KR102364062B1 - Composition for anti-inflammation containing active mountain-cultivated ginseng and preparation method thereof - Google Patents
Composition for anti-inflammation containing active mountain-cultivated ginseng and preparation method thereof Download PDFInfo
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- KR102364062B1 KR102364062B1 KR1020180166208A KR20180166208A KR102364062B1 KR 102364062 B1 KR102364062 B1 KR 102364062B1 KR 1020180166208 A KR1020180166208 A KR 1020180166208A KR 20180166208 A KR20180166208 A KR 20180166208A KR 102364062 B1 KR102364062 B1 KR 102364062B1
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- South Korea
- Prior art keywords
- wild ginseng
- inflammatory
- active
- extract
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Abstract
본 발명에서는 활성산양삼의 추출물을 유효성분으로 포함하여 고농도에서도 세포독성이 없이 증진된 염증억제 활성을 갖는 항염증 조성물이 개시된다.
본 발명에 따른 항염증 조성물은 고농도로 사용시에도 세포독성이 전혀 없고 질소산화물(NO) 생성 억제활성, 염증성 사이토카인 IL-6 및 MCP-1 억제활성을 통하여 현저히 증진된 항염증 효과를 나타내고, 인체 흡수형 진세노사이드 Rd2, Rg3, F2, Rh2와 컴파운드 케이, 클로르제닉산과 쿼르세틴, 페놀릭스, 플라보노이드스 및 갈변물질 함량도 현저히 증진되어 있어, 염증관련 질환의 예방 및 개선용 기능성 식품 및 화장품의 소재로 사용될 수 있다. In the present invention, an anti-inflammatory composition having enhanced anti-inflammatory activity without cytotoxicity even at high concentration by including an extract of active wild ginseng as an active ingredient is disclosed.
The anti-inflammatory composition according to the present invention has no cytotoxicity even when used at a high concentration and exhibits a significantly enhanced anti-inflammatory effect through nitric oxide (NO) production inhibitory activity, inflammatory cytokines IL-6 and MCP-1 inhibitory activity, and the human body Absorption-type ginsenosides Rd2, Rg3, F2, Rh2, compound K, chlorgenic acid, quercetin, phenolics, flavonoids and browning substances are also significantly increased, so it is a functional food and cosmetic product for the prevention and improvement of inflammation-related diseases. material can be used.
Description
본 발명은 활성산양삼을 함유하는 항염증 조성물 및 그 제조방법에 관한 것으로, 더 상세하게는 산양삼 전초를 락토바실러스 브레비스 BMK484 균주와 락토바실러스 플란타륨 P1201 균주로 복합발효하여 제조된 활성산양삼의 추출물을 유효성분으로 포함하여 고농도에서도 세포독성이 없이 증진된 염증 억제활성을 나타내는 항염증 조성물 및 그 제조방법에 관한 것이다. The present invention relates to an anti-inflammatory composition containing active wild ginseng and a method for producing the same, and more particularly, to an extract of active wild ginseng prepared by complex fermentation of wild ginseng outpost with Lactobacillus brevis BMK484 strain and Lactobacillus plantarum P1201 strain. It relates to an anti-inflammatory composition that includes an active ingredient and exhibits enhanced anti-inflammatory activity without cytotoxicity even at high concentrations, and a method for preparing the same.
산양삼은 오가피과에 속하며 다년생 초목인 인삼(Panax ginseng)이 산간의 삼림하의 야생상태에서 자연적으로 성장한 산삼의 씨앗이나 유삼을 인위적으로 산에서 재배한 것을 말한다. 산양삼은 여러 효능면에서 재배 인삼보다 효과가 더 높은 것으로 알려져 있으나 이를 뒷받침할 수 있는 과학적 근거가 인삼에 비해 그리 많지는 않은 것은 현 실정이다. 특히 산양삼 전초에 대한 효능적 연구는 부족한 실정이다. Wild ginseng belongs to the family Ogapiaceae and is a perennial plant called Panax . ginseng ) refers to the artificially cultivated mountain ginseng seeds or oil ginseng seeds grown naturally in the wild under mountain forests. Wild ginseng is known to be more effective than cultivated ginseng in several aspects, but the current situation is that there is not much scientific evidence to support this compared to ginseng. In particular, effective research on wild ginseng outpost is lacking.
또한 산양삼과 같은 삼은 중추신경계를 비롯하여 내분비계, 면역계, 대사계 등에 영향을 미쳐 신체기능 조절에 다양한 약리효과를 나타낸다고 알려져 있으나, 고농도로 사용시 세포독성이 발생하는 문제점이 있다. In addition, ginseng such as wild ginseng is known to exert various pharmacological effects on the regulation of body functions by affecting the central nervous system, endocrine system, immune system, and metabolic system, but there is a problem that cytotoxicity occurs when used in high concentrations.
삼의 약리효과는 인체 흡수형 진세노사이드의 기인하는데, 인체 흡수형 진세노사이드로는 진세노사이드 Rg3, Rd, F2, 컴파운드 케이(compound K), Rh 등이 있다. 진세노사이드 Rg3는 암세포 전이억제, 간보호 작용, 항암제 내성억제 작용, 진세노사이드 Rd 계열은 부신피질 호르몬 분비 촉진 작용, 진세노사이드 컴파운드 케이는 항암작용, 간보호 작용, 피부보호 작용, 종양증식억제 작용, 항산화 작용, 항알레르기 작용이 밝혀져 있다. 진세노사이드 F2는 아토피성 피부의 염증을 가라앉히고 가려움증을 억제시키는 효과가 최근에 알려져 있다. 진세노사이드 컴파운드 케이는 진세노사이드 대사체로서 특히 체내 흡수율이 탁월하고 약리학적 활성도 우수한 것으로 알려져 있으며, 최근에는 황반변성질환 치료, 신경변증성 통증 치료도 보고되고 있으며(등록특허 10-1539573호), 미백, 주름개선 및 항염증 등의 효과 역시 보고되어 있다.The pharmacological effects of ginseng are attributable to human absorption-type ginsenosides, such as ginsenosides Rg3, Rd, F2, compound K, and Rh. Ginsenoside Rg3 inhibits cancer cell metastasis, hepatoprotective action, anticancer drug resistance inhibitory action, ginsenoside Rd series promotes adrenocortical hormone secretion, ginsenoside compound K has anticancer action, hepatoprotective action, skin protective action, tumor growth It has been shown to have inhibitory, antioxidant, and anti-allergic effects. Ginsenoside F2 has recently been known to have the effect of alleviating inflammation of atopic skin and suppressing itching. Ginsenoside compound K is a ginsenoside metabolite, and it is known for its excellent absorption rate and pharmacological activity in the body. , whitening, wrinkle improvement and anti-inflammatory effects have also been reported.
염증(inflammation)은 물리적인 외상, 유해한 화학물질, 미생물에 의한 감염이나 생체 내 대사산물 중의 자극성 물질에 의하여 야기되는 조직손상에 대하여 국소적으로 나타나는 정상적이고 보호적인 생체 내 방어기전의 발현이다. 정상적인 경우에 생체는 염증반응을 통하여 발병 요인을 중화시키거나 제거하고 상한 조직을 재생시켜서 정상적인 구조와 기능을 회복시키지만, 그렇지 못한 경우에는 만성 염증과 같은 질병 상태로 진행되기도 한다. 또한, 꽃가루와 같이 무해한 물질이나 천식, 류마티스성 관절염과 같은 자가면역반응에 의해 부적절하게 염증이 촉발되는 경우에는 방어반응 자체가 오히려 조직을 손상시킴으로 항염증제가 필요하게 된다. 이러한 염증 질환을 치료하기 위한 가장 일반적인 항염증제는 크게 스테로이드성 및 비스테로이드성 항염증제로 구분되며, 이중 대부분의 합성 항염증제는 주작용 이외에 여러 가지 부작용을 수반하는 경우가 많으므로 효과가 탁월하며 부작용이 적은 천연 항염증제의 개발이 절실히 요구되고 있는 실정이다. Inflammation is the expression of a normal and protective in vivo defense mechanism that appears locally against tissue damage caused by physical trauma, harmful chemicals, infection by microorganisms, or irritants in in vivo metabolites. In a normal case, the living body neutralizes or removes the onset factor through an inflammatory reaction and regenerates damaged tissue to restore normal structure and function, but otherwise, it may progress to a disease state such as chronic inflammation. In addition, when inflammation is inappropriately triggered by a harmless substance such as pollen or an autoimmune reaction such as asthma or rheumatoid arthritis, the defense reaction itself rather damages the tissue, so an anti-inflammatory agent is needed. The most common anti-inflammatory agents for the treatment of such inflammatory diseases are largely divided into steroidal and non-steroidal anti-inflammatory agents, and most synthetic anti-inflammatory agents often have various side effects in addition to their main action, so they are excellent in effect and natural with few side effects. There is an urgent need for the development of anti-inflammatory drugs.
생체에서 염증의 발생에는 다양한 생화학적인 현상이 관여하고 있다. 염증반응은 체내에서 발생한 산화스트레스에 의해 촉진되는데, 산화스트레스는 세포사멸뿐만 아니라 퇴행성 질환을 일으키는 특정세포의 유전자 발현을 증가시켜 염증반응을 개시하거나 악화시킨다. 대식세포(macrophage)는 동물 체내 모든 조직에 분포하며 인체 내에서 선천적 면역반응을 담당하는 면역세포로, 인체 면역체계에서 중요한 역할을 하는 백혈구이다. 외부의 자극으로 인해 활성화된 대식세포는 염증매개물질 분비를 통해 염증반응을 유발함으로써 천식, 기관지염, 관절염, 다발성경화증, 동맥경화증, 뇌졸중, 알츠하이머병이나 파킨슨병과 같은 퇴행성뇌질환 및 바이러스 감염으로 인한 염증질환 등을 유발하고, 질환을 악화시키게 된다. 내독소의 하나인 리포폴리사카라이드(lipopolysaccharide; LPS)는 대식세포에서 인터루킨-6 (IL-6), MCP-1와 같은 염증성(pro-inflammatory) 사이토카인을 증가시키며, 질소산화물(NO) 등의 염증매개물질을 분비한다. 이들 염증성 사이토카인과 염증매개물질의 생성과 생성에 관여하는 효소의 발현을 조절할 수 있는 물질이 염증질환의 예방 및 치료제로서 주목을 받고 있다.Various biochemical phenomena are involved in the occurrence of inflammation in the living body. The inflammatory response is promoted by oxidative stress generated in the body, which initiates or worsens the inflammatory response by increasing gene expression of specific cells that cause not only apoptosis but also degenerative diseases. Macrophage is an immune cell that is distributed in all tissues of an animal and is responsible for the innate immune response in the human body, and is a white blood cell that plays an important role in the human immune system. Macrophages activated by external stimuli trigger an inflammatory response through the secretion of inflammatory mediators, thereby causing asthma, bronchitis, arthritis, multiple sclerosis, arteriosclerosis, stroke, degenerative brain diseases such as Alzheimer's disease or Parkinson's disease, and inflammation caused by viral infection. It causes disease and makes the disease worse. One of the endotoxins, lipopolysaccharide (LPS), increases pro-inflammatory cytokines such as interleukin-6 (IL-6) and MCP-1 in macrophages, nitric oxide (NO), etc. secretes inflammatory mediators of Substances capable of regulating the expression of enzymes involved in the production and production of these inflammatory cytokines and inflammatory mediators are attracting attention as preventive and therapeutic agents for inflammatory diseases.
이에 본 발명자들은 종래 기술의 요구에 부응하기 위한 연구를 지속한 결과, 산양삼 전초를 락토바실러스 브레비스 BMK484 균주와 락토바실러스 플란타륨 P1201 균주로 복합발효하여 제조한 활성산양삼은 고농도로 사용시에도 세포독성 없이 염증성 사이토카인과 질소산화물(NO)을 억제함을 확인하고 본 발명을 완성하였다.Accordingly, the present inventors continued research to meet the needs of the prior art, and as a result, active wild ginseng produced by complex fermentation of wild ginseng outpost with Lactobacillus brevis BMK484 strain and Lactobacillus plantarum P1201 strain without cytotoxicity even when used at high concentrations It was confirmed that it inhibits inflammatory cytokines and nitrogen oxides (NO) and completed the present invention.
따라서 본 발명의 목적은 활성산양삼의 추출물을 유효성분으로 포함하여 고농도에서도 세포독성이 없이 증진된 염증억제 활성을 갖는 항염증 조성물을 제공하는 것이다. Accordingly, it is an object of the present invention to provide an anti-inflammatory composition having enhanced anti-inflammatory activity without cytotoxicity even at high concentrations, including the extract of active wild ginseng as an active ingredient.
본 발명의 또 다른 목적은 고농도에서도 세포독성이 없이 증진된 염증억제 활성을 갖는 항염증 조성물의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing an anti-inflammatory composition having enhanced anti-inflammatory activity without cytotoxicity even at high concentrations.
본 발명의 또 다른 목적은 상기 항염증 조성물을 포함하는 염증 예방 및 개선용 기능성 식품을 제공하는 것이다. Another object of the present invention is to provide a functional food for preventing and improving inflammation comprising the anti-inflammatory composition.
본 발명의 또 다른 목적은 상기 항염증 조성물을 포함하는 피부염증 예방 및 개선용 기능성 화장품을 제공하는 것이다. Another object of the present invention is to provide a functional cosmetic for preventing and improving skin inflammation comprising the anti-inflammatory composition.
상기 목적을 달성하기 위하여, 본 발명은 활성산양삼 추출물을 유효성분으로 포함하고, 고농도에서도 세포독성이 없는 증진된 염증 억제활성을 갖는 항염증 조성물을 제공한다. In order to achieve the above object, the present invention provides an anti-inflammatory composition comprising an active wild ginseng extract as an active ingredient, and having enhanced anti-inflammatory activity without cytotoxicity even at high concentrations.
본 발명에서 '항염증' 또는 '염증억제'는 인체에서의 염증반응을 억제하는 것을 의미한다.In the present invention, 'anti-inflammatory' or 'inflammation inhibition' means suppressing an inflammatory response in the human body.
본 발명에서 '활성산양삼'은 인체 흡수형 진세노사이드 Rd2, Rg3, F2, Rh2와 컴파운드 케이, 클로르제닉산 및 쿼르세틴, 페놀릭스, 플라보노이드스 및 갈변물질의 함량이 증진되도록 산양삼을 가공된 것을 의미한다. In the present invention, 'active wild ginseng' refers to ginsenoside Rd2, Rg3, F2, Rh2, compound K, chlorgenic acid and quercetin, phenolics, flavonoids, and processed wild ginseng to increase the content of browning substances. it means.
본 발명에서 '산양삼'은 산양삼 또는 산양새싹삼을 의미한다. In the present invention, 'mountainous ginseng' means wild ginseng or wild ginseng sprout.
본 발명에서 '산양새싹삼'은 해발 500m 이상의 산지에서 재배된 3년근 이상의 5월 ~ 7월 사이 산양삼 혹은 2년근 이상 산양삼의 묘삼을 인공 환경에서 90일 이내로 수경 혹은 토경 재배한 것를 의미한다. 한편 산양새싹삼은 재배 동안에는 농약이 전혀 사용되지 않은 친환경 농산물로 수경 혹은 토경 재배, 연중생산 가능 및 재배 기간의 단축으로 인해 종래에 이용되는 산양삼보다 낮은 가격을 형성하고 있어 산양삼 보다 장점이 있다. In the present invention, 'goat sprout ginseng' refers to cultivating wild ginseng seedlings of 3 years or more between May and July, or 2 years or more of mountain ginseng grown in a mountainous area over 500 m above sea level, hydroponically or soil culture within 90 days in an artificial environment. On the other hand, goat sprout ginseng is an eco-friendly agricultural product that does not use any pesticides during cultivation. It has an advantage over wild ginseng because it forms a lower price than the conventionally used wild ginseng due to hydroponics or soil cultivation, year-round production possible, and shortened cultivation period.
활성산양삼 추출물은 산양삼 전초를 증숙과 고온숙성을 1~5회 반복한 후 복합발효하고 추출하여 제조할 수 있다.Active wild ginseng extract can be prepared by repeating steam and high-
본 발명에서 전초(全草)는 뿌리, 잎, 줄기의 전체를 의미한다. In the present invention, the whole plant means the entire root, leaf, and stem.
본 발명에서 '증숙'은 100℃에서 30~60분간 수증기로 찌는 것을 의미한다. 증숙시간이 30분 미만인 경우 충분한 증숙이 진행되지 않아 숙성과 같은 후속 단계가 원활하지 않을 수 있고 잡균의 오염이 발생될 수 있으며, 60분 초과하여 증숙될 경우는 오랜 열처리로 산양삼 전초의 생리활성성분이 파괴될 수 있다.In the present invention, 'steaming' means steaming with steam at 100° C. for 30 to 60 minutes. If the steaming time is less than 30 minutes, sufficient steaming does not proceed, so subsequent steps such as ripening may not be smooth, and contamination of various bacteria may occur. This can be destroyed.
본 발명에서 '고온숙성'은 70~90℃에서 2~4일간 유지하는 것을 의미한다. 숙성온도가 70℃ 미만이거나 숙성기간이 2일 미만인 경우 숙성이 원활히 이루어지지 않으며, 숙성온도가 90℃ 초과하거나 숙성기간이 4일 초과의 경우 생산된 생리활성물질이 분해되어 함량이 감소될 수 있다.In the present invention, 'high temperature aging' means maintaining for 2 to 4 days at 70-90°C. If the aging temperature is less than 70℃ or the aging period is less than 2 days, the aging is not performed smoothly. .
본 발명에서 '복합발효'는 증숙과 고온숙성된 산양삼 전초를 락토바실러스 브레비스 BMK484 균주와 락토바실러스 플란타륨 P1201 균주의 복합 종균으로 발효하는 것으로 수행된다.In the present invention, 'complex fermentation' is performed by fermenting steamed and high-temperature aged wild ginseng whole plant with a complex seed of Lactobacillus brevis BMK484 strain and Lactobacillus plantarum P1201 strain.
본 발명에서 복합 종균을 구성하는 하나의 균주인 락토바실러스 브레비스 BMK484 균주는 본 발명자들이 김치로부터 분리/동정하여 국립농업과학원 농업유전자원센터(KACC)에 2016년 12월 12일에 기탁한 균주(수탁번호 KACC92157P)로, 가바(GABA)의 생산성 및 진세노사이드 컴파운드 케이(compound K) 생산성이 탁월하다 (특허출원 10-2017-0152033).In the present invention, the Lactobacillus brevis BMK484 strain, which is one strain constituting the complex seed strain in the present invention, was isolated/identified from kimchi, and deposited on December 12, 2016 at the Agricultural Genetic Resources Center (KACC) of the National Academy of Agricultural Science (KACC). No. KACC92157P), the productivity of GABA and the ginsenoside compound K productivity are excellent (Patent Application 10-2017-0152033).
본 발명에서 복합종균을 구성하는 또 다른 하나의 균주인 락토바실러스 플란타륨 P1201 균주는 본 발명자들이 발효식품으로부터 분리/동정하여 국립농업과학원 농업유전자원센터(KACC)에 2013년 7월 19일에 기탁한 균주(수탁번호 KACC91848P)로서, 생균제제능이 우수하고 생리활성물질 생산성이 우수한 특성을 갖는다 (등록특허 10-154418호).Another strain constituting the complex spawn in the present invention, the Lactobacillus plantarum P1201 strain, was isolated/identified from the fermented food by the present inventors and transferred to the Agricultural Genetic Resources Center (KACC) of the National Academy of Agricultural Sciences on July 19, 2013 As the deposited strain (Accession No. KACC91848P), it has excellent probiotic ability and excellent productivity of bioactive substances (Registration Patent No. 10-154418).
본 발명의 복합종균을 구성하는 락토바실러스 브레비스 BMK484 균주와 락토바실러스 플란타륨 P1201 균주는 발효 특성이 상호 보완되어 상승작용을 나타낸다. 락토바실러스 브레비스 BMK484 균주는 이형유산발효 (hetero-lactic acid fermentation) 유산균으로 단독 발효 시 유산 생성이 충분하지 않고, 락토바실러스 플란타륨 P1201 균주는 동형유산발효(homo-lactic acid fermentation) 유산균으로 단독 발효 시 유산이 과다하게 생성되는데, 놀랍게도 산양삼 전초를 이들 두 가지 종균을 함께 사용하여 복합발효시 양 균주의 발효 특성이 상호 보완되고 인체 흡수형 진세노사이드함량이 현저히 증진되었다.The Lactobacillus brevis BMK484 strain and the Lactobacillus plantarum P1201 strain constituting the composite seed strain of the present invention exhibit a synergistic effect by complementing the fermentation characteristics. Lactobacillus brevis BMK484 strain is hetero-lactic acid fermentation lactic acid bacteria, and lactic acid production is not sufficient during single fermentation, and Lactobacillus plantarium P1201 strain is single fermentation with homo-lactic acid fermentation lactic acid bacteria. Surprisingly, when wild ginseng outpost was used together with these two spawns, the fermentation characteristics of both strains were complemented and the content of ginsenosides absorbed by the human body was significantly improved during complex fermentation.
본 발명에서 복합 종균은 락토바실러스 브레비스 BMK484 균주와 락토바실러스 플란타륨 P1201 균주 각각 별도의 배양액으로서 첨가될 수도 있으며, 혼합 배양된 혼합 배양액으로서 첨가될 수도 있다. 복합종균에서 상기 2종의 균주의 혼합 비율은 3 : 1 ~ 1 : 3(v/v)으로 혼합될 수 있다.In the present invention, the complex spawn may be added as a separate culture medium for each of the Lactobacillus brevis BMK484 strain and the Lactobacillus plantarum P1201 strain, or may be added as a mixed culture medium. The mixing ratio of the two types of strains in the composite seed culture may be 3 : 1 to 1 : 3 (v/v).
발효는 상기 복합 종균의 배양액을 증숙과 고온숙성된 산양삼 전초에 2~10% (v/w) 농도로 접종하여 25~40℃에서 2~7일간 발효시키는 것으로 수행될 수 있다. 복합 종균 접종량이 2%(v/w) 미만일 경우에는 발효 속도가 지연될 수 있고 10%(v/w) 초과 시에는 균체 증식 속도가 빨라 활성 진세노사이드의 전환율이 낮으며 신맛이 등이 과다할 수 있으며, 발효 온도가 25℃ 미만일 경우 발효기간이 길어져 잡균의 오염을 초래하고 40℃를 초과할 경우에는 균주의 생육이 정지될 수 있고, 발효 기간이 2일 미만일 경우 발효가 충분하지 않아 생리활성물질 등의 생성이 저조하게 될 수 있으며, 7일을 초과한 경우는 과발효에 의해 생리활성물질이 분해될 수 있다.Fermentation can be carried out by inoculating the culture medium of the complex seed strain at a concentration of 2 to 10% (v/w) in the steamed and high temperature aged wild ginseng outpost and fermenting it at 25 to 40° C. for 2 to 7 days. If the complex seed inoculation amount is less than 2% (v/w), the fermentation rate may be delayed. If the fermentation temperature is less than 25 ℃, the fermentation period is prolonged, causing contamination of various bacteria, and if it exceeds 40 ℃, the growth of the strain may be stopped. The production of active substances, etc. may be low, and if it exceeds 7 days, physiologically active substances may be decomposed by overfermentation.
또한 발효시, 필요에 따라서, 증숙과 고온숙성된 산양삼 전초에 물을 첨가하여 수분이 40~50% (v/w)가 되도록 조정 및/또는 살균한 후, 복합종균을 접종하여 발효할 수 있다. 수분이 함량이 상기 범위일 때 발효 효율이 가장 높다. 40% 미만의 수분함량일 경우 미생물이 생존 혹은 생육을 위한 수분이 충분하지 않아 원활한 발효가 진행되지 않고 50% 초과의 경우 발효 종료 후 건조 시간과 비용이 많이 들 수 있으며, 살균 처리를 하지 않을 경우 잡균의 오염 등으로 원활한 발효가 진행되지 않을 수 있다.In addition, during fermentation, if necessary, water is added to the steamed and high-temperature aged wild ginseng outpost to adjust and/or sterilize so that the moisture content is 40-50% (v/w), and then it can be fermented by inoculating a complex seed strain. . Fermentation efficiency is highest when the moisture content is within the above range. If the moisture content is less than 40%, smooth fermentation does not proceed because there is not enough moisture for the survival or growth of microorganisms. Fermentation may not proceed smoothly due to contamination of various bacteria.
본 발명에서 '추출'은 제조된 활성산양삼을 에탄올(주정)로 추출하는 것을 의미한다. 바람직하게는 50~70% 에탄올, 가장 바람직하게는 50% 에탄올을 사용한다. In the present invention, 'extraction' means extracting the prepared active wild ginseng with ethanol (alcohol). Preferably 50 to 70% ethanol, most preferably 50% ethanol is used.
본 발명의 활성산양삼 추출물은, 필요에 따라서, 여과하여 농축한다. 바람직하게는 활성산양삼의 중량의 1 ~ 3배로 농축한다. The active wild ginseng extract of the present invention, if necessary, is filtered and concentrated. Preferably, it is concentrated to 1 to 3 times the weight of the active wild ginseng.
활성산양삼의 중량의 1배 미만으로 농축 시 농축시간이 오래 걸려 진세노사이드 등의 생리활성물질 파괴될 수 있고 비용이 많이 발생하는 문제점이 있으며, 3배 초과로 농축 시 진세노사이드 등의 생리활성물질 충분히 함유되어 있지 않아 제품 제조 시 문제가 발생할 수 있다. 바람직하게는 2배로 농축하여 사용한다.When concentrated to less than 1 times the weight of active wild ginseng, it takes a long time to concentrate, so there is a problem that bioactive substances such as ginsenosides can be destroyed and cost is high. It may cause problems in product manufacturing because it does not contain enough substances. Preferably, it is used after concentration to 2 times.
본 발명에 따른 항염증 조성물은 0.2 brix 이상의 고농도 사용 시에도 세포독성 없고 염증성 사이토카인 IL-6, MCP-1와 질소산화물의 생성을 억제하여 현저히 증진된 항염증 활성 활성을 갖는다 (시험예 5 및 6, 도 3~도 6).The anti-inflammatory composition according to the present invention has no cytotoxicity even when used at a high concentration of 0.2 brix or more, and has significantly enhanced anti-inflammatory activity by inhibiting the production of inflammatory cytokines IL-6, MCP-1 and nitric oxide (Test Example 5 and 6, Figures 3-6).
본 발명에 따른 항염증 조성물은 또한 인체 흡수형 진세노사이드 Rd2 4.63 mg/g 이상, Rg3 2.90 mg/g 이상, F2 4.09 mg/g 이상, Rh2 0.8 mg/g 이상과 컴파운드 케이 6.30 mg/g 이상으로 함유하고, 클로르제닉산 0.47 mg/ml 이상 및 쿼르세틴 0.55 mg/ml 이상을 함유한다. 또한 현저히 증진된 페놀릭스, 플라보노이드 및 갈변물질을 함유한다 (표 3 및 표 4, 도 1).The anti-inflammatory composition according to the present invention is also human-absorbable ginsenoside Rd2 4.63 mg/g or more, Rg3 2.90 mg/g or more, F2 4.09 mg/g or more, Rh2 0.8 mg/g or more, and Compound K 6.30 mg/g or more It contains 0.47 mg/ml or more of chlorgenic acid and 0.55 mg/ml or more of quercetin. It also contains markedly enhanced phenolics, flavonoids and browning substances (Tables 3 and 4, Fig. 1).
본 발명의 또 다른 목적에 따라서, 본 발명은 According to another object of the present invention, the present invention provides
ⅰ) 산양삼 전초를 100℃에서 30~60분간 증숙과 증숙된 산양삼을 70~90℃에서 2~4일 고온숙성을 1~5회 반복한 후 락토바실러스 브레비스 BMK484 균주와 락토바실러스 플란타륨 P1201 균주의 복합 종균으로 발효하여 활성산양삼을 제조하는 단계; i) After steaming wild ginseng outpost at 100℃ for 30~60 minutes and high temperature aging of steamed wild ginseng at 70~90℃ for 2~4
ⅱ) 활성산양삼을 건조하여 분말화하는 단계; 및ii) drying and pulverizing active wild ginseng; and
ⅲ) 활성산양삼 분말에 50~70% 에탄올을 첨가하여 추출하는 단계iii) extracting by adding 50-70% ethanol to the active wild ginseng powder
를 포함하는 항염증 조성물의 제조방법을 제공한다. It provides a method for preparing an anti-inflammatory composition comprising a.
본 발명의 제조방법은, 필요에 따라서, 단계 ⅲ)으로부터의 활성산양삼 추출물을 여과하여 활성산양삼 분말 중량 대비 1~3배로 농축하는 단계를 추가로 포함할 수 있다. The manufacturing method of the present invention may further include, if necessary, filtering the active wild ginseng extract from step iii) and concentrating it to 1 to 3 times the weight of the active wild ginseng powder.
단계 ⅰ): Step i): 활성산양삼Active wild ginseng 제조 Produce
활성산양삼 제조공정은 상기에 기재된 바와 같다. The active wild ginseng manufacturing process is as described above.
본 발명에서 산양삼 전초 또는 산양새싹삼 전초를 흐르는 물에 3회 세척하고 물기를 제거한 후 사용한다. In the present invention, the wild ginseng or wild ginseng sprouts are washed three times in running water and dried after use.
단계 ⅱ) 건조 및 step ii) drying and 분말화powdered
단계 ⅰ)로부터의 활성산양삼을 48~52℃의 열풍으로 2~3일간 건조시킨 후, 100 메쉬 이상으로 분쇄하여 분말화한다. After drying the active wild ginseng from step i) for 2-3 days with hot air at 48-52°C, it is pulverized to 100 mesh or more.
상기와 같이 건조하여 분말화하면 인체 흡수형 진세노사이드 추출에 효율적이다.When dried and powdered as described above, it is effective for human body absorption type ginsenoside extraction.
단계 ⅲ) 추출step iii) extraction
활성산양삼 분말에 에탄올(주정) 10~20배(w/v)를 첨가하여 30~60℃에서 4~8시간 동안 추출하여 활성산양삼 추출액을 제조한다. Ethanol (alcohol) 10 to 20 times (w/v) is added to the active wild ginseng powder and extracted at 30 to 60° C. for 4 to 8 hours to prepare an active wild ginseng extract.
에탄올은 바람직하게는 50~70% 농도로, 가장 바람직하게는 50% 농도로 사용한다 (표 2). Ethanol is preferably used at a concentration of 50 to 70%, most preferably at a concentration of 50% (Table 2).
ⅳ) 여과 및 농축iv) Filtration and Concentration
활성산양삼 추출액을 여과한 후 활성산양삼 분말 중량 대비 1~3배로 농축한다. After filtering the active wild ginseng extract, it is concentrated to 1 to 3 times the weight of the active wild ginseng powder.
여과는 여과필터를 사용하고, 농축은 통상의 감압농축기를 사용하여 수행할 수 있다. Filtration may be performed using a filtration filter, and concentration may be performed using a conventional vacuum concentrator.
활성산양삼의 중량의 2배로 농축 시 진세노사이드 등의 생리활성물질 충분히 함유되어 있고 농축시간 등의 비용을 줄일 수 있어 가장 바람직하다. When concentrated to twice the weight of active wild ginseng, it is most preferable because it contains sufficient physiologically active substances such as ginsenosides and can reduce costs such as concentration time.
본 발명의 또 다른 목적에 따라서, 본 발명은 상기 항염증 조성물을 포함하는 염증 예방 및 개선용 기능성 식품을 제공한다.According to another object of the present invention, the present invention provides a functional food for preventing and improving inflammation comprising the anti-inflammatory composition.
본 발명의 조성물은 하루에 체중 1 ㎏당 0.1~500 ㎎, 바람직하게는 1~100 ㎎의 양으로 섭취되도록 상기 기능성 식품 등에 포함될 수 있다.The composition of the present invention may be included in the functional food and the like so as to be ingested in an amount of 0.1 to 500 mg, preferably 1 to 100 mg per 1 kg of body weight per day.
본 발명의 또 다른 목적에 따라서, 본 발명은 상기 항염증 조성물을 포함하는 피부염증 예방 및 개선용 기능성 화장품을 제공한다. According to another object of the present invention, the present invention provides a functional cosmetic for preventing and improving skin inflammation comprising the anti-inflammatory composition.
본 발명의 기능성 화장품은 마스크팩, 스킨, 크림, 로션 등의 형태의 제품으로 제조될 수 있다.The functional cosmetic of the present invention may be manufactured in the form of a mask pack, skin, cream, lotion, or the like.
본 발명에 따른 항염증 조성물은 고농도로 사용시에도 세포독성이 전혀 없고 질소산화물(NO) 생성 억제활성, 염증성 사이토카인 IL-6 및 MCP-1 억제활성을 통하여 현저히 증진된 항염증 효과를 나타내고, 인체 흡수형 진세노사이드 Rd2, Rg3, F2, Rh2와 컴파운드 케이, 클로르제닉산과 쿼르세틴, 페놀릭스, 플라보노이드스 및 갈변물질 함량도 현저히 증진되어 있어, 염증관련 질환의 예방 및 개선용 기능성 식품 및 화장품의 소재로 사용될 수 있다. The anti-inflammatory composition according to the present invention has no cytotoxicity even when used at a high concentration and exhibits a significantly enhanced anti-inflammatory effect through nitric oxide (NO) production inhibitory activity, inflammatory cytokines IL-6 and MCP-1 inhibitory activity, Absorption-type ginsenosides Rd2, Rg3, F2, Rh2, compound K, chlorgenic acid, quercetin, phenolics, flavonoids and browning substances content are also significantly increased. material can be used.
도 1은 본 발명에 따른 항염증 조성물의 진세노사이드 화합물 HPLC 크로마토그램을 나타낸 것이다. 도 1a는 진세노사이드 표준물질 21종의 HPLC 크로마토그램이며, 도 1b는 비교예 (산양삼 전초 추출물)의 진세노사이드 화합물 HPLC 크로마토그램이며, 도 1c는 실시예 (활성산양삼 추출물)의 진세노사이드 화합물 HPLC 크로마토그램이다.
도 2는 본 발명에 따른 항염증 조성물의 페놀릭스, 플라보노이드 및 갈변물질의 함량을 나타낸 그래프이다. 도 2a는 총 페놀릭스 함량을 나타낸 것이며, 도 2b는 총 플라보노이드스 함량을 나타낸 것이며, 도 3c는 갈변물질 함량을 나타낸 것이다.
도 3은 본 발명에 따른 항염증 조성물의 세포독성 시험결과를 나타낸 그래프이다. 도 3a는 비교예 (산양삼 전초 추출물)의 세포생존율을 나타낸 그래프이며, 도 3b는 실시예 (활성산양삼 추출물)의 세포생존율을 나타낸 그래프이다. CTL: 무처리구, LPS: LPS 처리구, 0.025, 0.05, 0.1, 0.2, 0.3: 시료 농도(brix)
도 4는 본 발명에 따른 항염증 조성물의 NO 생성 저해활성을 나타낸 그래프이다. 도 4a는 비교예 (산양삼 전초 추출물)의 NO 생성 저해활성을 나타낸 그래프이며, 도 4b는 실시예 (활성산양삼 추출물)의 NO 생성 저해활성을 나타낸 그래프이다. CTL: 무처리구, LPS: LPS 처리구, 0.025, 0.05, 0.1, 0.2, 0.3: 시료 농도(brix)
도 5는 본 발명에 따른 항염증 조성물의 염증성 사이토카인 IL-6 억제활성을 나타낸 그래프이다. 도 5a는 비교예 (산양삼 전초 추출물)의 염증성 사이토카인 IL-6 억제활성을 나타낸 그래프이며, 도 5b는 실시예 (활성산양삼 추출물)의 염증성 사이토카인 IL-6 억제활성을 나타낸 그래프이다. CTL: 무처리구, LPS: LPS 처리구, 0.025, 0.05, 0.1, 0.2, 0.3: 시료 농도(brix)
도 6은 본 발명에 따른 항염증 조성물의 염증성 사이토카인 MCP-1 억제활성을 나타낸 그래프이다. 도 6a는 비교예 (산양삼 전초 추출물)의 염증성 사이토카인 MCP-1 억제활성을 나타낸 그래프이며, 도 6b는 실시예 (활성산양삼 추출물)의 염증성 사이토카인 MCP-1 억제활성을 나타낸 그래프이다. CTL: 무처리구, LPS: LPS 처리구, 0.025, 0.05, 0.1, 0.2, 0.3: 시료 농도(brix) 1 shows the HPLC chromatogram of the ginsenoside compound of the anti-inflammatory composition according to the present invention. Figure 1a is an HPLC chromatogram of 21 kinds of ginsenoside standard materials, Figure 1b is a HPLC chromatogram of a ginsenoside compound of a comparative example (mountainous ginseng extract), Figure 1c is a ginsenoside of an example (active wild ginseng extract) Compound HPLC chromatogram.
2 is a graph showing the content of phenolics, flavonoids and browning substances in the anti-inflammatory composition according to the present invention. Figure 2a shows the total phenolics content, Figure 2b shows the total flavonoids content, Figure 3c shows the browning material content.
3 is a graph showing the cytotoxicity test results of the anti-inflammatory composition according to the present invention. Figure 3a is a graph showing the cell viability of the comparative example (sanyangsam whole plant extract), Figure 3b is a graph showing the cell viability of the example (active wild ginseng extract). CTL: no treatment group, LPS: LPS treatment group, 0.025, 0.05, 0.1, 0.2, 0.3: sample concentration (brix)
4 is a graph showing the NO production inhibitory activity of the anti-inflammatory composition according to the present invention. Figure 4a is a graph showing the NO production inhibitory activity of the comparative example (sanyangsam whole plant extract), Figure 4b is a graph showing the NO production inhibitory activity of the example (active wild ginseng extract). CTL: no treatment group, LPS: LPS treatment group, 0.025, 0.05, 0.1, 0.2, 0.3: sample concentration (brix)
5 is a graph showing the inflammatory cytokine IL-6 inhibitory activity of the anti-inflammatory composition according to the present invention. Figure 5a is a graph showing the inflammatory cytokine IL-6 inhibitory activity of Comparative Example (sanyangsam whole plant extract), Figure 5b is a graph showing the inflammatory cytokine IL-6 inhibitory activity of the Example (active wild ginseng extract). CTL: no treatment group, LPS: LPS treatment group, 0.025, 0.05, 0.1, 0.2, 0.3: sample concentration (brix)
6 is a graph showing the inflammatory cytokine MCP-1 inhibitory activity of the anti-inflammatory composition according to the present invention. Figure 6a is a graph showing the inflammatory cytokine MCP-1 inhibitory activity of the comparative example (mountainous ginseng extract), and Figure 6b is a graph showing the inflammatory cytokine MCP-1 inhibitory activity of the example (active wild ginseng extract). CTL: no treatment group, LPS: LPS treatment group, 0.025, 0.05, 0.1, 0.2, 0.3: sample concentration (brix)
다음의 실시예들에 의해 본 발명이 더 상세히 설명된다. 이들 실시예는 본 발명을 예시하기 위한 것이며, 본 발명의 범위가 이들에 의해 제한되어서는 안된다.The present invention is further illustrated by the following examples. These examples are for the purpose of illustrating the present invention, and the scope of the present invention should not be limited thereto.
제조예 1. 활성산양삼 제조Preparation Example 1. Preparation of Active Wild Ginseng
3년근 이상 산양삼 전초 1 kg을 흐르는 물에 3회 세척하고 완전히 물기를 제거한 후, 증자기에 적당량의 물과 함께 100℃에서 1시간 쪄서 증숙한 후 건조 채반에 담고 수분이 증발되게 하면서 75℃에서 3일간 숙성시키는 과정 (증숙과 숙성)을 3회 진행하여 증숙 및 숙성된 산양삼 전초를 제조하였다.After washing 1 kg of wild ginseng from 3 years old or more under running
제조된 증숙 및 숙성된 산양삼 전초 500 g을 발효용기에 넣고 2배의 정세수(1 L)를 첨가하고 1시간 동안 수화시켜 수분 함량을 약 50% 정도로 되게 조정한 후, 120℃에서 30분간 살균하고, 락토바실러스 브레비스 BMK484 및 락토바실러스 플란타륨 P1201 균주를 각각 맥아엑기스 액체배지에서 종균으로 배양하며 1:1 비율로 혼합한 복합종균을 5% (v/w)로 접종하고 30℃에서 7일간 발효시켜 활성산양삼을 제조하였다.Put 500 g of the prepared steamed and aged wild ginseng whole plant into a fermentation vessel, add 2 times of purified water (1 L), hydrate for 1 hour, adjust the moisture content to about 50%, and then sterilize at 120 ° C for 30 minutes. , Lactobacillus brevis BMK484 and Lactobacillus plantarum P1201 strains were each cultured as a starter in a malt extract liquid medium. to prepare active wild ginseng.
제조예 2. 활성산양삼 추출물 제조Preparation Example 2. Preparation of active wild ginseng extract
제조예 1에서 제조된 활성산양삼을 50℃에서 2일간 열풍 건조하고 분쇄기로 분쇄하여 100 메쉬 정도의 분말로 제조하였다.The active wild ginseng prepared in Preparation Example 1 was dried with hot air at 50° C. for 2 days and pulverized with a grinder to prepare a powder of about 100 mesh.
활성산양삼 건조 분말 20 g에 50% 주정을 20배인 400 ml을 가한 후 40℃에서 5시간 교반한 후 여과하여 활성산양삼 추출액을 제조하였다. 이 추출액을 감압농축기를 이용하여 건조 분말 대비 2배인 40 ml로 농축하여 활성산양삼 추출물(항염증 조성물)을 완성하였다 (실시예). To 20 g of dried active wild ginseng powder, 400 ml of 20
비교를 위하여 산양삼 전초 원료를 상기와 동일한 방식으로 열풍건조하여 분쇄한분말에 대해서 상기와 동일하게 추출, 농축하여 산양삼 전초 추출물을 제조하였가 (비교예).For comparison, the raw material of wild ginseng outpost was extracted and concentrated in the same manner as above for the powder pulverized by hot air drying in the same manner as above to prepare a wild ginseng outpost extract (Comparative Example).
<이화학적 특성><Physical and chemical properties>
제조된 실시예의 활성산양삼 추출물과 비교예의 산양삼 전초 추출물의 pH를 pH 미터기를 사용하여 측정하였고, 총산도는 중화적정법을 통해 수행하여 젖산으로 환산하여 표기하였고, 가용성 고형분은 당도계를 이용하여 측정하여 표 1에 나타냈다.The pH of the active wild ginseng extract of the prepared Example and the wild ginseng extract of the comparative example was measured using a pH meter, and the total acidity was expressed in terms of lactic acid by performing a neutralization titration method, and the soluble solid content was measured using a saccharometer. shown in 1.
상기 표 1에 나타낸 바에 의하면, 본 발명에 따라 제조된 활성산양삼 추출물(실시예, 항염증 조성물)은 비교예 (산양삼 전초 추출물) 보다 pH는 감소하였고 총산도는 증가하여서, 보존성과 기호성이 향상된 것으로 판단된다. 가용성 고형분 함량은 큰 차이는 없었다.As shown in Table 1, the active wild ginseng extract (Example, anti-inflammatory composition) prepared according to the present invention had a lower pH and an increased total acidity than the comparative example (Wild ginseng outpost extract), so that preservation and palatability were improved. is judged There was no significant difference in soluble solids content.
시험예 1: 활성산양삼 최적 추출 조건 확립Test Example 1: Establishment of optimal extraction conditions for active wild ginseng
제조예 1에서 제조된 활성산양삼의 인체 흡수형 진세노사이드의 최적 추출 용매(주정; 에탄올) 농도를 도출하기 위하여 분석하였다.Analysis was performed to derive the optimal concentration of the extraction solvent (ethanol; ethanol) of ginsenoside for human absorption of the active wild ginseng prepared in Preparation Example 1.
제조예 1에서 제조된 활성산양삼을 50℃에서 2일간 열풍 건조하고 분쇄기로 분쇄하여 100 메쉬 정도로 분말화한 후, 분말 5 g에 각각 주정 농도를 0%, 25%, 50%, 75% 및 95%로 하여 20배인 100 ml씩 첨가한 후 40℃에서 5시간 교반한 후 여과하여 주정 농도별 활성산양삼 추출액을 제조하였다. 이 추출액을 감압농축기를 이용하여 활성산양삼 분말 중량 대비 2배인 10 ml로 농축하여 주정농도별 활성산양삼 추출물을 제조하였다. 이들 활성산양삼 추출물 2 ml씩 0.45 ㎛ 멤브레인 필터로 여과한 후 진세노사이드 분석시료로 사용하였다.The active wild ginseng prepared in Preparation Example 1 was dried with hot air at 50° C. for 2 days, pulverized with a grinder, and powdered to about 100 mesh, and then the alcohol concentration was added to 5 g of the powder by 0%, 25%, 50%, 75% and 95, respectively. %, 20 times, 100 ml each was added, stirred at 40° C. for 5 hours, and filtered to prepare an active wild ginseng extract for each alcohol concentration. This extract was concentrated to 10 ml, twice the weight of the active wild ginseng powder, using a reduced pressure concentrator to prepare an active wild ginseng extract for each alcohol concentration. 2 ml of each of these active wild ginseng extracts were filtered through a 0.45 μm membrane filter and used as a ginsenoside analysis sample.
<진세노사이드 분석><Ginsenoside analysis>
상기 분석시료에 대해서 기능성식품 분석법에 기술된 방법을 변형하여 고압액체크로마토그래피(HPLC, high press liquid chromatograph)로 분석하였다. 진세노사이드 분석에 사용한 분석 컬럼은 TSKgel ODS-100Z을 사용하여 시료 주입량 10 ㎕, 온도는 30℃, 측정파장은 203 nm, 유속은 1.0 mL/min으로 하였고 이동상으로는 A용액은 HPLC water, B용액은 아세토니트릴을 사용하였다. HPLC 분석 조건은 이동상 용액은 0분에는 A용액 81% : B용액 19%로 흘려주고, 15분에는 A용액 80% : B용액 20%로 흘려주고, 40분에는 A용액 77% : B용액 23%, 42분에는 A용액 70% : B용액 30%, 75분에는 A용액 65% : B용액 35%, 80분에는 A용액 30% : B용액 70%, 90분에는 A용액 10% : B용액 90%로 이동상을 흘려주었다. 분석된 진세노사이드 함량은 표 2 에 나타내었다.The analysis sample was analyzed by high pressure liquid chromatography (HPLC) by modifying the method described in the functional food analysis method. The analytical column used for ginsenoside analysis was TSKgel ODS-100Z, with a sample injection volume of 10 μl, a temperature of 30°C, a measurement wavelength of 203 nm, and a flow rate of 1.0 mL/min. As the mobile phase, solution A was HPLC water and solution B. Acetonitrile was used. For HPLC analysis conditions, the mobile phase solution is 81% solution A: 19% solution B at 0 min, 80% solution A: 20% solution B at 15 min, and 77% solution A: solution B 23 at 40 min. %, solution A 70% at 42 minutes:
상기 표 2에 나타낸 바와 같이, 주정 농도 50~70%에서 인체 흡수형 진세노사이드 Rd2, F2, Rg3, Rh2 및 컴파운드 케이가 많이 추출되었으며, 특히 50% 주정농도에서 추출량이 가장 많았다.As shown in Table 2, human absorption-type ginsenosides Rd2, F2, Rg3, Rh2, and compound K were extracted a lot at 50-70% of the alcohol concentration, and the extraction amount was the highest especially at the 50% alcohol concentration.
시험예 2. 항염증 조성물의 인체 흡수형 진세노사이드 함량 분석Test Example 2. Analysis of human absorption-type ginsenoside content of anti-inflammatory composition
제조예 2에서 제조된 실시예의 활성산양삼 추출물(항염증 조성물)과 비교예의 산양삼 전초 추출물 각각 2 ml을 0.45 ㎛ 멤브레인 필터로 여과한 후 분석시료로 사용하여, 상기 시험예 2와 동일한 방식으로 인체 흡수형 진세노사이드 함량을 분석하였고, 그 결과를 하기 표 3과 도 1에 나타냈다.2 ml of each of the active wild ginseng extract (anti-inflammatory composition) of the Example prepared in Preparation Example 2 and the wild ginseng extract of the comparative example was filtered through a 0.45 μm membrane filter and used as an analysis sample, absorbed into the human body in the same manner as in Test Example 2 The type ginsenoside content was analyzed, and the results are shown in Table 3 and FIG. 1 below.
표 3과 도 1에 나타낸 바와 같이, 실시예의 활성산양삼 추출물의 경우에는, 비교예의 산양삼 전초 추출물에 비하여, 진세노사이드 Rd2는 약 1.3배(4.63 mg/g), 진세노사이드 F2는 약 1.1배(4.09 mg/g), 진세노사이드 Rg3는 약 2.9배(2.90 mg/g), 진세노사이드 Rh2는 약 3.1배(0.8 mg/g)로 증진되었고, 특히 진세노사이드 컴파운드 케이는 약 11.4배(6.30 mg/g)로 현저히 증진되었다 (도 1b, 도 1c).As shown in Table 3 and Figure 1, in the case of the active wild ginseng extract of the example, compared to the wild ginseng extract of the comparative example, the ginsenoside Rd2 is about 1.3 times (4.63 mg/g), and the ginsenoside F2 is about 1.1 times. (4.09 mg/g), ginsenoside Rg3 was enhanced by about 2.9 times (2.90 mg/g), ginsenoside Rh2 by about 3.1 times (0.8 mg/g), and especially, ginsenoside compound K was about 11.4 times (6.30 mg/g) was significantly improved (FIGS. 1b, 1c).
시험예 3. 활성산양삼 추출물의 페놀릭산과 플라보놀 화합물 함량 분석Test Example 3. Analysis of phenolic acid and flavonol compound content of active wild ginseng extract
제조예 2에서 제조된 실시예의 활성산양삼 추출물(항염증 조성물)과 비교예의 산양삼 전초 추출물에 대하여 생리활성성분인 페놀릭산(클로르제닉산) 및 플라보놀(쿼르세틴)의 함량을 분석하였다.The content of phenolic acid (chlorgenic acid) and flavonol (quercetin), which are physiologically active ingredients, was analyzed for the active wild ginseng extract (anti-inflammatory composition) of the Example and the wild ginseng extract of the comparative example prepared in Preparation Example 2.
페놀릭산과 플라보놀 함량 분석은 상기 시험예 2에서와 같이 준비된 각각의 분석시료에 대해 HPLC(high performance liquid chromatography)를 사용하여 분석하였다. 분석 컬럼은 XBridgeTM C18(4.6mm, 5 μm, Waters Corp., Milford, MA, USA) 컬럼을 사용하였고 0.5% 글라시알 아세트산 (glacial acetic acid, 이동상 용매 A)와 100% 메탄올(이동상 용매 B)을 0~100% 선형 구배(linear gradient)로 30℃에서 60분간 1분당 1 ml의 속도로 가동하면서 UV 검출기로 페놀산(280 nm)과 플라보놀(270 nm)을 검출하였고, 그 결과를 표 4에 나타내었다.The analysis of phenolic acid and flavonol content was analyzed using high performance liquid chromatography (HPLC) for each analysis sample prepared as in Test Example 2. As an analytical column, an XBridge TM C18 (4.6 mm, 5 μm, Waters Corp., Milford, MA, USA) column was used, and 0.5% glacial acetic acid (mobile phase solvent A) and 100% methanol (mobile phase solvent B) were used. phenolic acid (280 nm) and flavonol (270 nm) were detected with a UV detector while operating at a rate of 1 ml per minute at 30°C for 60 minutes with a 0-100% linear gradient, and the results are shown in the table. 4 is shown.
표 4에 나타낸 바와 같이, 실시예의 활성산양삼 추출물의 경우는, 비교예의 산양삼 전초 추출물에 비하여, 클로르제닉산(0.47 mg/ml)은 2.1배 증가하였고 쿼르세틴(0.55 mg/ml)은 1.3배 증가하였다.As shown in Table 4, in the case of the active wild ginseng extract of the example, compared to the wild ginseng extract of the comparative example, chlorgenic acid (0.47 mg/ml) increased by 2.1 times and quercetin (0.55 mg/ml) increased by 1.3 times did
시험예 4. 활성산양삼 추출물의 총 페놀릭스 및 총 플라보노이드스 함량 분석Test Example 4. Analysis of total phenolics and total flavonoids content of active wild ginseng extract
제조예 2에서 제조된 실시예의 활성산양삼 추출물(항염증 조성물)과 비교예의 산양삼 전초 추출물에 대하여 생리활성성분인 총 페놀릭스와 총 플라보노이드스 함량을 분석하였다.The contents of total phenolics and total flavonoids, which are physiologically active ingredients, were analyzed for the active wild ginseng extract (anti-inflammatory composition) of the Example prepared in Preparation Example 2 and the wild ginseng extract of the comparative example.
<총 페놀릭스 함량><Total phenolics content>
총 페놀릭스 함량은 Folin Denis법(1912)으로 측정하였다. Total phenolics content was measured by Folin Denis method (1912).
구체적으로는 상기 시험예 2에서와 같이 준비된 각각의 분석시료를 50% 에탄올에 100배 희석한 후, 시험관에 0.5 ml 분주하고 25% Na2CO3 용액 0.5 ml를 첨가하여 3분간 정치시켰다. 다시 2N-Folin-Ciocalteu 페놀 시약 0.25 ml를 첨가하여 혼합한 다음 30℃에서 1시간 동안 정치시킨 후 750 nm에서 흡광도를 측정하였다. 이때 총 페놀릭스 함량은 갈산(Gallic acid)을 이용하여 작성한 표준곡선으로부터 함량을 구하여 갈산에 상당하는 양으로 계산하였고 그 결과를 도 2a에 나타냈다. Specifically, after each analysis sample prepared as in Test Example 2 was diluted 100 times in 50% ethanol, 0.5 ml was dispensed into a test tube, 0.5 ml of a 25% Na 2 CO 3 solution was added, and left for 3 minutes. Again, 0.25 ml of 2N-Folin-Ciocalteu phenol reagent was added and mixed, and then the absorbance was measured at 750 nm after standing at 30° C. for 1 hour. At this time, the total phenolic content was calculated as an amount equivalent to gallic acid by obtaining the content from a standard curve prepared using gallic acid, and the results are shown in FIG. 2A.
도 2a에 나타낸 바와 같이, 총 페놀릭스 함량은 실시예의 활성산양삼 추출물이, 비교예의 산양삼 전초 추출물에 비하여 1.6배 이상 증진되었다.As shown in Figure 2a, the total phenolics content of the active wild ginseng extract of the example was improved by 1.6 times or more compared to the wild ginseng outpost extract of the comparative example.
<총 플라보노이드 함량><Total flavonoid content>
총 플라보노이드 함량은 Davis 변법으로 측정하였다. Total flavonoid content was determined by the Davis method.
구체적으로는 상기 시험예 2에서와 같이 준비된 각각의 분석시료를 50% 에탄올로 50배 희석한 후, 시험관에 0.1 ml 분주하고 1 N NaOH 0.01 ml를 첨가한 후, 디에틸렌글리콜 1.0 ml를 첨가하여 37℃에서 1시간 방치 후 420 nm 흡광도를 측정하였다. 이때 총 플라보노이드 함량은 루틴(rutin)의 최종 농도를 0, 0.25, 0.5, 1.0 mg/ml로 제조하여 작성한 표준곡선으로부터 함량을 구하였고 그 결과는 도 2b에 나타내었다. Specifically, after each analysis sample prepared as in Test Example 2 was diluted 50 times with 50% ethanol, 0.1 ml was dispensed into a test tube, 0.01 ml of 1 N NaOH was added, and 1.0 ml of diethylene glycol was added. After standing at 37° C. for 1 hour, absorbance at 420 nm was measured. At this time, the total flavonoid content was obtained from a standard curve prepared by preparing the final concentration of rutin at 0, 0.25, 0.5, and 1.0 mg/ml, and the results are shown in FIG. 2b.
도 2b에 나타낸 바와 같이, 총 플라보노이드 함량도, 실시예의 활성산양삼 추출물이, 비교예의 산양삼 전초 추출물에 비하여 1.5배 이상 증진되었다.As shown in Figure 2b, the total flavonoid content, the active wild ginseng extract of the example, was improved more than 1.5 times compared to the wild ginseng outpost extract of the comparative example.
<갈변물질 함량><Content of browning substances>
갈변물질의 함량은 비효소적 갈변도 측정 방법을 이용하였다.The content of the browning material was measured using a non-enzymatic browning degree measurement method.
구체적으로는 상기 시험예 2에서와 같이 준비된 각각의 분석시료를 50% 에탄올로 10배 희석한 후, 분광광도계(Spectronic 2D)를 이용하여 420 nm에서 측정하여 그 결과는 도 2c에 나타내었다. Specifically, after each analysis sample prepared as in Test Example 2 was diluted 10-fold with 50% ethanol, it was measured at 420 nm using a spectrophotometer (Spectronic 2D), and the results are shown in FIG. 2C.
도 2c에 나타낸 바와 같이, 갈변물질은 실시예의 활성산양삼 추출물이, 비교예의 산양삼 전초 추출물에 비하여 3.4배 이상 증진되었다.As shown in Figure 2c, the browning material was enhanced by more than 3.4 times in the active wild ginseng extract of the example, compared to the wild ginseng outpost extract of the comparative example.
시험예 5. 항염증 조성물의 세포독성 검증Test Example 5. Cytotoxicity verification of anti-inflammatory composition
본 발명의 항염증 조성물의 세포독성을 검증하기 위하여 RAW 264.7 대식세포주 대상으로 세포독성 시험을 수행하였다.In order to verify the cytotoxicity of the anti-inflammatory composition of the present invention, a cytotoxicity test was performed on the RAW 264.7 macrophage cell line.
<분석시료 준비><Preparation of analysis sample>
제조예 2에서 제조된 실시예의 활성산양삼 추출물(항염증 조성물)과 비교예의 산양삼 전초 추출물을 각각 3차 증류수로 0.025, 0.05, 0.1, 0.2 및 0.3 brix의 농도로 희석한 후 0.45 ㎛ 멤브레인 필터로 여과하여 분석시료로 사용하였다.The active wild ginseng extract (anti-inflammatory composition) of the Example prepared in Preparation Example 2 and the wild ginseng extract of the comparative example were diluted with tertiary distilled water to concentrations of 0.025, 0.05, 0.1, 0.2 and 0.3 brix, respectively, and then filtered through a 0.45 μm membrane filter. Thus, it was used as an analysis sample.
<세포독성 시험> <Cytotoxicity test>
MTT assay를 이용하여 측정하였다. RAW 264.7 대식세포는 10% 소태아혈청(FBS), 페니실린(100 U/mL) 및 스트렙토마이신(100 ㎍/mL)이 첨가된 DMEM (Dulbecco's modified Eagle medium) 배지를 사용하여 배양기에서 37℃와 5% CO2를 유지하며 배양하였고, 20 계대(passages)를 넘기지 않은 세포만 사용하였다. 96-웰 플레이트에 1×104 cells/well로 동일하게 분주하고 24시간 동안 배양하였다. 기존의 배지를 제거하고 새로운 배지를 넣어준 후 상기에서 준비된 각각의 농도별 분석시료를 처리한 후 다시 24시간 배양한 후에 MTT 시약(5 mg/mL)을 넣고, 4시간 동안 방치한 후 상등액을 제거하였다. 각 웰에 형성된 포마잔(formazan)은 DMSO 100 ㎕를 첨가하여 녹이고, 595 nm에서 흡광도를 측정하였다. LPS (리포폴리사카라이드)를 처리한 군의 흡광도 값을 기준으로 세포 생존율을 비교하여 도 3에 나타냈다. It was measured using the MTT assay. RAW 264.7 macrophages were cultured using DMEM (Dulbecco's modified Eagle medium) medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL) in an incubator at 37°C and 5 % CO 2 was maintained and cultured, and only cells that did not exceed 20 passages were used. 1×10 4 cells/well were equally distributed in a 96-well plate and cultured for 24 hours. After removing the old medium and adding a new medium, each concentration sample prepared above was treated, and after culturing for 24 hours, MTT reagent (5 mg/mL) was added, and the supernatant was left for 4 hours. removed. Formazan formed in each well was dissolved by adding 100 μl of DMSO, and absorbance was measured at 595 nm. The comparison of cell viability based on the absorbance value of the group treated with LPS (lipopolysaccharide) is shown in FIG. 3 .
대식세포 MTT 반응에서 80% 이상의 생존율을 보이는 경우 세포독성이 없는 것으로 판단하는데, 도 3에 나타낸 바와 같이, 비교예의 경우는 0.2 brix 이상의 고농도에서 세포생존율이 80% 미만으로 나타나 세포독성이 있는 것으로 확인되었으나(도 3a), 실시예의 경우에는 고농도로 처리시에도 세포생존율이 90% 이상으로 확인되어 세포독성이 없는 것을 알 수 있다 (도 3b).In the case of macrophage MTT response, it is judged that there is no cytotoxicity when it shows a survival rate of 80% or more. However (FIG. 3a), in the case of the example, it can be seen that the cell viability was confirmed to be 90% or more even when treated at a high concentration, and thus there was no cytotoxicity (FIG. 3b).
시험예 6. 활성산양삼 추출물의 항염증 효과 검증Test Example 6. Verification of anti-inflammatory effect of active wild ginseng extract
본 발명의 활성산양삼 추출물(실시예)의 항염증 효과 검증은 RAW 264.7 대식세포주를 대상으로 하여 NO 생성 억제활성 및 IL-6, MCP-1의 염증성 사이토카인 억제활성을 분석하여 수행하였다. The verification of the anti-inflammatory effect of the active wild ginseng extract (Example) of the present invention was performed by analyzing the NO production inhibitory activity and the inflammatory cytokine inhibitory activity of IL-6 and MCP-1 targeting RAW 264.7 macrophage cell line.
<NO 생성 억제활성 측정><Measurement of NO production inhibitory activity>
대식세포에서 염증유발물질인 LPS에 의해 유도된 NO 생성 억제활성을 측정하여 분석하였다. In macrophages, the inhibitory activity of NO production induced by LPS, an inflammatory substance, was measured and analyzed.
구체적으로는 RAW 264.7 대식세포를 24-웰 플레이트에 3×105 cells/well이 되도록 분주하여 상기 시험예 5와 같은 DMEM 배지에서 24시간 동안 배양하여 세포를 부착시킨 후 시험예 5에서와 동일한 방식으로 준비한 농도별 분석시료를 처리하고 1시간 배양 후 LPS(1 ㎍/ml)를 처리하고 24시간 배양하였다. 배양 후 상층액 100 ㎕를 취하여 동량의 Griess 시약을 첨가하고 10분간 방치한 후 570 nm에서 흡광도를 측정하였다. 생성된 아질산염(nitrite)의 농도는 아질산나트륨를 DMEM 배지에 용해한 표준곡선을 이용하여 계산하였으며 LPS를 처리한 대조군과 LPS를 처리하지 않은 대조군에서 생성된 아질산염의 양 차이를 기준으로 각 분석시료의 NO 생성 억제활성을 확인하여, 그 결과를 도 4에 나타냈다. Specifically, RAW 264.7 macrophages were seeded in a 24-well plate at 3×10 5 cells/well, cultured in the same DMEM medium as in Test Example 5 for 24 hours to attach the cells, and then in the same manner as in Test Example 5. was treated with the prepared concentration-specific analysis sample and cultured for 1 hour, then treated with LPS (1 μg/ml) and cultured for 24 hours. After incubation, 100 μl of the supernatant was taken, the same amount of Griess reagent was added, and the absorbance was measured at 570 nm after standing for 10 minutes. The concentration of the generated nitrite was calculated using a standard curve in which sodium nitrite was dissolved in DMEM medium. The inhibitory activity was confirmed, and the results are shown in FIG. 4 .
도 4에 도시된 바와 같이, 본 발명에 따른 활성산양삼 추출물(실시예)은 농도 의존적으로 NO 생성 억제활성이 증가하였다. 게다가 비교예에 비하여 NO 생성 억제활성은 현저히 증진되었음을 확인할 수 있다 (도 4b). 비교예의 경우는 실시예에 비하여 NO 생성 억제활성이 떨어지고 또한 0.2 브릭스 이상의 고농도에서는 세포독성으로 인하여 항염증 조성물로도 사용할 수 없다 (도 4a).As shown in Figure 4, the active wild ginseng extract (Example) according to the present invention increased the NO production inhibitory activity in a concentration-dependent manner. In addition, it can be confirmed that the NO production inhibitory activity was significantly improved compared to the comparative example (FIG. 4b). In the case of the comparative example, the NO production inhibitory activity is lower than that of the example, and at a high concentration of 0.2 brix or more, it cannot be used as an anti-inflammatory composition due to cytotoxicity (FIG. 4a).
<염증성 사이토카인 <Inflammatory cytokines ILIL -6 억제 활성 측정>-6 Measurement of inhibitory activity>
코팅버퍼(Coating buffer)에 캡처 항체를 1/250로 희석하여 각 웰에 50 ㎕씩 분주 후 냉장보관으로 오버나이트했다. 워시버퍼 100 ㎕를 이용하여 3회 세척하여 물기를 완전히 제거한 후, 분석 다일루언트(Assay Diluent)를 100 ㎕ 분주하여 실온에서 1시간 동안 블로킹했다. 워시버퍼 200 ㎕를 이용하여 3회 세척하여 물기를 완전히 제거 한 후, 분석시료 또는 LPS(1 ㎍/ml, 대조구)를 50 ㎕씩 각 웰에 분주하여 실온에서 2시간 방치했다. 워시버퍼 100 ㎕를 이용하여 5회 세척 후 작용검출제(working detector; Detecion Antibody + Sav-HRP reagent) 50 ㎕씩 각 웰에 분주하여 실온에서 1시간 방치했다. 워시버퍼 100 ㎕를 이용하여 7회 세척 후 기질 용액 50 ㎕씩 각 웰에 분주 후 암실에 30분 방치했다. 정지용액 20 ㎕씩 각 웰에 분주 후 흡광도 450 nm에서 측정하여, 그 결과를 도 5에 나타냈다.The capture antibody was diluted 1/250 in a coating buffer, and 50 μl of each was dispensed into each well, followed by overnight refrigeration. After washing 3 times using 100 μl of wash buffer to completely remove moisture, 100 μl of Assay Diluent was dispensed and blocking was performed at room temperature for 1 hour. After washing 3 times using 200 μl of wash buffer to completely remove moisture, 50 μl of an assay sample or LPS (1 μg/ml, control) was dispensed into each well and left at room temperature for 2 hours. After washing 5 times using 100 μl of wash buffer, 50 μl of a working detector (Detecion Antibody + Sav-HRP reagent) was dispensed into each well and left at room temperature for 1 hour. After washing 7 times using 100 μl of wash buffer, 50 μl of the substrate solution was dispensed into each well and left in the dark for 30 minutes. After dispensing 20 μl of the stop solution into each well, the absorbance was measured at 450 nm, and the results are shown in FIG. 5 .
도 5에 나타낸 바와 같이, 본 발명에 따른 활성산양삼 추출물(실시예)은 농도 의존적으로 높은 염증성 사이토카인 IL-6 억제활성을 나타냈고, 고농도에서 (세포독성 없이) 현저히 높은 IL-6 억제활성을 보였다 (도 5b). 이에 비하여 비교예의 경우는 0.2 brix 이상의 고농도에서는 세포독성을 나타낼 뿐만 아니라 IL-6 억제활성도 실시예에 비하여 현저히 떨어졌다 (도 5a).As shown in Figure 5, the active wild ginseng extract (Example) according to the present invention showed a high concentration-dependent high inflammatory cytokine IL-6 inhibitory activity, and at a high concentration (without cytotoxicity) significantly high IL-6 inhibitory activity. was seen (Fig. 5b). In contrast, in the case of Comparative Example, at a high concentration of 0.2 brix or more, not only showed cytotoxicity, but also IL-6 inhibitory activity was significantly lower than that of Example (FIG. 5a).
<염증성 사이토카인 <Inflammatory cytokines MCPMCP -1 억제활성 측정>-1 Measurement of inhibitory activity>
코팅버퍼(Coating buffer)에 캡처 항체를 1/250로 희석하여 각 웰에 50 ㎕씩 분주 후 냉장보관으로 오버나이트했다. 워시버퍼 100 ㎕를 이용하여 3회 세척하여 물기를 완전히 제거한 후, ELISA/ELISASPOT Diluent을 100 ㎕ 분주하여 실온에서 1시간 방치하여 블로킹했다. 워시버퍼 200 ㎕를 이용하여 3회 세척하여 물기를 완전히 제거한 후, 분석시료 또는 LPS(1 ㎍/ml, 대조구)를 50 ㎕씩 각 웰에 분주하여 실온에서 2시간 방치했다. 워시버퍼 100 ㎕를 이용하여 5회 세척 후 검출 항체 50 ㎕씩 각 웰에 분주하여 실온에서 1시간 방치했다. 워시버퍼 100 ㎕를 이용하여 5회 세척 후 avidin-HRP 50 ㎕씩 각 웰에 분주 후 실온에서 30분 방치했다. 워시버퍼 100 ㎕를 이용하여 5번 세척 후 TMB 용액 50 ㎕씩 각 웰에 분주 후 15분 방치했다. 정지용액 25 ㎕씩 각 웰에 분주 후 흡광도 450 nm에서 측정하여, 그 결과를 도 6에 나타냈다.The capture antibody was diluted 1/250 in the coating buffer, and 50 μl of each was dispensed into each well, followed by overnight refrigeration. After washing 3 times with 100 μl of wash buffer to completely remove water, 100 μl of ELISA/ELISASPOT Diluent was dispensed and left at room temperature for 1 hour to block. After washing 3 times using 200 μl of wash buffer to completely remove moisture, 50 μl of an assay sample or LPS (1 μg/ml, control) was dispensed into each well and left at room temperature for 2 hours. After washing 5 times using 100 μl of wash buffer, 50 μl of the detection antibody was dispensed into each well and left at room temperature for 1 hour. After washing 5 times using 100 μl of wash buffer, 50 μl of avidin-HRP was dispensed into each well and left at room temperature for 30 minutes. After washing 5 times using 100 μl of wash buffer, 50 μl of TMB solution was dispensed into each well and left for 15 minutes. After dispensing 25 μl of the stop solution into each well, absorbance was measured at 450 nm, and the results are shown in FIG. 6 .
도 6에 도시된 바와 같이, 본 발명에 따른 활성산양삼 추출물(실시예)은 농도 의존적으로 높은 염증성 사이토카인 MCP-1 억제활성을 나타냈고, 고농도에서 (세포독성 없이) 현저히 높은 MCP-1 억제활성을 보였다 (도 6b). 이에 비하여 비교예의 경우는 0.2 brix 이상의 고농도에서는 세포독성을 나타낼 뿐만 아니라 MCP-1 억제활성도 실시예에 현저히 못 미쳤다 (도 6a).As shown in FIG. 6 , the active wild ginseng extract (Example) according to the present invention exhibited a concentration-dependent high MCP-1 inhibitory activity, and at a high concentration (without cytotoxicity) significantly high MCP-1 inhibitory activity. was shown (Fig. 6b). In contrast, in the case of the comparative example, not only showed cytotoxicity at a high concentration of 0.2 brix or more, but also the MCP-1 inhibitory activity was significantly lower than that of the example (FIG. 6a).
Claims (6)
상기 활성산양삼은 산양삼 전초를 100℃에서 30~60분간 증숙과 증숙된 산양삼 전초를 70∼90℃에서 2~4일 고온숙성을 1~5회 반복한 후 수탁번호 KACC 92157P로 기탁된 락토바실러스 브레비스 BMK484 균주와 수탁번호 KACC91848P로 기탁된 락토바실러스 플란타륨 P1201 균주의 복합 종균으로 발효하여 제조된 것이고,
상기 추출은 활성산양삼을 50~70%(v/v) 농도의 에탄올로 추출하여 활성산양삼의 중량의 1 ~ 3배로 농축한 것이며,
상기 조성물에서 활성산양삼 추출물의 농도는 0.2 ~ 0.3 brix이고,
상기 활성산양삼 추출물은 클로로제닉산 0.47 mg/ml 이상 및 쿼르세틴 0.55 mg/ml 이상으로 함유하는 것인 항염증 조성물.
An anti-inflammatory composition comprising, as an active ingredient, an active wild ginseng extract having enhanced anti-inflammatory activity without cytotoxicity even at a concentration of 0.2 brix or more,
The active wild ginseng is obtained by repeating 1 to 5 times of steaming of wild ginseng outpost at 100 ° C. for 30 to 60 minutes and high temperature aging of the steamed wild ginseng outpost at 70 to 90 ° C. It is produced by fermentation with a complex seed of the BMK484 strain and the Lactobacillus plantarum P1201 strain deposited with accession number KACC91848P,
The extraction is to extract the active wild ginseng with ethanol at a concentration of 50 to 70% (v/v) and concentrate it to 1 to 3 times the weight of the active wild ginseng,
The concentration of the active wild ginseng extract in the composition is 0.2 ~ 0.3 brix,
The active wild ginseng extract is an anti-inflammatory composition containing chlorogenic acid 0.47 mg/ml or more and quercetin 0.55 mg/ml or more.
A functional food for preventing and improving inflammation comprising the anti-inflammatory composition according to claim 1.
A functional cosmetic for preventing and improving skin inflammation comprising the anti-inflammatory composition according to claim 1.
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