1 ~ 4회 추출하고, 냉각 및 여과하는 제1 단계와; 상기 제1 단계에서 얻어진 여액을 60 ~ 100℃로 감압 농축하는 제2 단계; 및 상기 제2 단계에서 얻어진 엑기스를 동결건조하고 분말엑기스를 제조하는 제3 단계를 포함하며, 상기 과정을 통해 추출된 지표물질인 루틴(rutin)이 0.1 ~ 20%이 되도록 함을 특징으로 한다.The preparation method for preparing a lettuce extract among the extracts contained in the anti-inflammatory functional food composition of the present invention is 2 to 8 at 30 to 80 ° C. with an aqueous solution of 0 to 99% ethanol in a weight ratio of 4 to 10 times after grinding the mulberry. A first step of extraction, cooling and filtration for 1 to 4 times; A second step of concentrating the filtrate obtained in the first step under reduced pressure at 60 to 100 占 폚; And a third step of lyophilizing the extract obtained in the second step and preparing a powder extract, wherein the index material extracted through the process is set to 0.1 to 20%.
본 발명의 항염증성의 경구 투여용 기능성 식품 조성물에 함유된 추출물 중 서리태 추출물을 얻기 위한 제조방법은, 서리태를 정제수로 2회 세척한 후 4 ~ 10배 중량비의 0 ~ 80% 알코올성 수용액으로 30 ~ 100℃에서 2 ~ 8시간, 1 ~ 4회 추출하고, 냉각 및 여과하는 제1 단계와; 상기 제1 단계에서 얻어진 여액을 60℃로 감압 농축하는 제2 단계; 및 상기 제2 단계에서 얻어진 엑기스를 동결건조하고, 분말엑기스를 제조하는 제3 단계를 포함하며, 상기 과정으로부터 추출된 지표물질인 총 사포닌(saponin)이 0.2 ~ 10%이 되도록 함을 특징으로 한다.The preparation method for obtaining the frost extract in the extract contained in the functional food composition for anti-inflammatory oral administration of the present invention, after washing twice with purified
본 발명의 기능성 식품 조성물에 함유된 추출물 중 양파 추출물을 얻기 위한 제조방법은, 양파를 껍질을 분리하지 않고 정제수로 2회 세척한 후 4 ~ 10배 중량비의 10 ~ 100% 알코올성 수용액으로 60 ~ 100℃에서 2 ~ 8시간, 1 ~ 4회 추출하고, 냉각 및 여과하는 제1 단계와; 상기 제1 단계에서 얻어진 여액을 60℃로 감압 농축하는 제2 단계; 및 상기 제2 단계에서 얻어진 엑기스를 동결건조하고 분말엑기스를 제조하는 제3 단계를 포함하며, 상기 과정으로부터 추출된 지표물질인 퀘르세틴(Quercetin)이 0.1 ~ 40%이 되도록 함을 특징으로 한다.Preparation method for obtaining onion extract of the extract contained in the functional food composition of the present invention, after washing the onion twice with purified water without separating the skin peel 4 to 10 times the weight ratio of 10 to 100% alcoholic
본 발명의 항염증성의 경구 투여용 기능성 식품 조성물에 의하면, 서로 상승 효과를 나타내는 천연 식물 추출물의 조성물을 이용하여 항산화능(DPPH 라디칼 소거능, SOD 유사활성, H2O2 소거능), 염증유발효소(COX-2, Lipoxygenase, iNOS, Type I collagenase)의 활성 저해 및 생체내 발현 억제, 염증유발물질(일산화질소, 프로스타그란딘, TNF-α, IL-1β, IL-6)의 생체내 생성억제, 염증성 부종억제 등에 효과가 우수한 기능성 식물 조성물을 제조함으로써 염증성 질환, 통증성 질환 및 관절염의 예방 및 증상 개선제로 유용한 효과를 나타낸다.According to the functional food composition for anti-inflammatory oral administration of the present invention, by using a composition of natural plant extracts showing synergistic effects, antioxidant activity (DPPH radical scavenging activity, SOD-like activity, H 2
도 1은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 DPPH 라디칼을 50% 소거하는 SC50값을 ppm의 농도로 나타낸 그래프도.
도 2는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 SOD 유사활성을 나타내는 SC50값을 ppm의 농도로 나타낸 그래프도.
도 3은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 H2O2를 50% 소거하는 SC50값을 ppm의 농도로 나타낸 그래프도.
도 4는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 마우스 대식세포에서 LPS에 유도되는 일산화질소의 생성을 저해하는 것을 나타낸 그래프도.
도 5는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 COX-1 효소를 50% 저해하는 IC50값을 ppm의 농도로 나타낸 그래프도.
도 6은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 COX-2 효소를 50% 저해하는 IC50값을 ppm의 농도로 나타낸 그래프도.
도 7은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA(mono sodium iodoacetate)에 의한 만성염증 동물모델의 혈액에서 리포옥시제나제 효소의 활성을 시료의 무처리군과 비교하여 저해하는 비율을 나타낸 그래프도.
도 8은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 부종이 일어난 족부위에서 TNF-α의 생성량을 나타낸 그래프도.
도 9는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 염증이 일어난 슬관절부위에서 TNF-α의 생성량을 나타낸 그래프도.
도 10은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 부종이 일어난 족부위에서 IL-1β의 생성량을 나타낸 그래프도.
도 11은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 염증이 일어난 슬관절부위에서 IL-1β의 생성량을 나타낸 그래프도.
도 12는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 부종이 일어난 족부위에서 IL-6의 생성량을 나타낸 그래프도.
도 13은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 염증이 일어난 슬관절부위에서 IL-6의 생성량을 나타낸 그래프도.
도 14는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 마우스 대식세포에서 LPS에 유도되는 프로스타그란딘의 생성을 저해하는 것을 나타낸 그래프도.
도 15는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 염증이 일어난 슬관절부위에서 프로스타그란딘의 생성을 저해하는 것을 나타낸 그래프도.
도 16은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 Type I collagenase를 50% 저해하는 IC50값을 ppm의 농도로 나타낸 그래프도.
도 17은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 부종이 일어난 족부위에서 iNOS, COX-1, COX-2 효소의 발현을 억제하는 것을 나타낸 것이다.(Ibu: Ibuprofen, 실1-100: 실시예1 100mg/체중 Kg 처리, 실1-400: 실시예1 400mg/체중 Kg, Joi: 조인스정 400mg/체중1Kg 처리)
도 18은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 LPS에 의한 마우스 대식세포의 염증반응에서 발현이 유도되는 iNOS, COX-1, COX-2 효소의 발현을 억제하는 것을 나타낸 도면.(Ibu: Ibuprofen, 실1-200: 실시예1 200ppm 처리, 실1-1K: 실시예1 1000ppm 처리, Joi: 조인스정 1000ppm 처리)
도 19는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 발부종 억제효과를 시간대별로 나타낸 도면이다.1 is a graph showing the SC50 value in ppm concentration of 50% scavenging DPPH radicals of the mixed extracts prepared by Reference Examples 1 to 3 and Example 1 of the present invention.
Figure 2 is a graph showing the SC50 value showing the SOD-like activity of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 in a concentration of ppm.
Figure 3 is a graph showing the SC50 value in ppm concentration of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the
Figure 4 is a graph showing that the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits the production of nitrogen monoxide induced by LPS in mouse macrophages.
Figure 5 is a graph showing the IC50 value in ppm concentration of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits COX-1 enzyme by 50%.
Figure 6 is a graph showing the IC50 value in ppm concentration of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits COX-2 enzyme by 50%.
Figure 7 is a mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention, the group treated with lipooxygenase enzyme activity in the blood of chronic animal model of chronic inflammation by MIA (mono sodium iodoacetate) A graph showing the rate of inhibition in comparison with.
8 is a graph showing the amount of production of TNF-α in the foot portion where the edema of the acute inflammatory animal model caused by carrageenan occurred in the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention.
9 is a graph showing the amount of production of TNF-α in the knee joint site of the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention chronic inflammation induced animal model by MIA.
10 is a graph showing the amount of production of IL-1β in the foot portion of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention caused carrageenan swelling.
Figure 11 is a graph showing the amount of IL-1β production in the knee joint site where the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention inflamed chronic animal model by MIA.
12 is a graph showing the amount of IL-6 produced in the foot portion of the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention caused by carrageenan swelling animal model.
Figure 13 is a graph showing the amount of IL-6 produced in the knee joint site where the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inflamed chronic animal model by MIA.
14 is a graph showing that the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits the production of prostaglandin induced by LPS in mouse macrophages.
15 is a graph showing that the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits the production of prostaglandin in the knee joint site where inflammation of the chronic inflammation animal model by MIA occurred.
Figure 16 is a graph showing the IC50 value in ppm concentration of the mixed extract prepared by Reference Examples 1-3 and Example 1 of the present invention inhibits Type I collagenase by 50%.
17 is a mixture extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention to inhibit the expression of iNOS, COX-1, COX-2 enzymes in the foot area where edema of acute inflammatory animal model by carrageenan occurred (Ibu: Ibuprofen, Thread 1-100: Example 1 100 mg / weight Kg treatment, Thread 1-400: Example 1 400 mg / weight Kg, Joi: Joining
18 is a mixture extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits the expression of iNOS, COX-1, COX-2 enzymes induced expression in the inflammatory response of mouse macrophages by LPS (Ibu: Ibuprofen, Thread 1-200: Example 1 200ppm treatment, Thread 1-1K: Example 1 1000ppm treatment, Joi: Joining tablet 1000ppm treatment)
19 is a view showing the effect of inhibiting the paw edema of the animal model of acute inflammation caused by carrageenan of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention.
이하 첨부된 도면을 참조하여 본 발명을 더욱 상세하게 설명하기로 한다.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings.
본 발명은 양파 추출물과 상심자 추출물 및 서리태 추출물을 적정 비율로 혼합하여, 소정량의 범위로 최적화시킴으로써 각각의 추출물보다 우수한 진통효과, 염증유발효소인 Type I collagenase, lipoxygenase, COX-1 및 COX-2의 저해효과, 염증성 부종 완화효과, 염증유발인자인 일산화질소(NO) 및 프로스타그란딘(prostaglandins)의 생성억제효과, 염증유발효소인 iNOS, COX-1 및 COX-2의 생체내 발현억제효과, 염증유발 사이토카인인 TNF-α, IL-1β 및 IL-6의 생성억제, 관절염의 잠재적인 유발요인이며 이 질환의 심화 요인으로 지목되는 유해활성산소 라디칼 소거효과 및 항산화 효과를 가짐으로써 보다 뛰어난 염증성 질환, 통증성 질환 및 관절염의 예방 및 증상 개선에 유용한 항염증성의 경구 투여용 기능성 식품 조성물에 관한 것이다.The present invention is mixed with onion extract, lettuce extract and frost extract in an appropriate ratio, by optimizing in a predetermined amount of analgesic effect than each extract, Type I collagenase, lipoxygenase, COX-1 and COX- 2 inhibitory effect, inflammatory edema alleviation effect, inhibitory effect of production of inflammatory factors nitrogen monoxide (NO) and prostaglandins (inhibition effect of in vivo expression of inflammatory enzymes iNOS, COX-1 and COX-2, inflammation Inflammatory disease that is more prominent by inhibiting the production of the induced cytokines TNF-α, IL-1β and IL-6, as a potential inducer of arthritis, and having a radical free radical scavenging effect and an antioxidant effect that are considered as a serious factor of the disease The present invention relates to a functional food composition for anti-inflammatory oral administration useful for the prevention and improvement of symptoms of painful diseases and arthritis.
이와 같은 복합 식품 조성물은 양파(적양파 혹은 흑양파 포함)와 상심자 및 서리태를 각각 물 또는 알코올성 수용액으로 추출하여 조성물을 제조함에 있어서 다음과 같은 방법을 사용하였다. Such a composite food composition was used to prepare the composition by extracting onion (including red onions or black onions), lettuce and frosted with water or alcoholic aqueous solution, respectively.
1) 먼저, 양파를 추출하여 조성물을 제조하는 방법은 다음과 같다.1) First, the method of preparing the composition by extracting the onion is as follows.
양파를 껍질을 분리하지 않고 정제수로 2회 세척 후 식품용 분쇄기를 사용하여 500 ~ 700rpm으로 10 ~ 20분간 분쇄 후 4 ~ 10배량의 10 ~ 100% 에탄올 수용액으로 60 ~ 100℃에서 2 ~ 8시간, 1 ~ 4회 추출 및 냉각하고, 0.125mm(80mesh) 규격의 스테인레스 스틸 거름망으로 여과하는 제1 단계를 실시한다. 상기 제1 단계에서 얻어진 여액을 60℃로 20brix까지 감압 농축하는 제2 단계를 실시한다. 상기 제2 단계에서 얻어진 엑기스를 동결건조하고 분말엑기스를 제조하는 제3 단계를 실시한다.After washing the onion twice with purified water without separating the skin, grind it for 10 to 20 minutes at 500 to 700 rpm using a food grinder, and then use it for 4 to 10 times at 10 to 100% ethanol aqueous solution at 60 to 100 ° C for 2 to 8 hours. Extraction, cooling 1 to 4 times, and a first step of filtration with a stainless steel strainer of 0.125mm (80mesh) specifications. The filtrate obtained in the first step is subjected to a second step of concentrating the filtrate under reduced pressure at 60 DEG C to 20brix. A third step of lyophilizing the extract obtained in the second step and producing a powdery extract is carried out.
상기의 방법으로 얻어진 양파 분말엑기스에는 특히, 퀘르세틴(quercetin)이 대개 0.1 ~ 40 중량%로 함유되어 있다.In particular, the onion powder extract obtained by the above method contains quercetin in an amount of usually 0.1 to 40% by weight.
2) 다음, 상심자를 추출하여 조성물을 제조하는 방법은 다음과 같다.2) Next, the method of preparing a composition by extracting the loser is as follows.
상심자를 식품용 분쇄기를 사용하여 500 ~ 1000rpm으로 분쇄한 후, 4 ~ 10배량의 0 ~ 99% 에탄올 수용액으로 30 ~ 80℃에서 2 ~ 8시간, 1 ~ 4회 추출한 다음 냉각하고 0.125mm(80 mesh) 규격의 스테인레스 스틸 거름망을 사용하여 여과하는 제1 단계를 실시한다.After grinding the heartache at 500 ~ 1000rpm using a food grinder, extract 4 ~ 10 times with 0 ~ 99% ethanol aqueous solution at 30 ~ 80 ℃ for 2 ~ 8 hours, 1 ~ 4 times, and then cool it. A first step of filtration is carried out using a stainless steel strainer of mesh size.
상기 제1 단계에서 얻어진 여액을 20 ~ 100℃로 20brix까지 감압 농축하는 제2 단계를 실시한다. 상기 제2 단계에서 얻어진 엑기스를 동결건조하고 분말엑기스를 제조하는 제3 단계를 실시한다.The filtrate obtained in the first step is subjected to a second step of concentrating the filtrate under reduced pressure at 20 to 100 占 폚 to 20 byrix. A third step of lyophilizing the extract obtained in the second step and producing a powdery extract is carried out.
상기의 방법으로 얻어진 상심자 분말엑기스에는 특히, 루틴(rutin)이 0.01 ~ 20 중량%로 함유되어 있다.The core powder extract obtained by the above method contains, in particular, rutin in an amount of 0.01 to 20% by weight.
3) 다음, 서리태를 추출하여 조성물을 제조하는 방법은 다음과 같다.3) Next, the method of preparing the composition by extracting the frost is as follows.
서리태를 정제수로 2회 세척한 후 4 ~ 10배량의 물 또는 5 ~ 80% 에탄올 수용액으로 30 ~ 100℃에서 2 ~ 8시간, 2 ~ 4회 추출하고, 냉각 후 0.074mm(200 mesh) 규격의 스테인레스 스틸 거름망으로 여과하는 제1 단계를 시행한다.After washing twice with purified water, extracted 2-4 times at 30-100 ℃ for 2 ~ 4 hours with 4 ~ 10 times of water or 5 ~ 80% aqueous ethanol solution, and cooled it to 0.074mm (200 mesh) standard. Perform the first step of filtration with a stainless steel strainer.
상기 제1 단계에서 얻어진 여액을 60℃로 15brix까지 감압 농축하는 제2 단계를 실시한다. 상기 제2 단계에서 얻어진 엑기스를 동결건조하고 분말엑기스를 제조하는 제3 단계를 시행한다.The second step of concentrating the filtrate obtained in the first step under reduced pressure up to 15brix at 60 ° C. The extract obtained in the second step is lyophilized and a third step of preparing a powder extract is performed.
상기의 방법으로 얻어진 서리태 분말엑기스에는 특히, 사포닌(saponin)이 0.2 ~ 10 중량%로 함유되어 있다.The frosted powder extract obtained by the above method contains, in particular, 0.2 to 10% by weight of saponin.
이상 상기한 1) ∼3)의 과정을 통해 양파 추출물과 상심자 추출물 및 서리태 추출물이 1 : 0.2 ~ 1의 중량비로 혼합된 것은 루틴이 0.01 ~ 10 중량%로 함유되어 있다.In the above 1) to 3) process, the onion extract, the lettuce extract and the frost extract are mixed at a weight ratio of 1: 0.2 to 1, which contains 0.01 to 10% by weight of rutin.
참고로, 본 발명에서 사용된 양파의 원산지는 경상북도 창녕이며 만생종을 사용하였고, 상심자의 원산지는 전라북도 고창이고 초여름에 수확한 것을 사용하였으며, 서리태의 원산지는 경상북도 안동 및 강원도 원주지역이며 11월 중순에 수확한 것을 사용하였다. 그렇다고 하여 각 식물의 원산지나 품종 때문에 본 발명의 범위가 한정되는 것은 아니다.For reference, the origin of the onion used in the present invention is Changnyeong, Gyeongsangbuk-do, and the native species was used, and the origin of the heartache was harvested in early summer, Gochang, Jeollabuk-do. Harvest was used. However, the scope of the present invention is not limited because of the origin or variety of each plant.
상기 식물추출물은 그 자체로도 사용할 수 있지만 분말, 타정, 과립 또는 겔 등으로 제조하기 위하여 식품학적으로 허용되는 흡습제(moisture absorbent), 부형제(forming agent), 희석제(dilute), 담체(carrier) 등과 함께 혼합하여 사용 가능하다.The plant extract may be used as such, but food-acceptable moisture absorbents, excipients, forming agents, diluents, carriers, and the like, for preparing powders, tablets, granules, or gels may be used. Can be mixed together.
또한, 본 발명은 상기 방법으로 수득한 양파와 상심자 및 서리태 추출물로 이루어진 군으로부터 선택된 조합을 주성분으로 하고, 비타민군과 퀘르세틴(quercetin) 등으로 이루어진 군으로부터 선택된 조합을 부가성분으로 포함하는 염증성 질환, 통증성 질환 및 관절염용 기능성 조성물 외에 기타 식품 성분을 함유하는 식품을 제공한다.In addition, the present invention is an inflammatory disease comprising as a main ingredient a combination selected from the group consisting of onions, lettuce and frost extract, obtained from the above method, and a combination selected from the group consisting of a vitamin group and quercetin, etc. Provided are foods containing other food ingredients in addition to functional compositions for painful diseases and arthritis.
상기 식품은 음료류, 특수영양식품, 건강보조식품, 기능성 식품류 외 기타 식품류를 포함한다.The food includes beverages, special nutritional supplements, health supplements, functional foods and other foods.
한편, 상기 식품의 형태는 분말, 과립, 정제, 캡슐, 액상 또는 음료 형태를 포함한다.Meanwhile, the form of the food includes powder, granule, tablet, capsule, liquid or beverage form.
또한, 본 발명은 상기의 식품첨가제를 식품에 살균제, 향신료, 조미제, 여러가지 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, 알콜, 탄산 음료에 사용되는 탄산화제 등 또는 식품소재의 필수원료로 사용하는 것을 특징으로 하는 식품첨가제의 이용방법을 제공한다. 이때, 식품첨가제는 식품을 침지, 분무 또는 혼합하여 상기 식품에 첨가할 수 있으며, 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 기능성 조성물 100 중량% 당 0 내지 약 20 중량%의 범위에서 선택되는 것이 일반적이다.In addition, the present invention, the food additives in the food, such as fungicides, spices, seasonings, various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as colorants and neutralizing agents (cheese, chocolate, etc.) ), Pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, or the like as essential raw materials for food materials It provides a method of using a food additive characterized in that. At this time, the food additive may be added to the food by dipping, spraying or mixing the food, the ratio of these additives is not so important but selected from the range of 0 to about 20% by weight per 100% by weight of the functional composition of the present invention Is common.
또한, 염증성 질환, 통증성 질환 및 관절염 예방과 증상 개선을 목적으로 식품 또는 음료에 첨가될 수 있다. 이때, 식품 또는 음료 중의 상기 조성물의 양은, 일반적으로 본 발명의 건강기능식품 조성물의 경우는 전체 식품 중량의 10 내지 80 중량%, 바람직하게는 20 내지 70 중량%로 가할 수 있으며, 건강 음료 조성물에는 100ml를 기준으로 0.1 ∼ 30g, 바람직하게는 1 ∼ 30g의 비율로 가할 수 있다.It may also be added to foods or beverages for the purpose of preventing and improving symptoms of inflammatory diseases, painful diseases and arthritis. In this case, the amount of the composition in the food or beverage is generally 10 to 80% by weight, preferably 20 to 70% by weight of the total food weight in the case of the health functional food composition of the present invention, It can be added in the ratio of 0.1-30 g, Preferably it is 1-30 g based on 100 ml.
이하, 본 발명을 실시예에 의거하여 상세히 설명하면 다음과 같으나, 본 발명의 범위가 아래의 실시예에 의해 한정된 것은 아니다.Hereinafter, the present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited by the following examples.
참조예 1 : 양파 추출물의 제조Reference Example 1 Preparation of Onion Extract
양파를 식품용 분쇄기를 사용하여 500 ~ 700rpm으로 10 ~ 20분간 분쇄 후 4 ~ 10배량의 10 ~ 100% 에탄올 수용액으로 60 ~ 100℃에서 2 ~ 8시간, 1 ~ 4회 추출 및 냉각하고, 0.125mm(80mesh) 규격의 스테인레스 스틸 거름망으로 여과한 후 여기에서 얻어진 여액을 60 ~ 100℃로 20brix까지 감압 농축한다. 상기 농축단계에서 얻어진 엑기스를 동결건조하고 분말 엑기스를 제조한다.Grind the onion for 10-20 minutes at 500 ~ 700rpm using a food grinder, extract and cool 2 to 8 hours, 1 to 4 times at 60 to 100 ℃ in 10 to 100% aqueous ethanol solution of 4 to 10 times, 0.125 After filtering with a stainless steel strainer of mm (80mesh) standard, the filtrate obtained here is concentrated under reduced pressure to 20brix at 60 ~ 100 ℃. The extract obtained in the concentration step is lyophilized to prepare a powder extract.
참조예 2 : 상심자 추출물의 제조REFERENCE EXAMPLE 2 Preparation of a Lettuce Extract
정제수로 세척한 상심자는 식품용 분쇄기를 사용하여 500 ~ 1000rpm으로 20 ~ 60분간 분쇄 후 4 ~ 10배량의 0 ~ 35% 에탄올 수용액으로 50 ~ 60℃에서 2 ~ 8시간, 1 ~ 4회 추출 및 냉각하고 0.125mm(80mesh) 규격의 스테인레스 스틸 거름망으로 여과한 후 여기에서 얻어진 여액을 60 ~ 100℃로 20brix까지 감압 농축한다. 상기 농축단계에서 얻어진 엑기스를 동결건조하고 분말 엑기스를 제조한다.The fresh heart washed with purified water was crushed for 20 to 60 minutes at 500 to 1000 rpm using a food grinder, and then extracted 4 to 10 times at 0 to 35% aqueous ethanol solution for 2 to 8 hours and 1 to 4 times at 50 to 60 ° C. After cooling and filtering with a stainless steel strainer of 0.125mm (80mesh) standard, the filtrate obtained here is concentrated under reduced pressure to 20brix at 60 ~ 100 ℃. The extract obtained in the concentration step is lyophilized to prepare a powder extract.
참조예 3 : 서리태 추출물의 제조Reference Example 3: Preparation of Seoritae Extract
정제수로 세척한 서리태는 4 ~ 10배량의 물 또는 20 ~ 50% 에탄올 수용액으로 60 ~ 100℃에서 2 ~ 8시간, 2 ~ 4회 추출 및 냉각하고 0.074mm(200mesh) 규격의 스테인레스 스틸 거름망으로 여과하고 여기에서 얻어진 여액을 60℃로 15brix까지 감압 농축한다. 상기 농축단계에서 얻어진 엑기스를 동결건조하고 분말엑기스를 제조한다.The frosted washed with purified water was extracted and cooled 2-4 times at 60-100 ℃ for 2 ~ 4 hours with 4 ~ 10 times of water or 20 ~ 50% ethanol aqueous solution, and filtered by 0.074mm (200mesh) stainless steel strainer. And the filtrate obtained here is concentrated under reduced pressure at 60 ℃ to 15brix. The extract obtained in the concentration step is lyophilized to prepare a powder extract.
실시예 1 : 혼합 추출물의 제조Example 1: Preparation of Mixed Extract
상기 참조예 1과 참조예 2 및 참조예 3에서 수득한 양파 추출물과 상심자 추출물 및 서리태 추출물이 5 : 5 : 1.2 중량비로 분말 혼합기를 이용하여 혼합하였다. The onion extract obtained from Reference Example 1, Reference Example 2, and Reference Example 3, and the extract from the lettuce and the frost extract were mixed using a powder mixer in a 5: 5: 1.2 weight ratio.
실험예 1 : DPPH 라디칼 소거능 실험Experimental Example 1: DPPH radical scavenging experiment
매우 안정한 free radical인 DPPH(2,2-Diphenyl-1-picrylhydrazyl, Sigma, MO, USA)가 hydrogen proton-radical scavenger에 의해 보라색에서 노란색의 diphenylpicrylhydrazine으로 변색되는 현상을 이용하여 각 샘플의 전자 공여능을 측정하였다. 그 결과는 첨부한 도 1 및 <표 1>에 나타낸 바와 같다.DPPH (2,2-Diphenyl-1-picrylhydrazyl, Sigma, MO, USA), a very stable free radical, was used to determine the electron donating ability of each sample by using a hydrogen proton-radical scavenger that discolors purple to yellow diphenylpicrylhydrazine. It was. The results are as shown in FIG. 1 and Table 1.
도 1은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 DPPH 라디칼을 50% 소거하는 SC50값을 ppm의 농도로 나타낸 그래프도이다.1 is a graph showing the SC50 value in ppm concentration of 50% scavenging DPPH radicals of the mixed extracts prepared according to Reference Examples 1 to 3 and Example 1 of the present invention.
[실험방법]Experimental Method
각 샘플을 농도별로 용해한 후 각각 75ml를 DPPH(0.2 mM in ethanol) 750 ml와 3차 정제수 675ml으로 혼합한다. 상온에서 30분간 반응하고 96 웰플레이트(well plate)에 반응액 200ml를 옮긴 후 ELISA reader 기기(Molecular Devices, CA, USA)로 520nm의 파장에서 흡광도를 측정한다. DPPH 라디칼 소거활성(%)은 (1-시료 첨가구의 흡광도/시료 무첨가구의 흡광도)X100의 계산식을 사용하였다. SC50 값은 발생한 라디칼 50%를 소거하는데 필요한 최소농도를 ppm 단위로 표시한 것이다.After dissolving each sample by concentration, 75 ml of each was mixed with 750 ml of DPPH (0.2 mM in ethanol) and 675 ml of tertiary purified water. After reacting for 30 minutes at room temperature, 200 ml of the reaction solution was transferred to a 96 well plate, and the absorbance was measured at a wavelength of 520 nm with an ELISA reader device (Molecular Devices, CA, USA). The DPPH radical scavenging activity (%) was calculated using the formula ((absorbance of 1-sample added / absorbed sample-free)) X100. The SC50 value represents the minimum concentration, in ppm, required to eliminate 50% of the generated radicals.
<표 1>TABLE 1
상기 <표 1> 및 도 1에서 보는 바와 같이, 본 발명에 의하여 제조된 실시예 1에 대한 DPPH 라디칼의 소거능은 각각의 추출물을 사용한 경우 또는 SK제약의 조인스정을 사용한 경우보다 뛰어난 것을 알 수 있다. 양성 대조군으로는 vitamin C를 사용하였다.As shown in Table 1 and Figure 1, it can be seen that the scavenging ability of the DPPH radical for Example 1 prepared by the present invention is superior to the case of using each extract or the joining tablet of SK Pharmaceuticals. . Vitamin C was used as a positive control.
실험예 2 : SOD 유사활성 실험Experimental Example 2: SOD-like activity test
각각의 식물 추출 조성물이 가지고 있는 유해활성산소 라디칼(superoxide radical)의 소거활성을 측정하기 위해서 잔틴 옥시다제(xanthine oxidase)에 의해 잔틴(xanthine)에서 유리된 산소라디칼의 소거활성을 비교하였고 그 결과는 첨부한 도 2 및 <표 2>에 나타낸 바와 같다.The scavenging activity of oxygen radicals liberated from xanthine by xanthine oxidase was compared to determine the scavenging activity of the superoxide radical of each plant extract composition. As shown in FIG. 2 and <Table 2> which were attached.
도 2는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 SOD 유사활성을 나타내는 SC50값을 ppm의 농도로 나타낸 그래프도이다. Figure 2 is a graph showing the SC50 value in ppm concentrations showing the SOD-like activity of the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention.
[실험방법]Experimental Method
37℃로 완충용액(0.1M phosphate buffer, pH 8.0)에 용해한 잔틴 옥시다제 용액(0.045U/ml)에 잔틴용액(0.4mM)과 NBT용액(0.24mM, nitro blue tetrazolium)용액으로 이루어진 발색시약을 첨가하고 각각의 농도별로 용해한 샘플용액을 첨가한 후 20분간 반응시키고 1ml의 SDS(70mM)용액의 첨가로 반응을 정지시킨 후 560nm에서 시토크롬-c의 산화에 따른 변색의 정도를 측정하였다.A coloring reagent consisting of xanthine solution (0.4mM) and NBT solution (0.24mM, nitro blue tetrazolium) solution in xanthine oxidase solution (0.045U / ml) dissolved in buffer solution (0.1M phosphate buffer, pH 8.0) at 37 ℃ After adding the sample solution dissolved in each concentration, the reaction was stopped for 20 minutes, and the reaction was stopped by the addition of 1 ml of SDS (70 mM) solution, and then the degree of discoloration due to oxidation of cytochrome-c was measured at 560 nm.
<표 2><Table 2>
상기 <표 2> 및 도 2에서 보는 바와 같이, 본 발명에 의하여 제조된 실시예 1에 대한 SOD 유사활성은 각각의 추출물을 사용한 경우 또는 SK제약의 조인스정을 사용한 경우보다 뛰어난 것을 알 수 있다. 양성 대조군으로는 vitamin C를 사용하였다.As shown in Table 2 and Figure 2, it can be seen that the SOD-like activity for Example 1 prepared by the present invention is superior to the case of using the respective extracts or using the joining tablet of SK Pharmaceutical. Vitamin C was used as a positive control.
실험예 3 : H2O2 소거능 실험Experimental Example 3 H2O2 Scavenging Capacity Experiment
일반적으로 생체내에서 발생한 유해활성산소 라디칼인 초과산화수소이온이 SOD(Super Oxide Dismutase)에 의해서 분해된 후 발생되는 산물인 과산화수소를 더욱 안전한 물과 산소로 바꾸는 항산화 활성을 측정하기 위하여 Fenton반응에 의해 생성된 hydroxyl radical이 2-deoxyribose를 산화하여 발생하는 MDA(malondialdehyde)를 520nm에서 흡광의 정도를 측정하는 방법을 이용하였고 그 결과는 도 3 및 <표 3>에 나타낸 바와 같다.In general, the Fenton reaction is used to measure the antioxidant activity of converting hydrogen peroxide, a product generated after the decomposition of superoxide dismutase (SOD) into safer water and oxygen. The hydroxyl radical was measured by the degree of absorption of MDA (malondialdehyde) generated by oxidizing 2-deoxyribose at 520nm and the results are shown in Figure 3 and Table 3.
도 3은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 H2O2를 50% 소거하는 SC50값을 ppm의 농도로 나타낸 그래프도이다.Figure 3 is a graph showing the SC50 value in ppm concentration of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention to remove H2O2 by 50%.
[실험방법]Experimental Method
FeSO4·7H2O(50mM)-EDTA(50mM)용액 10ml에 2-deoxyribose(50mM)용액 10ml를 혼합한 용액에 각각의 농도로 용해한 샘플을 20ml씩 첨가한 후 완충용액(0.2M sodium phosphate buffer, pH 7.0) 30ml과 H2O2(50mM) 10ml 및 증류수 20ml를 넣고 37℃에서 4시간 동안 반응시키고 2.8% trichloroacetic acid 100 ml와 NaOH(50mM)용액에 녹인 1% 2-thiobarbituric acid 100ml를 첨가하고 100℃에서 15분간 가열하고 급속 냉각한 다음 분광광도계를 이용하여 530nm에서 흡광도를 측정하였다. Hydroxy radical 소거활성(%)은 (1 - 시료 첨가구의 흡광도/시료 무첨가구의 흡광도) X 100의 계산식으로 구하였다.10 ml of FeSO4 · 7H2O (50mM) -EDTA (50mM) solution and 10ml of 2-deoxyribose (50mM) solution were added to each solution, and 20ml of each sample was added to each concentration.Then, 0.2M sodium phosphate buffer, pH 7.0 ) 30 ml, 10 ml of H2O2 (50 mM) and 20 ml of distilled water were added and reacted at 37 ° C. for 4 hours. Then, 100 ml of 2.8% trichloroacetic acid and 100 ml of 1% 2-thiobarbituric acid dissolved in NaOH (50 mM) solution were added. After heating and rapid cooling, the absorbance was measured at 530 nm using a spectrophotometer. Hydroxy radical scavenging activity (%) was calculated by the formula (1-absorbance of sample added / absorbance of sample free)
<표 3><Table 3>
상기 <표 3> 및 도 3에서 보는 바와 같이, 본 발명에 의하여 제조된 실시예 1에 대한 H2O2 소거능은 각각의 추출물을 사용한 경우 또는 SK제약의 조인스정을 사용한 경우보다 뛰어난 것을 알 수 있다. 양성 대조군으로는 vitamin C를 사용하였다.As shown in Table 3 and Figure 3, it can be seen that the H 2
실험예 4 : LPS 유도 NO 생성량 억제 실험Experimental Example 4: Inhibition of LPS induced NO production
각 샘플이 염증유발 물질인 NO(nitric oxide,일산화질소)의 생성을 억제하는 작용효과는 마우스 대식세포에 LPS(lipopolysaccharide)를 처리함으로써 생성되는 배양액내의 nitric oxide 농도를 Griess 반응을 이용하여 측정하였고 그 결과는 도 4 및 <표 4>에 나타낸 바와 같다.The effect of inhibiting the production of NO (nitric oxide), which is an inflammation-causing substance, was measured by Griess reaction of nitric oxide concentration in culture medium produced by treating LPS (lipopolysaccharide) on mouse macrophages. The results are shown in FIG. 4 and <Table 4>.
도 4는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 마우스 대식세포에서 LPS에 유도되는 nitric oxide의 생성을 저해하는 것을 나타낸 그래프도이다.4 is a graph showing that the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits the production of nitric oxide induced in LPS in mouse macrophages.
[실험방법]Experimental Method
NO의 농도는 배양액 내의 nitric oxide 농도를 Griess 반응을 이용하여 측정하였다. 먼저, FBS와 항생제가 함유된 DMEM 배지를 이용하여 96 웰 컬쳐 플레이트(well culture plate)에 1×104cells/well의 마우스 대식세포 유래의 RAW 264.7 세포를 분주 후 37℃, 5% CO2 항온기에서 24시간동안 배양하였다. 24시간 후, 이전 배양에 사용된 배지를 제거하고 FBS와 항생제가 함유되지 않은 새로운 DMEM 배지를 분주한 후 실시예1에 의해 제조된 식물조성물을 농도별(mg/mL)로 처리하였다. 1시간 후 1mg/mL의 LPS를 처리하여 24시간 배양하였다. 배양 동안 생성된 NO는 Griess시약을 이용하여 세포배양액 중에 존재하는 전체 NO2-의 농도로 측정하였는데, 세포배양 상층액 50μL와 Griess시약 50μL를 혼합하여 96 웰플레이트(well plate)에서 10분 동안 반응시킨 후 ELISA reader를 이용하여 540nm에서 흡광도를 측정하였다. Nitric oxide 소거능(%)은 (1-시료를 첨가한 반응군의 흡광도/시료를 첨가하지 않은 대조군의 흡광도)×100의 계산식을 이용하여 나타내었다.NO concentration was measured by Griess reaction of nitric oxide concentration in the culture. First, dispensing RAW 264.7 cells derived from 1 × 10 4 cells / well of mouse macrophages into a 96 well culture plate using DMEM medium containing FBS and antibiotics, and then 24 hours at 37 ° C. and a 5
<표 4>TABLE 4
상기 <표 4> 및 도 4에서 보는 바와 같이, 본 발명에 의하여 제조된 식물추출 조성물을 사용한 경우 마우스 대식세포에서 LPS에 의해 유도된 염증유발물질의 하나인 NO의 생성이 현저히 억제됨을 알 수 있었으며, 특히 실시예 1을 처리한 경우에서 NO의 생성 억제가 아주 우수한 것으로 나타났다.As shown in Table 4 and Figure 4, the use of the plant extract composition prepared according to the present invention was found to significantly inhibit the production of NO, one of the LPS-induced inflammatory substances in mouse macrophages. In particular, when Example 1 was treated, NO production was found to be excellent.
실험예 5 : COX-1 및 COX-2의 활성저해 실험Experimental Example 5: Inhibition of activity of COX-1 and COX-2
각 샘플이 염증유발 효소인 COX-1(cyclooxygenase-1) 또는 COX-2(cyclooxygenase-2)의 활성을 저해하는 효능을 알아보기 위해서 이러한 효소의 기질인 아라키돈산(arachidonic acid)을 사용하여 hematin의 산화를 유도하여 변색되는 현상을 이용하였고, 그 결과는 도 5 ~ 6 및 <표 5>에 나타낸 바와 같다. To determine the efficacy of each sample to inhibit the activity of the pro-inflammatory enzymes COX-1 (cyclooxygenase-1) or COX-2 (cyclooxygenase-2), the substrates of these enzymes, using arachidonic acid, The phenomenon of discoloration by using oxidation was used, and the results are shown in FIGS. 5 to 6 and Table 5.
도 5는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 COX-1 효소를 50% 저해하는 IC50값을 ppm의 농도로 나타낸 그래프도이고,Figure 5 is a graph showing the IC50 value in ppm concentration of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits COX-1 enzyme by 50%,
도 6은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 COX-2 효소를 50% 저해하는 IC50값을 ppm의 농도로 나타낸 그래프도이다.Figure 6 is a graph showing the IC50 value in ppm concentration of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits COX-2 enzyme by 50%.
[실험방법]Experimental Method
COX-1 또는 COX-2 억제효능은 96 웰플레이트(well plate)에 한 웰(well)당 60units/ml의 COX-1 또는 30units/ml의 COX-2를 40μl씩 넣고, 100mM Tris-HCl buffer(pH 8.0) 90μl, 30μM EDTA 20μL, 150μM hematin 20μl 그리고 시료 20μl를 혼합하여 25℃에서 5분간 반응시키고 5mM TMPD 5μl와 20mM 아라키돈산 5μl를 첨가하여 25℃에서 5분간 반응시킨 후 ELISA reader 기기를 사용하여 590nm에서 흡광도를 측정하였다. COX-1 또는 COX-2의 억제율(%)은 다음과 같은 식을 이용하여 50% inhibitory concentration(IC50) 값으로 나타내었다. COX 억제율(%)=[(대조군의 흡광도-실험군의 흡광도)/대조군의 흡광도]×100COX-1 or COX-2 inhibitory effect was measured in a 100-well Tris-HCl buffer (40 μl) with 60 units / ml of COX-1 or 30 units / ml of COX-2 per well in a 96 well plate. pH 8.0) 90μl, 30μM EDTA 20μL, 150μM hematin 20μl and sample 20μl were mixed and reacted at 25 ° C for 5 minutes, 5mM TMPD 5μl and 20mM arachidonic acid were added for 5 minutes at 25 ° C. Absorbance was measured at 590 nm. The percentage inhibition of COX-1 or COX-2 was expressed as a 50% inhibitory concentration (IC50) using the equation COX inhibition rate (%) = [(absorbance of the control group-absorbance of the experimental group) / absorbance of the control group] × 100
<표 5><Table 5>
상기 <표 5> 및 도 5 ~ 6에서 보는 바와 같이, 본 발명에 의하여 제조된 식물추출 조성물을 사용한 경우 염증관련 효소인 COX-1과 COX-2의 활성이 현저히 억제됨을 알 수 있었으며, 특히 실시예 1을 처리한 경우에서 COX-1과 COX-2의 활성 억제가 아주 우수한 것으로 나타났다.As shown in Table 5 and Figures 5 to 6, the use of the plant extract composition prepared according to the present invention was found to significantly inhibit the activity of inflammation-related enzymes COX-1 and COX-2, particularly In case of treatment of Example 1, the inhibition of COX-1 and COX-2 activity was very good.
실험예 6 : MIA(Mono sodium iodoacetate)유도 퇴행성관절염 동물모델 제작Experimental Example 6 Manufacture of MIA (Mono sodium iodoacetate) induced degenerative arthritis animal model
SD계 흰쥐에 MIA를 관절강내 주사(intra-articular injection)함으로써 관절연골세포(chondrocyte)의 대사(metabolism)를 저해해서 연골(cartilage), 인대(ligament) 및 힘줄(tendon)의 손상을 유발해서 골관절염을 발생시키는 원리를 이용하였다. 그 결과는 <표 6>에 나타낸 바와 같다.Intra-articular injection of MIA into SD rats inhibits metabolism of chondrocytes, causing cartilage, ligament, and tendon damage to osteoarthritis Was used to generate the principle. The results are as shown in Table 6.
[실험방법]Experimental Method
체중 300g 이상의 SD계 흰쥐의 관절강내로 27 게이지(gauge)의 주사바늘이 있는 1ml 주사기를 이용하여 MIA(40mg/ml)를 50ml씩 주사하고 각 실험군을 10 마리로 해서 각각의 샘플 또는 식염수 또는 Ibuprofen을 14일간 매일 정해진 투여량으로 경구투여 하였다.Inject 50 ml of MIA (40mg / ml) using a 1ml syringe with a 27 gauge needle into the joint cavity of SD rats weighing more than 300g. Each sample, 10 saline, or Ibuprofen The dose was administered orally at daily doses for 14 days.
<표 6><Table 6>
실험예 7 : 리포옥시제나제(LOs, Lipoxygenases) 활성저해 실험Experimental Example 7: Lipooxygenase (LOs, Lipoxygenases) inhibitory activity
각 샘플이 MIA를 이용한 퇴행성관절염 동물모델에서 염증 및 통증유발효소인 리포옥시제나제를 저해하는 효능을 측정하기 위해 Lipoxygenase Inhibitor Screening Assay Kit(Cayman Chemical Com, MI, USA)를 사용하여 리포옥시제나제에 의한 lipoxygenation 반응의 결과산물인 과산화수소의 양을 측정하는 원리를 이용하였고, 그 결과는 도 7 및 <표 7>에 나타낸 바와 같다.Lipogenasease using Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical Com, MI, USA) to measure the efficacy of each sample to inhibit lipooxygenase, an inflammation and pain-causing enzyme, in a degenerative arthritis animal model using MIA. The principle of measuring the amount of hydrogen peroxide as a result of the lipoxygenation reaction by using was used, the results are shown in Figure 7 and Table 7.
도 7은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 혈액에서 Lipoxygenase 효소의 활성을 시료의 무처리군과 비교하여 저해하는 비율을 나타낸 그래프도이다.Figure 7 shows the ratio of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits Lipoxygenase enzyme activity in the blood of chronic inflammatory animal model by MIA compared to the untreated group of samples. It is a graph figure.
[실험방법]Experimental Method
샘플 10μl에 5-LO(220units/ml) 90μl와 1 mM linoleic acid 10μl를 첨가하여 5분간 상온에서 반응시킨 후, 색원체(chromogen) 100μl를 가하여 상온에서 5분 동안 반응시키고 ELISA reader 기기로 490nm 파장에서 흡광도를 측정하였다. 그 결과는 50% inhibitory concentration(IC50) 값으로 나타내었고 5-LO 억제율(%)은 [(대조군의 흡광도-실험군의 흡광도)/대조군의 흡광도]×100 식을 이용하여 IC50값으로 나타내었다.After 10 μl of sample, 90 μl of 5-LO (220 units / ml) and 10 μl of 1 mM linoleic acid were reacted at room temperature for 5 minutes, and then 100 μl of chromogen was added for 5 minutes at room temperature, followed by 490 nm wavelength using an ELISA reader. Absorbance was measured at. The results were expressed as 50% inhibitory concentration (IC50) and the 5-LO inhibition rate (%) was expressed as IC50 using [(absorbance of control-absorbance of control group) / absorbance of control group] × 100.
<표 7><Table 7>
상기 <표 7> 및 도 7에서 보는 바와 같이, 본 발명에 의하여 제조된 식물추출 조성물을 사용한 경우 염증관련 효소인 리포옥시제나제의 활성이 억제됨을 알 수 있었으며, 특히 실시예 1를 처리한 경우에서 리포옥시제나제의 활성 억제가 아주 우수한 것으로 나타났다. 또한 양성 대조군으로 사용한 Ibuprofen은 리포옥시제나제의 활성저해에 영향을 주지 못하는 것으로 나타났다.As shown in Table 7 and Figure 7, when using the plant extract composition prepared according to the present invention was found that the activity of the inflammation-related enzyme lipooxygenase is inhibited, especially in the case of treating Example 1 Showed a very good inhibition of the activity of lipooxygenase. In addition, Ibuprofen, which was used as a positive control, did not appear to affect the inhibition of lipooxygenase activity.
실험예 8 : TNF-α 생성저해 실험Experimental Example 8: TNF-α production inhibition experiment
각 샘플이 염증유발인자인 TNF-α의 생체내 발현을 감소시키는 효능을 알아보기 위해서 카라기난 유도 발부종 동물모델 및 MIA유도 퇴행성관절염 동물모델을 대상으로 TNF-α sandwich ELISA kit(eBiosience, Vienna, Austria)을 사용하여 측정하였고, 그 결과는 도 8~9 및 <표 8~9>에 나타낸 바와 같다.To evaluate the efficacy of each sample in reducing the expression of TNF-α, an inflammation-inducing factor, the TNF-α sandwich ELISA kit (eBiosience, Vienna, Austria) was studied in carrageenan-induced edema and MIA-induced degenerative arthritis ), And the results are shown in FIGS. 8-9 and <Table 8-9>.
도 8은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 부종이 일어난 족부위에서 TNF-α의 생성량을 나타낸 그래프도이고,FIG. 8 is a graph showing the amount of TNF-α production in the foot region in which the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention caused swelling of an acute inflammatory animal model caused by carrageenan.
도 9은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 부종이 일어난 슬관절부위에서 TNF-α의 생성량을 나타낸 그래프도이다.9 is a graph showing the amount of production of TNF-α in the knee joint where the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention caused edema of a chronic inflammation animal model by MIA.
[실험방법]Experimental Method
각 샘플을 <표 8 ~ 9>에서 제시한 기간 및 용량으로 매일 1회씩 경구투여한 카라기난 유도 발부종 동물모델 및 MIA유도 퇴행성관절염 동물모델을 희생하고 채혈 및 혈장분리를 한 다음 혈장내에 존재하는 TNF-α의 농도를 ELISA kit의 제조회사가 제시한 방법으로 측정하였다. 먼저, 96 웰 플레이트(well plate)에 코팅 되어 있는 anti-rat TNF-α antibody에 혈장 중의 TNF-α를 결합시키고 각 웰(well)을 5번 세척하고 여기에 biotin-conjugated anti-rat TNF-α antibody를 반응시킨 후 각 웰(well)을 5번 세척하고 streptavidin-HRP를 처리하고 최종적으로 tetramethyl-benzidine으로 발색하여 ELISA reader 기기를 이용해서 450nm의 파장에서 흡광도를 측정하였다. TNF-α의 양은 ELISA kit에 포함된 표준 재조합 TNF-α의 반응표준곡선을 이용하여 산술비례적으로 구하였다.Each sample was sacrificed by carrageenan-induced edema animal model and MIA-induced degenerative arthritis animal model, which were orally administered once daily at the period and dose shown in Tables 8 to 9, and blood and plasma were separated. The concentration of -α was measured by the method suggested by the manufacturer of the ELISA kit. First, TNF-α in plasma was bound to anti-rat TNF-α antibody coated on a 96 well plate, and each well was washed five times, followed by biotin-conjugated anti-rat TNF-α. After the reaction of the antibody, each well was washed five times, treated with streptavidin-HRP, and finally colored with tetramethyl-benzidine, and the absorbance was measured at 450 nm using an ELISA reader. The amount of TNF- [alpha] was calculated in an arithmetic proportion using the standard curve of the standard recombinant TNF- [alpha] contained in the ELISA kit.
<표 8><Table 8>
<표 9><Table 9>
상기 <표 8 ~ 9> 및 도 8 ~ 9에서 보는 바와 같이, 카라기난에 의한 급성염증 동물모델 및 MIA에 의한 만성염증 동물모델에서 본 발명에 의하여 제조된 식물추출 조성물을 사용한 경우 염증관련 인자인 TNF-α의 생체내 생성이 억제됨을 알 수 있었으며, 특히 실시예 1를 처리한 경우에서 TNF-α의 생체내 생성 억제가 아주 우수한 것으로 나타났다. 또한 양성 대조군으로 사용한 Ibuprofen은 TNF-α의 생체내 생성에 아주 미미한 영향만 주는 것으로 나타났다.As shown in Tables 8 to 9 and 8 to 9, TNF is an inflammation-related factor when the plant extract composition prepared by the present invention is used in an acute inflammatory animal model by carrageenan and a chronic inflammatory animal model by MIA. In vivo production of -α was suppressed. In particular, when Example 1 was treated, it was found that the inhibition of in vivo production of TNF-α was excellent. In addition, Ibuprofen used as a positive control was found to have only a slight effect on the in vivo production of TNF-α.
실험예 9 : IL-1β 생성저해 실험Experimental Example 9 IL-1β Production Inhibition Experiment
각 샘플이 염증유발인자인 IL-1β의 생체내 발현을 감소시키는 효능을 알아보기 위해서 카라기난 유도 발부종 동물모델 및 MIA유도 퇴행성관절염 동물모델을 대상으로 IL-1β sandwich ELISA kit(eBiosience, Vienna, Austria)을 사용하여 측정하였고, 그 결과는 도 10 ~ 11 및 <표 10 ~ 11>에 나타낸 바와 같다.To evaluate the efficacy of each sample to reduce the in vivo expression of IL-1β, an inflammation-inducing factor, the IL-1β sandwich ELISA kit (eBiosience, Vienna, Austria) was studied in carrageenan-induced edema and MIA-induced degenerative arthritis. ), And the results are shown in FIGS. 10 to 11 and <Table 10 to 11>.
도 10은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 부종이 일어난 족부위에서 IL-1β의 생성량을 나타낸 그래프도이고,FIG. 10 is a graph showing the amount of IL-1β production in the foot region in which the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention caused swelling of an acute inflammatory animal model caused by carrageenan,
도 11은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 부종이 일어난 슬관절부위에서 IL-1β의 생성량을 나타낸 그래프도이다.11 is a graph showing the amount of IL-1β production in the knee joint where the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention caused edema of a chronic inflammation animal model by MIA.
[실험방법] Experimental Method
각 샘플을 표 10 ~ 11에서 제시한 기간 및 용량으로 매일 1회씩 경구투여한 카라기난 유도 발부종 동물모델 및 MIA유도 퇴행성관절염 동물모델을 희생하고 채혈 및 혈장분리를 한 다음 혈장내에 존재하는 IL-1β의 농도를 ELISA kit의 제조회사가 제시한 방법으로 측정하였다. 먼저, 96 웰 프레이트(well plate)에 코팅 되어 있는 anti-rat IL-1β antibody에 혈장 중의 IL-1β를 결합시키고 각 웰(well)을 5번 세척하며 여기에 biotin-conjugated anti-rat IL-1β antibody를 반응시킨 후 각 웰(well)을 5번 세척하고 streptavidin-HRP를 처리하고 최종적으로 tetramethyl-benzidine으로 발색하여 ELISA reader 기기를 이용해서 450nm의 파장에서 흡광도를 측정하였다. IL-1β의 양은 ELISA kit에 포함된 표준 재조합 IL-1β의 반응표준곡선을 이용하여 산술비례적으로 구하였다.IL-1β present in plasma after sacrificing blood collection and plasma separation after sacrificing carrageenan-induced edema animal model and MIA-induced degenerative arthritis animal model orally administered once daily with the periods and doses shown in Tables 10-11. The concentration of was measured by the method suggested by the manufacturer of the ELISA kit. First, IL-1β in plasma is bound to anti-rat IL-1β antibody coated on a 96 well plate, and each well is washed five times, followed by biotin-conjugated anti-rat IL-1β. After the reaction of the antibody, each well was washed five times, treated with streptavidin-HRP, and finally colored with tetramethyl-benzidine, and the absorbance was measured at 450 nm using an ELISA reader. The amount of IL-1β was calculated in an arithmetic proportion using the standard curve of the standard recombinant IL-1β contained in the ELISA kit.
<표 10><Table 10>
<표 11><Table 11>
상기 <표 10 ~ 11> 및 도 10 ~ 11에서 보는 바와 같이, 카라기난에 의한 급성염증 동물모델 및 MIA에 의한 만성염증 동물모델에서 본 발명에 의하여 제조된 식물추출 조성물을 사용한 경우 염증관련 인자인 IL-1β의 생체내 생성이 억제됨을 알 수 있었으며, 특히 실시예 1를 처리한 경우에서 IL-1β의 생체내 생성 억제가 아주 우수한 것으로 나타났다. 또한 양성 대조군으로 사용한 Ibuprofen은 IL-1β의 생체내 생성에 아주 미미한 영향만 주는 것으로 나타났다.As shown in Tables 10 to 11 and FIGS. 10 to 11, IL-related inflammation factors when the plant extract composition prepared according to the present invention is used in an acute inflammatory animal model by carrageenan and a chronic inflammatory animal model by MIA It was found that the in vivo production of -1β was suppressed. In particular, when Example 1 was treated, the inhibition of in vivo production of IL-1β was very excellent. In addition, Ibuprofen, used as a positive control, was found to have only a minor effect on the in vivo production of IL-1β.
실험예 10 : IL-6 생성저해 실험Experimental Example 10 IL-6 Production Inhibition Experiment
각 샘플이 염증유발인자인 IL-6의 생체내 발현을 감소시키는 효능을 알아보기 위해서 카라기난 유도 발부종 동물모델 및 MIA유도 퇴행성관절염 동물모델을 대상으로 IL-6 sandwich ELISA kit(eBiosience, Vienna, Austria)을 사용하여 측정하였고, 그 결과는 도 12 ~ 13 및 <표 12 ~ 13>에 나타낸 바와 같다.IL-6 sandwich ELISA kit (eBiosience, Vienna, Austria) for carrageenan-induced edema and MIA-induced degenerative arthritis animal models ), And the results are shown in FIGS. 12 to 13 and <Table 12 to 13>.
도 12는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 부종이 일어난 족부위에서 IL-6의 생성량을 나타낸 그래프도이고,12 is a graph showing the amount of IL-6 production in the foot region where edema of acute inflammatory animal model caused by carrageenan occurred in the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention.
도 13은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 부종이 일어난 슬관절부위에서 IL-6의 생성량을 나타낸 그래프이다.FIG. 13 is a graph showing the amount of IL-6 production in the knee joint where the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention caused edema of a chronic inflammation animal model by MIA.
[실험방법]Experimental Method
각 샘플을 <표 12 ~ 13>에서 제시한 기간 및 용량으로 매일 1회씩 경구투여한 카라기난 유도 발부종 동물모델 및 MIA유도 퇴행성관절염 동물모델을 희생하고 채혈 및 혈장분리를 한 다음 혈장내에 존재하는 IL-6의 농도를 ELISA kit의 제조회사가 제시한 방법으로 측정하였다. IL was present in plasma after sacrificing blood collection and plasma separation at the expense of carrageenan-induced edema animal model and MIA-induced degenerative arthritis animal model, which were orally administered once daily at the periods and doses shown in Tables 12-13. The concentration of -6 was measured by the method suggested by the manufacturer of the ELISA kit.
먼저, 96 웰 플레이트(well plate)에 코팅되어 있는 anti-rat IL-6 antibody에 혈장 중의 IL-6를 결합시키고 각 웰(well)을 5번 세척하고 여기에 biotin-conjugated anti-rat IL-6 antibody를 반응시킨 후 streptavidin-HRP를 처리하고 최종적으로 tetramethyl-benzidine으로 발색하여 ELISA reader 기기를 이용해서 450nm의 파장에서 흡광도를 측정하였다. IL-6의 양은 ELISA kit에 포함된 표준 재조합 IL-6의 반응표준곡선을 이용하여 산술비례적으로 구하였다.First, IL-6 in plasma was bound to anti-rat IL-6 antibody coated on a 96 well plate, and each well was washed five times, followed by biotin-conjugated anti-rat IL-6. After reacting the antibody, streptavidin-HRP was treated, and finally, tetramethyl-benzidine was developed and the absorbance was measured at 450 nm using an ELISA reader. The amount of IL-6 was calculated arithmetic proportionally using the standard reaction curve of the standard recombinant IL-6 included in the ELISA kit.
<표 12><Table 12>
<표 13><Table 13>
상기 <표 12 ~ 13> 및 도 12 ~ 13에서 보는 바와 같이, 카라기난에 의한 급성염증 동물모델 및 MIA에 의한 만성염증 동물모델에서 본 발명에 의하여 제조된 식물추출 조성물을 사용한 경우 염증관련 인자인 IL-6의 생체내 생성이 억제됨을 알 수 있었으며, 특히 실시예 1를 처리한 경우에서 IL-6의 생체내 생성 억제가 아주 우수한 것으로 나타났다. 또한 양성 대조군으로 사용한 Ibuprofen은 IL-6의 생체내 생성에 아주 미미한 영향만 주는 것으로 나타났다.As shown in Tables 12 to 13 and Figures 12 to 13, IL is an inflammation-related factor when a plant extract composition prepared by the present invention is used in an acute inflammatory animal model by carrageenan and a chronic inflammatory animal model by MIA. In vivo production of -6 was found to be suppressed. In particular, when Example 1 was treated, it was found that the inhibition of in vivo production of IL-6 was very excellent. In addition, Ibuprofen, used as a positive control, was found to have only a minor effect on the in vivo production of IL-6.
실험예 11 : 프로스타그란딘(PGs,Prostaglandins) 생성억제 실험 Experimental Example 11: Prostaglandin (PGs, Prostaglandins) production inhibition experiment
각 샘플이 LPS를 이용한 염증 세포모델과 MIA를 이용한 퇴행성관절염 동물모델에서 염증 및 통증유발인자인 프로스타그란딘(Prostaglandins)의 생성을 억제하는 효능을 측정하기 위해 Ptostaglandin Screening EIa Kit(Cayman Chemical Co., MI, USA)를 사용하여 프로스타그란딘과 Tracer(acetylcholinesterase를 인위적으로 결합시킨 프로스타그란딘)가 서로 경쟁적으로 anti-PGs-antibody에 결합하는 원리를 이용하였고, 그 결과는 도 14 ~ 15 및 <표 14 ~ 15>에 나타낸 바와 같다.To evaluate the efficacy of each sample to inhibit the production of prostaglandins, inflammation and pain-causing factors, in inflammatory cell models using LPS and animal models of degenerative arthritis using MIA, Ptostaglandin Screening EIa Kit (Cayman Chemical Co., MI, USA) using the principle of prostaglandin and tracer (prostaglandin artificially coupled to acetylcholinesterase) competitively binds to the anti-PGs-antibody, the results are shown in Figures 14-15 and Tables 14-15 As shown.
도 14는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 마우스 대식세포에서 LPS에 유도되는 프로스타그란딘의 생성을 저해하는 것을 나타낸 그래프도이고,14 is a graph showing that the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits the production of prostaglandin induced by LPS in mouse macrophages,
도 15는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 MIA에 의한 만성염증 동물모델의 부종이 일어난 슬관절부위에서 프로스타그란딘의 생성을 저해하는 것을 나타낸 그래프도이다.15 is a graph showing that the mixed extract prepared according to Reference Examples 1 to 3 and Example 1 of the present invention inhibits the production of prostaglandin in the knee joint site where edema of a chronic inflammation animal model by MIA occurred.
[실험방법]Experimental Method
마우스 대식세포 유래의 RAW 264.7 세포를 FBS와 항생제가 함유된 DMEM 배지를 사용하여 96 웰 컬처 플레이트(well culture plate)에 1×104cells/well의 분주 후 37℃, 5% CO2 항온기에서 24시간동안 배양하였다. 24시간 후, 이전 배양에 사용된 배지를 제거하고 FBS와 항생제가 함유되지 않은 새로운 DMEM 배지를 분주한 후 각 샘플을 농도별로 처리하였다. 1시간 후 1mg/mL의 LPS를 처리하여 24시간 배양하였다. Prostaglandin AChE Tracer와 antiserum을 anti-PGs antibody가 코팅된 96 웰프레이트(well plate)에 넣고 표준 프로스타그란딘(prostaglandin) 또는 각 샘플을 처리한 배지를 50 μl씩 첨가한 후 18시간 상온에서 반응시킨 후 각 웰(well)을 5번 세척하고 Ellman's Reagent를 200 ml씩 넣고 70분간 발색하고 ELISA reader를 이용하여 420 nm의 파장에서 흡광도를 측정한다. 프로스타그란딘의 양은 ELISA kit에 포함된 표준곡선을 이용하여 산술비례적으로 구하였다.RAW 264.7 cells derived from mouse macrophages were cultured in a 96 well culture plate using DMEM medium containing FBS and antibiotics for 1 hour after dispensing 1 × 10 4 cells / well in 37 ° C., 5
<표 14>TABLE 14
<표 15><Table 15>
상기 <표 14 ~15>에서 보는 바와 같이, 본 발명에 의하여 제조된 식물추출 조성물을 사용한 경우 마우스 대식세포에서 LPS에 의해서 유도된 염증유발물질의 하나인 프로스타그란딘의 생성이 억제됨을 알 수 있으며, 특히 실시예 1에서 프로스타그란딘 생성 억제율이 아주 우수한 것으로 나타났다.As shown in Tables 14 to 15, when the plant extract composition prepared according to the present invention is used, the production of prostaglandin, which is one of LPS-induced inflammatory substances in mouse macrophages, is inhibited. In Example 1, the inhibition of prostaglandin production was found to be excellent.
실험예 12 : Type I Collagenase 활성저해 실험Experimental Example 12: Type I Collagenase Inhibition Experiment
각 샘플이 관절연골 건강에 주요 악영향을 미치는 Type I Collagenase의 활성을 저해하는 효과를 보기 위하여 재조합 Collagenase enzyme(Sigma, MO, USA)이 기질인 PZ-peptide (4-phenylazobenzyloxycarbonyl-Pro-Lue-Gly-Pro-D-Arg, Fluka Chemie GmbH, Buchs, Switzerland)를 분해할 때의 변색현상을 이용하여 측정하였고 그 결과는 도 16 및 <표 16>에 나타낸 바와 같다.PZ-peptide (4-phenylazobenzyloxycarbonyl-Pro-Lue-Gly-), a substrate of recombinant Collagenase enzyme (Sigma, MO, USA), was used to examine the effect of each sample on the activity of Type I Collagenase, which has a major adverse effect on articular cartilage health. Pro-D-Arg, Fluka Chemie GmbH, Buchs, Switzerland) was measured using the discoloration phenomenon when decomposing the results are shown in Figure 16 and Table 16.
도 16은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 Type I collagenase를 50% 저해하는 IC50값을 ppm의 농도로 나타낸 그래프도이다.Figure 16 is a graph showing the IC50 value of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibiting Type I collagenase 50% by the concentration of ppm.
[실험방법]Experimental Method
Collagenase enzyme 0.2mg/ml의 농도로 용해하고 PZ-peptide는 0.3mg/ml의 농도로 완충용액에 희석한 후 농도별 샘플 100ml과 collagenase 150ml 및 PZ-peptide 250ml을 순서대로 혼합하여 vortex 후 37℃, 30분간 반응한 후 Citric acid(25mM) 0.5ml을 첨가하고 ethyl acetate 1.5ml을 첨가한 후 상온에서 10분간 반응시키고 석영 cuvette을 이용하여 320nm 파장에서 흡광도를 측정한다.After dissolving collagenase enzyme at a concentration of 0.2mg / ml and diluting PZ-peptide in buffer at a concentration of 0.3mg / ml, vortex after mixing 100ml of sample by concentration, 150ml of collagenase and 250ml of PZ-peptide, and vortex After reacting for 30 minutes, 0.5 ml of citric acid (25 mM) is added, ethyl acetate 1.5 ml is added, and then reacted at room temperature for 10 minutes. Absorbance is measured at 320 nm using a quartz cuvette.
<표 16><Table 16>
상기 <표 16> 및 도 16에서 보는 바와 같이, 본 발명에 의하여 제조된 실시예 1에 대한 Type I Collagenase저해능은 각각의 추출물을 사용한 경우 또는 SK제약의 조인스정을 사용한 경우보다 뛰어난 것을 알 수 있다. As shown in Table 16 and Figure 16, it can be seen that Type I Collagenase inhibitory ability for Example 1 prepared by the present invention is superior to the case of using each extract or the joining tablet of SK Pharmaceuticals. .
실험예 13 : iNOS, COX-1, COX-2의 생체내 발현억제 실험Experimental Example 13: In vivo expression inhibition experiment of iNOS, COX-1, COX-2
각 샘플이 LPS를 이용한 염증 세포모델과 카라기난을 이용한 급성염증 동물모델에서 염증유발효소인 iNOS, COX-1, COX-2의 생체내 발현을 억제하는 효능을 측정하기 위해 웨스턴 블랏(Western blot)을 실시하였고, 그 결과는 도 17 ~ 18에 나타낸 바와같다.Western blot was used to determine the efficacy of each sample to inhibit the in vivo expression of pro-inflammatory enzymes iNOS, COX-1, and COX-2 in inflammatory cell models using LPS and acute inflammatory animal models using carrageenan. The results are as shown in Figs. 17-18.
도 17은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 부종이 일어난 족부위에서 iNOS, COX-1, COX-2 효소의 발현을 억제하는 것을 나타낸 도면(Ibu: Ibuprofen, 실1-100: 실시예1 100mg/체중Kg 처리, 실1-400: 실시예1 400mg/체중Kg, Joi: 조인스정 400mg/체중Kg 처리)이고,17 is a mixture extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention to inhibit the expression of iNOS, COX-1, COX-2 enzymes in the foot area where edema of acute inflammatory animal model by carrageenan occurred (Ibu: Ibuprofen, Thread 1-100: Example 1 100 mg / weight Kg treatment, Thread 1-400: Example 1 400 mg / weight Kg, Joi: Joining
도 18은 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 LPS에 의한 마우스 대식세포의 염증반응에서 발현이 유도되는 iNOS, COX-1, COX-2 효소의 발현을 억제하는 것을 나타낸 도면이다.(Ibu: Ibuprofen, 실1-200: 실시예1 200ppm 처리, 실1-1K: 실시예1 1000ppm 처리, Joi: 조인스정 1000ppm 처리)18 is a mixture extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention inhibits the expression of iNOS, COX-1, COX-2 enzymes induced expression in the inflammatory response of mouse macrophages by LPS (Ibu: Ibuprofen, Thread 1-200: Example 1 200ppm treatment, Thread 1-1K: Example 1 1000ppm treatment, Joi: Joining tablet 1000ppm treatment)
[실험방법]Experimental Method
염증반응에 의한 단백질 발현량의 변화는 <표 17 ~ 18>에서 나타낸 바와 같이 카라기난에 의한 급성염증 동물모델 및 LPS에 의한 염증 세포모델을 제작하였고 각 실험군별로 동물조직 및 세포를 용해(Lysis)시켜 모은 총 단백질을 정량하고 각 웰(well)당 20mg의 총 단백질을 전기영동(SDS-PAGE)한 후 nitro cellulose 막에 전환(Transfer)한 다음, iNOS(Santa Cruz Biotechnology, CA, USA), COX-1(Cell Signaling Technology, MA, USA), COX-2(Cell Signaling Technology, MA, USA)의 1차 항체와 2차 항체(Santa Cruz Biotechnology, CA, USA) 및 enhanced chemiluminescence(ECL, Pierce, IL, USA)를 이용하여 X-ray필름에 표현하였고 총 단백질량의 동일성은 β-actin(Santa Cruz Biotechnology, CA, USA)의 양으로 검증하였다.As shown in <Table 17 ~ 18>, the protein expression level caused by the inflammatory response was prepared by carrageenan-induced acute inflammation animal model and LPS-induced inflammatory cell model. Quantify the total protein collected, electrophoresis (SDS-PAGE) of 20 mg total protein per well, transfer to nitro cellulose membrane, and then iNOS (Santa Cruz Biotechnology, CA, USA), COX- 1 (Cell Signaling Technology, MA, USA), COX-2 (Cell Signaling Technology, MA, USA) primary and secondary antibodies (Santa Cruz Biotechnology, CA, USA) and enhanced chemiluminescence (ECL, Pierce, IL, USA) was expressed on the X-ray film and the identity of the total protein was verified by the amount of β-actin (Santa Cruz Biotechnology, CA, USA).
<표 17>TABLE 17
<표 18><Table 18>
상기 <표 17 ~ 18> 및 도 17 ~ 18에서 보는 바와 같이, 본 발명에 의하여 제조된 실시예 1에 대한 염증관련 효소인 iNOS, COX-2의 생체내 발현이 저해된 것을 알 수 있다. 또한 Ibuprofen은 iNOS의 발현은 저해하였으나 COX-1 및 COX-2의 발현에는 영향을 주지 않는 것으로 나타난다.As shown in Tables 17 to 18 and 17 to 18, it can be seen that the expression of iNOS, COX-2, which is an inflammation-related enzyme for Example 1 prepared by the present invention, was inhibited. Ibuprofen also inhibited the expression of iNOS but did not affect the expression of COX-1 and COX-2.
실험예 14 : 카라기난(carrageenan) 유도 급성 염증에 대한 부종(edema)억제 실험Experimental Example 14 edema inhibition experiment for carrageenan-induced acute inflammation
상기 실시예 1에서 제조한 식물조성물에 의한 카라기난 유도 급성 염증반응에서 발 부종의 억제정도를 대조군과 비교하여 시간대별로 관찰하여 그 결과 값으로 하여 <표 17> 및 도 19에 나타내었다. In the carrageenan-induced acute inflammatory response by the plant composition prepared in Example 1, the degree of inhibition of foot edema was observed by time period compared to the control group, and the results are shown in Table 17 and FIG. 19.
도 19는 본 발명의 참조예 1 ~ 3 및 실시예 1에 의해 제조된 혼합추출물이 카라기난에 의한 급성염증 동물모델의 발부종 억제효과를 시간대별로 나타낸 도면이다.19 is a view showing the effect of inhibiting the paw edema of the animal model of acute inflammation caused by carrageenan of the mixed extract prepared by Reference Examples 1 to 3 and Example 1 of the present invention.
[실험방법]Experimental Method
SD(Spraque-Dqwley)계 흰쥐에 각 군의 개체수를 10으로 하여 다음 <표 19>의 농도로 경구투여하고 1% 카라기난을 용해한 생리식염수를 100ml의 양으로 좌측 족저부에 주사하여 족 부종을 유도한 다음 Digimatic micrometer(Mitutoyo Corporation, Japan)를 이용하여 6시간 동안 각 시간별로 족부종의 정도를 측정하였다.Spraque-Dqwley (SD) rats were inoculated with oral administration at the concentration of 10 in the following <Table 19>, and 1% carrageenan-dissolved saline solution was injected into the left plantar portion in an amount of 100 ml to induce foot edema. Then, the degree of foot edema was measured at each hour for 6 hours using a Digimatic micrometer (Mitutoyo Corporation, Japan).
<표 19><Table 19>
제조예: 본 발명의 추출물을 포함하는 식품 또는 건강 기능 식품의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Preparation Example: Although the preparation example of the food or health functional food containing the extract of the present invention will be described, the present invention is not intended to limit this, but is intended to explain in detail.
제조예 1: 산제의 제조Preparation Example 1: Preparation of powder
실시예 1의 기능성 조성물 300mg, 유당 100mg, 탈크 10mg300 mg of the functional composition of Example 1, 100 mg of lactose, 10 mg of talc
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled in an airtight cloth to prepare a powder.
제조예 2: 정제의 제조Production Example 2: Preparation of tablets
실시예 1의 기능성 조성물 50mg, 옥수수전분 100mg, 유당 100mg, 스테아린산 마그네슘 2mg50 mg of the functional composition of Example 1, 100 mg of corn starch, 100 mg of lactose, 2 mg of magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.
제조예 3: 캅셀제의 제조Preparation Example 3: Preparation of capsule
실시예 1의 기능성 조성물 50mg, 옥수수전분 100mg, 유당 100mg, 스테아린산 마그네슘 2mg50 mg of the functional composition of Example 1, 100 mg of corn starch, 100 mg of lactose, 2 mg of magnesium stearate
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
제조예 4: 액제의 제조Production Example 4: Preparation of liquid agent
실시예 1의 기능성 조성물 100mg, 이성화당 10g, 만니톨 5g, 정제수 적량100 mg of the functional composition of Example 1, 10 g of isomerized sugar, 5 g of mannitol,
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고, 레몬향을 적량 가한 다음, 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the appropriate amount, the above components are mixed, purified water is added, and the whole is adjusted to 100 ml by adding purified water to a brown bottle. Fill and sterilize to prepare a liquid.
제조예 5: 건강 기능 식품의 제조Production Example 5: Preparation of Health Functional Foods
실시예 1의 기능성 조성물 1000㎎, 비타민 혼합물 적량, 비타민 A 아세테이트 70㎍, 비타민 E 1.0㎎, 비타민 B10 13㎎, 비타민 B20 15㎎, 비타민 B6 0.5㎎, 비타민 B12 0.2㎍, 비타민 C 10㎎, 비오틴 10㎍, 니코틴산아미드 1.7㎎, 엽산 50㎍, 판토텐산 칼슘 0.5㎎, 무기질 혼합물 적량, 황산 제1철 1.75㎎, 산화아연 0.82㎎, 탄산마그네슘 25.3㎎, 제1 인산칼륨 15㎎, 제2 인산칼슘 55㎎, 구연산칼륨 90㎎, 탄산칼슘 100㎎, 염화마그네슘 24.8㎎1000 mg of the functional composition of Example 1, a proper amount of vitamin A, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 13 mg of vitamin B10, 15 mg of vitamin B20, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of niacinamide, 1.7 mg of nicotinic acid amide, 50 g of folic acid, 0.5 mg of calcium pantothenate, an appropriate amount of inorganic mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic 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.Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.
제조예 6: 건강 음료의 제조 Preparation Example 6: Preparation of health drinks
실시예 1의 기능성 조성물 1000㎎, 구연산 1000㎎, 올리고당 100g, 매실농축액 2g, 타우린 1g, 정제수를 가하여 전체 900㎖으로 조정.1,000 mg of the functional composition of Example 1, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of a plum concentrate, 1 g of taurine and purified water were added to adjust the total amount to 900 ml.
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 사용한다.The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >
상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.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 (12)
상기 항염증용 식품 조성물이 투여되는 경구 투여용 기능성 식품은 캡슐, 환, 과립, 분말, 캔디, 껌, 정제, 음료, 시럽 중 어느 하나인 것을 특징으로 하는 항염증용 식품 조성물.The method according to claim 2,
Functional food for oral administration to which the anti-inflammatory food composition is administered is any one of capsules, pills, granules, powders, candy, gum, tablets, beverages, syrups.
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