KR101908862B1 - Method for manufacturing fermented Cordyceps militaris extract grown on Rhynchosia Nulubilis Soybean and its use for prevention and treatment of non-alcoholic fatty liver disease - Google Patents
Method for manufacturing fermented Cordyceps militaris extract grown on Rhynchosia Nulubilis Soybean and its use for prevention and treatment of non-alcoholic fatty liver disease Download PDFInfo
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- KR101908862B1 KR101908862B1 KR1020170140177A KR20170140177A KR101908862B1 KR 101908862 B1 KR101908862 B1 KR 101908862B1 KR 1020170140177 A KR1020170140177 A KR 1020170140177A KR 20170140177 A KR20170140177 A KR 20170140177A KR 101908862 B1 KR101908862 B1 KR 101908862B1
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- extract
- fermented
- cordyceps
- fatty liver
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Abstract
Description
본 발명은 간에서 지방 축적으로 발생하는 지방간 질환(fatty liver disease)에 대해 지방산 산화 활성화를 통해 중성지방 축적 저해 효능을 갖는 서목태 동충하초 발효추출물의 제조 방법과 이를 유효성분으로 하는 지방간 억제용 약학 조성물에 관한 것으로, 보다 상세하게는 스핑고신 일인산 인산화 효소의 활성화를 통해 스핑고신 일인산의 증가가 간 내 지방산 산화를 증가시키도록 한 서목태 동충하초 발효 추출물의 제조 방법 및 이를 포함하는 지방간 억제용 약학 조성물에 관한 것이다.The present invention relates to a method for producing a fermented extract of Cordyceps mellitus Worticulture having the effect of inhibiting the accumulation of triglycerides by fatty acid oxidation activation against fatty liver disease caused by fat accumulation in the liver and a pharmaceutical composition for inhibiting fatty liver comprising the active ingredient More particularly, the present invention relates to a method for producing a fermented extract of Cordyceps mellitus Wort, which causes increase of sphingosine monophosphate and the increase of sphingosine monophosphate through the activation of sphingophospholipase, and a pharmaceutical composition for inhibiting fatty liver comprising the same .
현대사회에서 생활수준의 향상으로 과다영양섭취가 발생하고 있으나 운동부족과 소비열량이 적어 체내 과다 영양분의 축적으로 비만, 지방간, 인슐린 저항성, 심혈관 질환 등의 각종 대사이상 성인병의 발생이 증가하고 있다. 과영양으로 인한 성인병 중 지방간은 과도한 지방 및 알코올 섭취, 간의 중성지방 생합성 증가, 간으로부터의 중성지방 분비 감소 등으로 인해 간에서 중성지방의 축적으로 발생하며 간에서 축적된 지방이 5% 이상일 때 지방간으로 진단된다.In the modern society, due to the improvement of living standards, excessive nutrient intake occurs. However, due to lack of exercise and low calorie consumption, accumulation of excess nutrients in the body increases the incidence of various metabolic disorders such as obesity, fatty liver, insulin resistance and cardiovascular disease. Among the adult diseases caused by overeating, fatty liver is caused by accumulation of triglyceride in the liver due to excessive fat and alcohol consumption, increase of triglyceride biosynthesis in the liver, decrease of triglyceride from the liver, Is diagnosed.
지방간 질환은 알코올 과다 섭취로 발생하는 알코올성 지방간 질환과 비만, 당뇨병, 고지혈증, 또는 약물섭취로 인한 비알코올성 지방간 질환으로 분류된다. 비알코올성 지방간질환은 음주 여부와 상관없이 간 내에 중성지방이 축적되는 질환으로 초기에 지방간증 (steatosis)로 나타나며 발전 시 지방간염 (steatohepatitis)으로 발전된다. Fatty liver disease is classified as alcoholic fatty liver disease caused by excessive alcohol consumption and nonalcoholic fatty liver disease caused by obesity, diabetes, hyperlipidemia, or drug consumption. Nonalcoholic fatty liver disease is an accumulation of triglycerides in the liver, regardless of whether it is alcoholic or not. It appears as steatosis in the early stage and develops as steatohepatitis during development.
비알코올성 지방간증은 병리학적으로 간세포에 발생하는 지방적 (lipid droplet)의 크기에 따라 거대소포성 지방간증과 미세소포성 지방간증으로 구분되며 대부분의 비알코올성 지방간증은 거대 소포성으로 초기에는 일반적으로 특이한 증상이 없으나 만성적으로 서서히 진행되어 간기능이 점차적으로 나빠진다. 비알코올성 지방간증은 에너지소모의 불균형으로 인해 지방조직으로부터 간으로 이동되는 지방산이 증가하여 발생하며 지방산 산화능 감소와 중성지방 생합성 증가로 인한 간 내 지방축적이 원인이다. 따라서 지방산 산화 및 생합성 관련 효소의 발현을 조절하는 PPARα와 SREBP-1과 같은 핵수용체의 변화가 중요한 역할을 한다. 지방축적이 심한 지방간증은 지방간염으로 발전하며 염증반응이 심해지면서 간섬유화 (fibrosis)와 간경화 (cirrhosis)로 발전하게 된다. 따라서 초기 예후인 지방간증에서 간경변으로 발전하는 것을 예방하기 위해서는 간 내 지방적의 축적을 예방해야 하나 마땅한 치료법이 없다. 알코올성 지방간증으로 판명된 환자의 50%, 그리고 비알코올성 지방간으로 진단된 환자의 30%가 간경화로 발전하다는 사실을 감안한다면 지방간증은 매우 심각한 간질환으로 간주될 수 있다. Nonalcoholic lipidemia is classified into giant bacilli lipid and small bacilli lipid according to the size of lipid droplet which occurs in hepatocyte pathologically. Most nonalcoholic lipid bacilli are giant bacillary, There is no unusual symptom, but it progresses chronically and the liver function is gradually deteriorated. Nonalcoholic lipidemia is caused by an increase in fatty acid transported from adipose tissue to the liver due to unbalanced energy consumption, and is caused by a decrease in fatty acid oxidation ability and an accumulation of fat in the liver due to an increase in triglyceride biosynthesis. Thus, changes in nuclear receptors such as PPARα and SREBP-1, which regulate the expression of fatty acid oxidation and biosynthesis-related enzymes, play an important role. Fatty fat accumulation in the province of lipid hepatitis develops as the inflammatory response to the deepening of fibrosis (fibrosis) and cirrhosis (cirrhosis) will develop. Therefore, in order to prevent the development of liver cirrhosis from the initial prognosis, lipid-lowering, there is no proper treatment to prevent accumulation in the liver. Given that 50% of patients with alcoholic lipidemia and 30% of those diagnosed with nonalcoholic fatty liver develop into cirrhosis, lipidemia can be considered a very serious liver disease.
현재 특정적으로 지방간만을 약학적으로 치료하는 유효약제는 거의 없는 상태이며 운동과 식이요법을 추천하고 있으나 치료효율은 매우 낮아 다국적 제약회사에 의한 지방간 치료제 개발이 계속적으로 진행되고 있다. 당뇨병과 비만상태에서 관찰되는 인슐린저항성과 지방간과의 상관성이 보고되면서 당뇨병치료제 및 고중성지방치료제인 메트포민 (metformin)이나 PPARα활성화제인 fenofibrate가 지방간 치료에 처방되고 있다. Currently, there are few effective medicines that specifically treat fatty liver disease, and exercise and diet therapy are recommended. However, treatment efficiency is very low, and the development of therapeutic agents for fatty liver disease by multinational pharmaceutical companies is continuing. As a result of the reported correlation between insulin resistance and fatty liver observed in diabetes and obesity, metformin or fenofibrate, a PPARα activator, is prescribed for the treatment of fatty liver.
동충하초 (Cordyceps militaris)는 사상균의 일종인 동충하초균이 곤충에 감염되어 성장한 약용버섯으로 한국, 일본, 중국, 네팔 등에 분포되어 있다. 동충하초 추출물은 항염증 및 림프종, 백혈병, 방광암 등 항암활성과 면역력 조절 및 강화에 효과적이다. 그러나 자연산 동충하초는 희귀하고 자연에서 대량 채취가 어렵기 때문에 발아시킨 서목태 (쥐눈이콩, Rhynchosia Nulubilis Soybean)를 기주로 하여 동충하초를 재배하였다. 이렇게 기주를 달리하여 배양된 서목태 동충하초 (Cordyceps militaris grown on Rhynchosia Nulubilis Soybean)는 곤충을 기주로 하여 생장하는 자연 채취 동충하초와 비교했을 때 생리활성에 대한 차이점이 아직 규명되어 있지 않다. Cordyceps militaris (Cordyceps militaris) is a medicinal mushroom grown by insect infestation, a kind of filamentous fungus, distributed in Korea, Japan, China and Nepal. Cordyceps extracts are effective for anti-inflammatory and lymphatic, leukemic, bladder cancer, anti-cancer activity and immunity control. However, because the wild Chinese caterpillar fungus is rare and it is difficult to collect large quantities in nature, it was cultivated with Rhizopus nulubilis Soybean as a host. Cordyceps militaris grown on Rhynchosia Nulubilis Soybean cultivated in different host cultivars has not yet been clarified in terms of physiological activity as compared with natural harvesting caterpillar fungus growing on insect host.
우리나라는 된장, 간장 및 김치 등과 같은 발효식품을 많이 섭취하고 있으며 전통발효식품은 많은 건강 효과가 있다고 알려져 왔다. 특히 유산균에 의한 식품의 발효는 보존, 맛, 향, 질감 뿐 아니라 소화능과 천연대사산물과 같은 생리활성을 상승시키며 유산균으로 발효한 식품은 기존의 식품보다 생리활성 물질이 증가되고 면역력을 높인다는 연구결과가 있다. 그러나 서목태 동충하초를 유산균으로 발효한 추출물의 비알코올성 지방간 경감 및 치료 효능에 대해서는 아직까지 알려진 바 없다.Korea is consuming a lot of fermented foods such as miso, soy sauce, and kimchi, and traditional fermented foods have been known to have many health effects. In particular, the fermentation of food by lactic acid bacteria increases the physiological activities such as digestibility and natural metabolites, as well as preservation, taste, aroma and texture. Foods fermented with lactic acid bacteria increase the physiologically active substances and increase immunity There are research results. However, the nonalcoholic fatty liver alleviation and therapeutic efficacy of extracts fermented with Lactobacillus acidophilus were not known.
관련 종래기술로서 미국공개특허 제2013-0259894호(2014.02.15.)는 중국에서만 시판이 승인되는 동충하초인 Cordyceps synensis 균사체 분말에 대한 간섬유증 및 비알코올성 지방간 질환 방지 효능에 대한 조성물을 개시한 바 있다.As related related art, U.S. Published Patent Application No. 2013-0259894 (Feb. 21, 2014) discloses a composition for preventing hepatic fibrosis and non-alcoholic fatty liver disease diseases on Cordyceps synensis powder, a Chinese lichen- .
본 발명은 상기한 종래 기술의 요망에 부응하기 위하여 발명된 것으로서, 본 발명은 서목태 동충하초 미생물 발효 추출물이 스핑고신 인산화 효소의 발현을 상승시키고, 간세포 내 지방산 산화를 증가시켜 지방간 치료에 유용한 서목태 동충하초 발효 추출물의 비알코올성 지방간 예방 및 개선용 식품 조성물 및 이의 제조방법을 제공하는 것을 목적으로 한다.The present invention has been made in order to meet the above-mentioned demand of the prior art. The present invention relates to a fermented extract of Cordyceps sinensis microflora, which increases the expression of sphingosine phosphorylase and increases fatty acid oxidation in hepatocytes, And a food composition for preventing and alleviating the non-alcoholic fatty liver of the extract and a process for producing the same.
본 발명의 제1 실시예에 의한 서목태 동충하초 발효 추출물의 비알코올성 지방간 예방 및 개선용 식품 조성물 및 이의 제조방법은, a) 서목태 동충하초를 증류수로 105℃에서 2시간 열수 추출한 다음, 그 추출액에 유산균(Pediococcus pentosaceus ON188)을 접종하여 24시간 동안 발효한 후 100℃의 열탕 조건에서 10분간 가열한 후 3분간 초음파 처리하는 단계; 및 b) 상기 발효된 서목태 동충하초 발효 추출물을 열수로 추출하는 단계로 이루어진 것을 특징으로 한다. A food composition for prevention and improvement of nonalcoholic fatty liver disease and a method for producing the same according to the first embodiment of the present invention is characterized by a) a method of hot water extraction of caterpillar fungus ciliaris extracts at 105 DEG C for 2 hours with distilled water, Pediococcus pentosaceus ON188), fermented for 24 hours, heated for 10 minutes at a heating temperature of 100 ° C, and sonicated for 3 minutes; And b) extracting the fermented Pseudomonas aeruginosa fermented extract of fermented West Indies with hot water.
본 발명의 서목태 동충하초 발효 추출물의 제조 방법에 의하면, 지방산 산화 유전자의 발현을 증가시키면서도 세포독성은 나타내지 않으므로, 지방간 치료를 목적으로 유용하게 사용될 수 있는 효과를 가지고 있다. According to the method of the present invention for producing a fermented extract of Cordyceps mellitus, the present invention has an effect of increasing the expression of a fatty acid oxidizing gene and showing no cytotoxicity, and thus being useful for the purpose of treatment of fatty liver.
도 1은 본 발명의 간세포에 (A) 자연산 동충하초(C. militaris), (B) 서목태 동충하초(GSC) 열수 추출물 또는 (C) 서목태 동충하초 발효 추출물(GSC-ON188)의 세포 생존율을 도시한 막대그래프이고,
도 2는 본 발명의 간세포에 (A) 자연산 동충하초(C. militaris), (B) 서목태 동충하초(GSC) 열수 추출물 또는 (C) 서목태 동충하초 발효 추출물(GSC-ON188)의 지방산 산화 유전자의 발현 변화를 도시한 막대그래프이며,
도 3은 본 발명의 간세포에 (A) 자연산 동충하초(C. militaris), (B) 서목태 동충하초(GSC) 열수 추출물 또는 (C) 서목태 동충하초 발효 추출물(GSC-ON188)의 생합성 유전자의 발현 변화와, (D) 세포 내 S1P의 농도 변화 및, (E) 세포 내의 단백질 발현 변화를 도시한 막대그래프이며,
도 4는 본 발명의 간세포에 지방산 처리하여 세포 내 지방적을 발생시키고 서목태 동충하초 미생물 발효 추출물을 처리하여 지방적의 축적을 염색으로 관찰한 사진이며,
도 5는 본 발명의 서목태 동충하초 미생물 발효추출물을 용량별로 고지방식이에 혼합하여 마우스에 4주간 섭취시켰을 때 (A) 마우스의 체중변화와 (B) 식이섭취율을 도시한 그래프이며,
도 6은 본 발명의 서목태 동충하초 발효추출물을 용량별로 고지방식이에 혼합하여 마우스에 4주간 섭취시키고 나서 분리한 (A) 간 조직의 지방적 변화와 (B) 지방조직 내 지방세포의 크기를 염색으로 나타낸 사진이다.Brief Description of the Drawings Fig. 1 is a bar graph showing the cell survival rate of (A) wild-type Cordyceps mellifera, (B) Seomotai caterpillar fungus (GSC) hot water extract or (C) Seedlings caterpillar fungus extract (GSC-ON188) ego,
2 is a graph showing changes in the expression of the fatty acid oxidized genes of (A) wild-type C. militaris, (B) seaweed, or (GSC) hydrothermal extract or (C) fermented extract of caterpillar fungus (GSC-ON188) A bar graph is shown,
FIG. 3 is a graph showing changes in expression of biosynthetic genes of (A) wild-type C. militaris, (B) Pseudomonas aeruginosa (GSC) hot water extract or (C) Pseudomonas aeruginosa extract (GSC-ON188) (D) a change in S1P concentration in a cell, and (E) a change in protein expression in a cell,
FIG. 4 is a photograph showing fatty acid in the hepatocyte of the present invention to generate intracellular lipid, treating lipid peroxidized microorganism fermented extract, and observing accumulation of lipid by dyeing.
FIG. 5 is a graph showing changes in body weight of a mouse (A) and an intake rate of a dietary composition (B) when the fermented extract of the present invention was mixed with a high fat diet for 4 weeks,
FIG. 6 is a graph showing the results of (A) liver lipid change and (B) fat fat mass in the fat tissue obtained by mixing the fermented extract of the present invention with the high fat diet for 4 weeks, This is the picture shown.
이하, 본 발명의 바람직한 실시예를 첨부한 도면을 참조하여 당해 분야의 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 설명하기로 한다. 첨부된 도면들에서 구성에 표기된 도면번호는 다른 도면에서도 동일한 구성을 표기할 때에 가능한 한 동일한 도면번호를 사용하고 있음에 유의해야 한다. 또한, 본 발명을 설명함에 있어 관련된 공지의 기능 또는 공지의 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략하기로 한다. 그리고 도면에 제시된 어떤 특징들은 설명의 용이함을 위해 확대 또는 축소 또는 단순화된 것이고, 도면 및 그 구성요소들이 반드시 적절한 비율로 도시되어 있지는 않다. 그러나 당업자라면 이러한 상세 사항들을 쉽게 이해할 것이다.Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be noted that the drawings denoted by the same reference numerals in the drawings denote the same reference numerals whenever possible, in other drawings. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. And certain features shown in the drawings are to be enlarged or reduced or simplified for ease of explanation, and the drawings and their components are not necessarily drawn to scale. However, those skilled in the art will readily understand these details.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.
이하, 본 발명의 바람직한 실시예를 첨부된 도면을 참조하여 상세히 설명하기로 한다. 아울러 본 발명을 설명함에 있어서, 관련된 공지 구성 또는 기능에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략한다.With reference to the accompanying drawings, preferred embodiments of the present invention will be described in detail. In the following description of the present invention, detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.
[제1실시예][First Embodiment]
도 1은 AML-12 간세포에 (A) 자연산 동충하초(C. militaris), (B) 서목태 동충하초(GSC) 열수 추출물 또는 (C) 서목태 동충하초 발효 추출물 (GSC-ON188)을 처리한 후 24시간 경과 후에 측정한 세포 생존율을 나타낸다. 결과는 매개체 대조군(vehicle-treated control)과의 상대적인 백분율로 표시하였으며 평균 ㅁ 표준오차로 나타내었다. Figure 1 shows the results obtained after 24 hours after treatment of AML-12 hepatocytes with (A) wild-caught C. militaris, (B) hot-water extract of Seomotai caterpillar fungus (GSC) or (C) And the cell viability measured. Results were expressed as a percentage relative to the vehicle-treated control and expressed as mean ㅁ standard error.
도 2는 AML-12 간세포에 (A) 자연산 동충하초(C. militaris), (B) 서목태 동충하초(GSC) 열수 추출물 또는 (C) 서목태 동충하초 발효 추출물 (GSC-ON188)을 처리한 후 24시간 후 지방산 산화 유전자인 CPT1의 mRNA 발현 변화를 real-time PCR로 측정한 결과를 나타낸다. 양성대조군으로 PPARα 활성화제인 WY-14643을 처리하였다. 데이터는 평균 ㅁ 표준오차로 표시하였다(*p<0.05 vs. 매개체 대조군).FIG. 2 shows the results of treatment of AML-12 hepatocyte with (A) wild-type C. militaris, (B) hot water extract of Seomotai caterpillar fungus (GSC) or (C) fermented caterpillar fungus extract (GSC-ON188) The change in mRNA expression of the oxidized gene, CPT1, was measured by real-time PCR. WY-14643, a PPARα activator, was treated as a positive control. Data were expressed as mean ㅁ standard error ( * p <0.05 vs. vehicle control).
도 3은 AML-12 간세포에 (A) 자연산 동충하초(C. militaris), (B) 서목태 동충하초(GSC) 열수 추출물 또는 (C) 서목태 동충하초 발효 추출물 (GSC-ON188)을 처리한 후 24 시간 후 sphingosine 1-phosphate (S1P) 생합성 유전자인 SPHK2의 mRNA 발현 변화를 real-time PCR로 측정한 결과를 나타낸다. GSC-ON188 24시간 처리 후 (D) 세포 내 S1P의 농도 변화와 (E) 세포 내 CPT1, SPHK2, PPARα의 단백질 발현 변화를 나타낸다. 양성대조군으로 PPARα 활성화제인 WY-14643을 처리하였다. 데이터는 평균 ㅁ 표준오차로 표시하였다(*p<0.05 vs. 매개체 대조군).FIG. 3 is a graph showing the effect of sphingosine (A) on the AML-12 hepatocyte after 24 hours after (A) wild-caught C. militaris treatment, (B) hot water extract of Seok-Tae Cordyceps (GSC) 1-phosphate (S1P) biosynthesis gene SPHK2 mRNA expression was measured by real-time PCR. GSC-ON188 (D) Changes in S1P concentration in cells after 24-hour treatment and (E) Changes in protein expression of intracellular CPT1, SPHK2 and PPARα. WY-14643, a PPARα activator, was treated as a positive control. Data were expressed as mean ㅁ standard error ( * p <0.05 vs. vehicle control).
도 4는 AML-12 간세포에 지방산인 oleic acid를 처리하여 세포 내 지방적을 발생시키고 서목태 동충하초 미생물 발효 추출물을 처리하여 지방적의 축적을 Oil Red O 염색으로 관찰하였다. 양성대조군으로 PPARα 활성화제인 WY-14643을 처리하였다.FIG. 4 shows lipid accumulation of AML-12 hepatocyte treated with oleic acid, which is a fatty acid, and lipid accumulation was observed by Oil Red O staining. WY-14643, a PPARα activator, was treated as a positive control.
도 5는 서목태 동충하초 미생물 발효추출물을 용량별로 고지방식이에 혼합하여 8주령 C57BL6 마우스에 4주간 섭취시켰을 때 (A) 마우스의 체중변화와 (B) 식이섭취율을 나타낸다. 데이터는 평균 ㅁ 표준오차로 표시하였다(*p<0.05 vs. 고지방식이 대조군).FIG. 5 shows (A) mice body weight change and (B) dietary intake when 8 weeks old C57BL6 mice were fed the fermented extracts of P. japonicum microorganism fermented in a high fat diet for 4 weeks. Data were expressed as mean ㅁ standard error ( * p <0.05 vs. high-fat diet control group).
도 6은 서목태 동충하초 발효추출물을 용량별로 고지방식이에 혼합하여 8주령 C57BL6 마우스에 4주간 섭취시키고 나서 분리한 (A) 간 조직의 지방적 변화와 (B) 지방조직 내 지방세포의 크기를 Hematoxylin & Eosin 염색으로 나타내었다.FIG. 6 shows the results of (A) liver lipid changes and (B) fat cells in the adipose tissues after 8 weeks old C57BL6 mice, Eosin staining.
본 발명의 제1실시예에 따라, a) 서목태 동충하초를 증류수로 105℃에서 2시간 열수 추출한 다음, 그 추출액에 유산균(Pediococcus pentosaceus ON188)을 접종하여 24시간 동안 발효한 후 100℃의 열탕 조건에서 10분간 가열한 후 3분간 초음파 처리하는 단계; 및 b) 상기 발효된 서목태 동충하초 발효 추출물을 열수로 추출하는 단계를 포함하는 열수 추출물의 제조 방법이 제공된다.According to a first embodiment of the present invention, there is provided a method for producing a fermented soybean curd according to the first embodiment of the present invention, comprising the steps of: a) Heating for 10 minutes and ultrasonication for 3 minutes; And b) extracting the fermented endemic caterpillar fungus fermentation extract with hot water.
상기 단계 a)의 발효는 2 ~ 10시간 동안 수행될 수 있고, 상기 단계 b)의 추출은 30분 ~ 5시간 동안 수행될 수 있다.The fermentation of step a) may be carried out for 2 to 10 hours, and the extraction of step b) may be carried out for 30 minutes to 5 hours.
본 발명의 일 태양에 따라, 상기 서목태 동충하초의 발효 열수 추출물을 포함하는 식품 조성물이 제공된다.According to one aspect of the present invention, there is provided a food composition comprising a fermented hot-water extract of the above-mentioned Cordyceps mellon.
본 발명의 일 태양에 따라, 서목태 동충하초의 발효 추출물의 열수 추출물을 포함하는 지방간 치료용 식품 및 약학 조성물이 제공된다.According to one aspect of the present invention, there is provided a food and pharmaceutical composition for the treatment of fatty liver comprising a hydrothermal extract of a fermented extract of Cichoraceae Cordyceps.
상기 열수 추출물은 SPHK1, SPHK2, CPT1, ACOX1 및 PPARα로 이루어진 군에서 선택되는 하나 이상의 발현을 증가시킬 수 있고, 간조직 내 지방적(lipid droplet)의 감소를 가져올 수 있으며, 지방조직에서 지방세포의 크기를 감소시킬수 있으며, 세포독성을 나타내지 않을 수 있다.The hot-water extract may increase expression of one or more selected from the group consisting of SPHK1, SPHK2, CPT1, ACOX1, and PPARa, may lead to a decrease in lipid droplet in liver tissue, , And may not exhibit cytotoxicity.
본 발명은 a) 서목태 동충하초를 고상 발효시키는 단계; 및 b) 상기 발효된 서목태 동충하초를 열수로 추출하는 단계를 포함하는 서목태 동충하초 발효 추출물의 제조 방법을 제공한다. 상기 발효 온도는 37 ℃, 발아시간은 48시간 일 때 바람직하게 수행될 수 있다.The present invention relates to a method for the fermentation of caterpillar fungus; And b) extracting the above-mentioned fermented caterpillar fungus extract with hot water. The fermentation temperature is preferably 37 ° C and the germination time is preferably 48 hours.
상기 단계 a)의 발효는 24 ~ 48시간 동안 수행될 수 있고, 48시간 동안 발효시킴으로써 바람직하게 수행될 수 있다. 상기 단계 b)의 추출은 6시간 ~ 24시간 동안 수행될 수 있고, 1시간 동안 가열함으로써 바람직하게 수행될 수 있다.The fermentation of step a) can be carried out for 24 to 48 hours and can be preferably carried out by fermentation for 48 hours. The extraction of step b) may be carried out for from 6 hours to 24 hours and may preferably be carried out by heating for 1 hour.
또한, 본 발명은 상기 발효된 서목태 동충하초 열수 추출물을 포함하는 식품 조성물을 제공한다.In addition, the present invention provides a food composition comprising the above-mentioned fermented water-extract of Cordyceps mellon.
또한, 본 발명은 발효된 서목태 동충하초 발효 추출물을 포함하는 지방간 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the treatment of fatty liver comprising fermented extract of Fermented Cordyceps mellifera from Fermented West Indies.
일 구현예에서, 상기 약학 조성물은 1종 이상의 약학적으로 허용 가능한 담체 또는 첨가제를 추가로 포함할 수 있다. 즉, 본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제제화될 수 있다. 상기 약제학적으로 허용가능한 담체는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한다. 또한, 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 포함한다. 경구용 고형 제제는 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하며, 이러한 고형제제는 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 포함할 수 있으며, 마그네슘 스테아레이트, 탈크 같은 윤활제 등을 포함할 수 있다. 경구용 액상 제제는 현탁제, 내용액제, 유제, 시럽제 등을 포함하며, 물, 리퀴드 파라핀 등의 희석제, 습윤제, 감미제, 방향제, 보존제 등을 포함할 수 있다. 비경구용 제제는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제를 포함하며, 비수성 용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르류 등을 포함한다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween), 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.In one embodiment, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers or additives. That is, the pharmaceutical composition of the present invention can be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like oral preparation, external preparation, suppository and sterilized injection solution according to a conventional method . The pharmaceutically acceptable carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. It also includes diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Solid form preparations for oral use include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like, and may include lubricants such as magnesium stearate and talc. Oral liquid preparations include suspensions, solutions, emulsions, syrups, and the like, and may contain diluents such as water and liquid paraffin, wetting agents, sweetening agents, fragrances, preservatives and the like. Examples of the non-aqueous solution include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. Non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, ethyl And injectable esters such as oleate. As a suppository base, witepsol, macrogol, tween, cacao butter, laurin, glycerogelatin and the like can be used.
본 발명의 약학 조성물에 함유되는 서목태 동충하초 발효 추출물의 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여 경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 예를 들면, 상기 서목태 동충하초 발효 추출물은 1일 0.0001 내지 1000mg/kg으로, 바람직하게는 0.01 내지 1000 mg/kg의 용량으로 투여할 수 있으며, 상기 투여는 하루에 한번 또는 수 회 나누어 투여할 수도 있다. 또한, 본 발명의 약학 조성물은 조성물 총 중량에 대하여 상기 서목태 동충하초 발효추출물을 0.001 내지 90% 중량백분율로 포함할 수 있다.The dosage of the Fusarium oxyspora fermented extract contained in the pharmaceutical composition of the present invention varies depending on the condition and body weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. For example, the fermented extract of Cordyceps mellitus may be administered at a dose of 0.0001 to 1000 mg / kg per day, preferably 0.01 to 1000 mg / kg per day, and the administration may be administered once or several times a day . In addition, the pharmaceutical composition of the present invention may contain 0.001 to 90% by weight of the above-mentioned Cordyceps mellitus fermented extract of the present invention for the total weight of the composition.
본 발명의 약학 조성물은 랫트, 마우스, 가축, 인간 등의 포유동물에 다양한 경로로, 예를 들면, 경구, 복강, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내(intracerebroventricular) 주사에 의해 투여될 수 있다.The pharmaceutical compositions of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes, such as oral, intraperitoneal, rectal or intravenous, intramuscular, subcutaneous, intracerebral, or intracerebroventricular ≪ / RTI >
일 구현예에서, 상기 열수 추출물은 TNFα, IL-1β 및 iNOS로 이루어진 군에서 선택되는 하나 이상의 발현을 증가시킬 수 있고, pJNK, pERK 및 pp38로 이루어진 군에서 선택되는 하나 이상의 발현을 증가시킬 수 있으며, 세포독성을 나타내지 않을 수 있다.In one embodiment, the hot-water extract can increase expression of one or more selected from the group consisting of TNF [alpha], IL-1 [beta] and iNOS and increase expression of one or more selected from the group consisting of pJNK, pERK and pp38 , And may not exhibit cytotoxicity.
이하, 본 발명을 실시예 및 시험예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예 및 시험예는 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이에 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples and test examples. However, the following examples and test examples are provided for illustrating the present invention, and the scope of the present invention is not limited thereto.
<실시예><Examples>
1. 서목태 동충하초 추출물의 준비1. Preparation of Seomyeongtae Cordyceps Extract
서목태 동충하초 (GRC. Cordyceps militaris grown on germinated Rhynchosia nulubilisa, Kucati: 0093)은 (주) 세포활성연구소 (성남시, 경기도)에서 재배한 것을 준비하였다. Cordyceps militaris grown on germinated Rhynchosia nulubilisa, Kucati: 0093) was cultivated in the Cell Activity Research Institute (Seongnam City, Gyeonggi Province).
열수 추출물의 제조를 위해, 서목태동충하초 원물을 121℃에서 15분간 멸균처리하여 발효에 이용하였다. 발효를 위해 양파로부터 유래한 유산균인 Pediococcus pentosaceus ON188를 접종하여 48시간 동안 37℃에서 발효하였다. 다음 서목태 동충하초 발효물 100g을 열수 500 ml에 넣고 24시간 동안 추출하였다. 이후 동결건조하여 최종 추출된 분말은 분석 시까지 -20 ℃에서 보관하였다.For the preparation of hot water extract, Seomyeongtae caterpillar fungus was sterilized at 121 ℃ for 15 minutes and used for fermentation. For fermentation, Pediococcus pentosaceus ON188, a lactic acid bacteria derived from onion, was inoculated and fermented at 37 ° C for 48 hours. Next, 100 g of the fermented product of Tae-kwanghaek, Seomyeongtaek was added to 500 ml of hot water and extracted for 24 hours. After the lyophilization, the final extracted powder was stored at -20 ° C until analysis.
2. 세포 배양 및 세포 생존율 분석2. Cell Culture and Cell Survival Analysis
AML-12 간세포는 ATCC(American Type Culture Collection, Manassas, VA)로부터 획득하였다. 세포는 10 % FBS(fetal bovine serum), 100 U/mL의 페니실린 및 100 μg/mL의 스트렙토마이신으로 보충된 고함량 글루코스(glucose)의 DMEM 배지(Dulbecco's Modified Eagle's Medium)에서 5 % CO2 조건하의 37 ℃의 습한 기온 조건하에서 6-웰 플레이트에 8ㅧ105 cells 접종하여 24시간 동안 배양하여 준비하였다. 다양한 농도의 서목태 동충하초 발효추출물을 세포에 처리하고 24시간 후에 세포에 XTT(2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)를 처리하여 450㎚에서 흡광도를 분광기에서 측정하여 세포 생존율을 측정하였다. AML-12 hepatocytes were obtained from ATCC (American Type Culture Collection, Manassas, Va.). Cells is 10% FBS (fetal bovine serum) , 100 U / mL of penicillin and 100 μg / in mL DMEM medium in a high content of glucose (glucose) supplemented with streptomycin (Dulbecco's Modified Eagle's Medium) 5
3. RNA 분리 및 실시간 RT-PCR 방법3. RNA isolation and real-time RT-PCR method
RNA 추출 키트(intron Biotechnology, Korea)를 사용하여 AML-12 간세포로부터 총 RNA를 분리하였다. iScript cDNA 생합성 키트(BioRad, Hercules, CA)를 사용하여 PCR 유전자 증폭기(thermal cycler)로 cDNA(First-strand cDNA)를 합성하였다. PCR 조건은 25℃에서 5분, 42℃에서 30분, 8℃에서 5분 그리고 이후 4℃에서 유지하는 것으로 하였다. RT-PCR 분석은 PCR 시스템(Step-One Plus Real-Time PCR System, Applied Biosystems Ins, Carlsbad, CA)에서 프라이머 및 SYBR 그린 마스터 믹스(SYBR Green Master Mix, TaKaRa, Japan)를 사용하여 수행하였다. RT-PCR의 조건은 초기 95℃에서 5분을 유지한 다음 95 ℃에서 5분, 60℃에서 15분, 72℃에서 45분, 95℃에서 15분, 60℃에서 1분을 40주기로 하여 수행하였다. 본 발명에서 사용한 프라이머의 서열은 하기 표 1에 정리하였다. 상기 유전자들의 발현은 β-actin을 기준으로 정상화하였다.Total RNA was isolated from AML-12 hepatocytes using an RNA extraction kit (intron Biotechnology, Korea). cDNA (first-strand cDNA) was synthesized with PCR cyclase using an iScript cDNA biosynthesis kit (BioRad, Hercules, Calif.). The PCR conditions were 5 min at 25 ° C, 30 min at 42 ° C, 5 min at 8 ° C and then kept at 4 ° C. RT-PCR analysis was performed using a primer and a SYBR Green Master Mix (TaKaRa, Japan) in a PCR system (Step-One Plus Real-Time PCR System, Applied Biosystems Ins, Carlsbad, CA). RT-PCR was performed at 95 ° C for 5 minutes, followed by 5 cycles of 95 ° C for 15 minutes, 60 ° C for 15 minutes, 72 ° C for 45 minutes, 95 ° C for 15 minutes, and 60 ° C for 1 minute for 40 cycles Respectively. The sequences of the primers used in the present invention are summarized in Table 1 below. Expression of these genes was normalized on the basis of β-actin.
4. 4. 스핑고신Sphingosine 일인산Monophosphate 측정 방법 How to measure
AML-12 세포에 다양한 농도의 서목태 동충하초 발효추출물을 처리하고 24시간 동안 배양 후 세포를 분쇄한 후 스핑고지질을 분리한 후 고속액체크로마토그래피-질량분석기(LC-MS/MS)를 활용하여 정량하였다.AML-12 cells were treated with various concentrations of Fusarium oxysporum miltiorrhizae extract and cultured for 24 hours. After the cells were pulverized, the sphingolipids were separated and quantified using a high performance liquid chromatography-mass spectrometer (LC-MS / MS) Respectively.
5. 웨스턴 블로팅 분석 방법5. Western blotting analysis method
세포를 10 mM Tris (pH 7.8), 1 mM EDTA, 150 mM NaCl, 인산가수분해효소 억제제(phosphatase inhibitors) 및 단백질가수분해효소 억제제(protease inhibitors)를 포함하는 용균 버퍼에서 균질화시킨 후 용해물(lysate)을 원심분리하여 세포 파쇄물 및 파쇄되지 않은 세포를 제거하였다. 단백질 분석 키트(BioRad, Hercules, CA)를 사용하여 750nm에서 흡광도를 측정함으로써 단백질의 농도를 정량하였다. 단백질 30㎍을 10% SDS-PAGE 겔을 이용하여 분리하였고, 상기 겔 내의 단백질은 PDVF 멤브레인으로 이동시켰다. 상기 단백질에 특이적인 1차 항체를 연결시키고, 쥐-특이적 또는 토끼-특이적 2차 항체를 인큐베이션하였다. 상기 단백질에 대한 관점을 강화된 화학발광 키트(BioRad, Hercules, CA) 및 화학발광 영상화 장치(Vilber Lourmat, Sint-Denijs-Westrem, Belgique)를 사용하여 시각화하였다. CPT1, SPHK2, PPARα, β-actin (Cell signaling, Danvers, MA)에 대한 1차 항체가 사용되었다.Cells were homogenized in a lysis buffer containing 10 mM Tris (pH 7.8), 1 mM EDTA, 150 mM NaCl, phosphatase inhibitors and protease inhibitors, and lysates ) Were centrifuged to remove cell lysates and non-lysed cells. The protein concentration was quantified by measuring the absorbance at 750 nm using a protein assay kit (BioRad, Hercules, CA). 30 [mu] g of the protein was separated using a 10% SDS-PAGE gel, and proteins in the gel were transferred to the PDVF membrane. A primary antibody specific for the protein was ligated and a mouse-specific or rabbit-specific secondary antibody was incubated. A view of the protein was visualized using an enhanced chemiluminescence kit (BioRad, Hercules, CA) and a chemiluminescent imaging device (Vilber Lourmat, Sint-Denijs-Westrem, Belgique). Primary antibodies to CPT1, SPHK2, PPARα, β-actin (Cell signaling, Danvers, MA) were used.
6. 마우스 동물실험6. Mouse animal experiment
8주령 C57BL6 마우스에 2주간 60% kcal 고지방식이를 섭식시킨 후, 60% 고지방식이에 다양한 농도 (50, 100, 200 mg/kg/day, n=7)로 서목태 동충하초 발효추출물을 혼합하여 4주간 섭식시켰다. 이때 대조군으로서 일반식이 (NC)와 PPARα활성화제인 fenofibrate(20mg/kg/day)를 혼합한 60% kcal 고지방식이를 섭식시켰다. 4주 섭식 동안 매주 체중과 식이섭취량을 측정하였으며 4주 섭식후 마우스에서 간조직과 지방조직을 분리하여 병리분석에 사용하였다. 8 weeks old C57BL6 mice were fed a 60% kcal high fat diet for 2 weeks and then mixed with 60% high fat diet (50, 100, 200 mg / kg / day, n = 7) 4 weeks. At this time, a 60% kcal high-fat diet was fed with a combination of a normal diet (NC) and fenofibrate (20 mg / kg / day) as a PPARα activator. Body weight and dietary intake were measured weekly during 4 weeks feeding. Liver and adipose tissues were separated from mice after 4 weeks feeding and used for pathological analysis.
7. 병리 분석7. Pathology Analysis
마우스에서 분리한 간 조직과 지방조직은 포르말린에 24시간 고정시킨 후 파라핀 블록을 만들고 5 μm 두께의 절편을 준비하여 Hematoxylin & eosin에 염색하였다. 이때 간조직의 지방적 축적과 지방조직의 세포크기를 현미경으로 관찰하였다. Liver and adipose tissues from mouse were fixed in formalin for 24 hours. Paraffin blocks were prepared and 5 μm thick sections were stained with hematoxylin and eosin. At this time, lipid accumulation of liver tissue and cell size of adipose tissue were observed under a microscope.
8. 통계 처리8. Statistical processing
모든 결과는 평균 ㅁ 표준오차(SEM)로 표현하였다. 시험은 적어도 세 번씩 독립적으로 수행하였다. 결과는 t 테스트(Student's t-test)를 사용하여 분석하였고 p 값(p value)이 0.05보다 적으면 통계적으로 유의성이 있는 것으로 간주하였다.All results were expressed as mean ㅁ standard error (SEM). The test was performed at least three times independently. Results were analyzed using a Student's t-test and were considered statistically significant if the p-value was less than 0.05.
<시험예><Test Example>
1. 서목태 동충하초 발효추출물의 세포생존율1. Cell survival rate of Fermented Extract of Cordyceps sinensis
서목태 동충하초 추출물(GSC)과 서목태 동충하초 발효추출물(GSC-ON188)의 세포독성을 평가하기 위해 GSC와 GSC-ON188이 세포 생존율에 영향을 미치는 지를 시험하였다. AML-12 간세포를 0, 5, 10, 25, 50 및 100㎍/㎖의 동충하초 추출물과 함께 24시간 동안 배양하였다. 세포 생존율은 세포 증식 분석 키트(Wel gene Inc, Korea)를 사용하여 XTT 분석을 통해 측정하였다. 대조군인 자연산 동충하초 (C. militaris) 추출물과 GSC 추출물은 모두 세포독성을 나타내지 않았다(도 1의 A와 B). To investigate the cytotoxicity of GSC and GSC-ON188, we investigated whether GSC and ON188 affect cell viability. AML-12 hepatocytes were cultured with 0, 5, 10, 25, 50 and 100 μg / ml of Cordyceps sinensis extract for 24 hours. Cell viability was measured by XTT analysis using a cell proliferation assay kit (Wel gene Inc, Korea). The control group, C. militaris extract and GSC extract, showed no cytotoxicity (Fig. 1, A and B).
또한 GSC-ON188 추출물도 세포독성을 나타내지 않았다(도 1의 C). 이러한 결과로 서목태 동충하초 추출물과 서목태 동충하초 발효추출물이 모두 세포독성을 전혀 나타내지 않는다는 것을 제시할 수 있다.The GSC-ON188 extract also did not show cytotoxicity (FIG. 1C). As a result, it can be suggested that the extracts of Seomotai mushroom extract and Seomyeongtae mushroom extract do not show cytotoxicity at all.
2. 서목태 동충하초 발효추출물의 지방산 산화 유전자 발현 증가 효능2. Increase of expression of fatty acid oxidized gene in fermented extract of Cordyceps mellon
서목태 동충하초 발효추출물의 세포독성이 없는 것을 관찰하고 간세포에서 지방산산화를 증가시키는지에 대해 확인하고자 하였다. AML-12 간세포에 C. militaris, GSC 및 GSC-ON188 추출물을 다양한 농도로 처리하고 24 시간 후 mRNA를 분리하여 발현변화를 관찰하였다. 이때 대조군으로는 아무것도 처리하지 않은 control(Con)과 양성대조군인 PPARα 활성화제 WY-14643(WY)을 처리한 군을 사용하였다. WY-14643을 처리한 간세포에서 지방산 산화 유전자인 CPT1이 증가하는 것을 관찰할 수 있었다(도 2). To investigate the effect of fermented extracts of P. vannamei on cell viability and fatty acid oxidation in hepatocytes. AML-12 hepatocytes were treated with C. militaris, GSC and GSC-ON188 extracts at various concentrations, and mRNA was isolated after 24 hours. Control group (control) (Con) and positive control group PPARα activator WY-14643 (WY) were used as the control group. It was observed that CPT1, a fatty acid oxidizing gene, was increased in hepatocytes treated with WY-14643 (FIG. 2).
C. militaris와 GSC를 처리한 간세포에서는 CPT1의 mRNA 발현이 증가하지 않은 반면(도 2의 A와 B), GSC-ON188을 처리한 간세포에서는 CPT1의 발현이 대조군에 비해 4-5배 증가하였다(도 2의 C). 이러한 결과는 자연산 동충하초, 서목태 동충하초와 달리 서목태 동충하초 발효추출물이 간세포에서 지방산 산화를 크게 증가시킨다는 것을 증명하고 있다.CPT1 mRNA expression was not increased in C. militaris and GSC-treated hepatocytes (Fig. 2, A and B), whereas GSC-ON188-treated hepatocytes increased CPT1 expression 4-5 times 2C). These results demonstrate that the fermented extracts of P. vannamei, unlike the wild - harvested caterpillar fungus, do not significantly increase fatty acid oxidation in hepatocytes.
어떤 기전을 통해 GSC-ON188이 지방산 산화능을 증가시키는지 규명하기 위해 sphingosine 1-phosphate(S1P) 합성 효소인 SPHK2의 발현을 측정하였다. Expression of SPHK2, a synthetic enzyme for sphingosine 1-phosphate (S1P), was measured in order to investigate the mechanism by which GSC-ON188 increases fatty acid oxidizing ability.
C. militaris와 GSC를 처리한 간세포에서는 SPHK2의 mRNA 발현이 증가하지 않은 반면(도 3의 A와 B), GSC-ON188을 처리한 간세포에서는 SPHK2의 발현이 대조군에 비해 5배 정도 증가하였다(도 3의 C).In the hepatocytes treated with C. militaris and GSC, mRNA expression of SPHK2 was not increased (Fig. 3, A and B), whereas expression of SPHK2 was increased 5-fold in hepatocytes treated with GSC-
또한 세포 내 S1P를 측정한 결과 GSC-ON188 처리시 대조군에 비해 72% 증가한 것을 관찰하였다(도 3의 D). 또한 CPT1, SPHK2, PPARα의 단백질 발현증가가 GSC-ON188에 의해 일어나는 것을 관찰하였다. 이러한 결과는 GSC-ON188이 SPHK2의 발현 증가를 통해 간에서 지방산 산화를 증가하는 신호전달물질인 S1P를 증가시키고 지방산 산화 유전자인 CPT1의 발현이 증가된다는 것을 증명하고 있다(Hepatology. 2015.62(1):135-46). In addition, S1P in the cells was observed to be increased by 72% when treated with GSC-ON188 (FIG. 3 D). It was also observed that increased expression of CPT1, SPHK2 and PPARα was induced by GSC-ON188. These results demonstrate that GSC-ON188 increases S1P, a signal transducer that increases fatty acid oxidation in the liver through increased expression of SPHK2, and increases expression of the fatty acid oxidant CPT1 (Hepatology. 2015.62 (1): 135-46).
3. 서목태 동충하초 발효추출물에 의한 간세포 내 지방축적 억제 효능 3. Effect of Fermented Extract of Cordyceps sinensis on the Fat Storage Accumulation in Hepatocytes
지방산 산화 유전자의 발현증가가 정말 세포내 지방축적을 저해하는지 규명하기 위해 AML-12 간세포에 oleic acid 지방산을 처리하였다. Oleic acid 처리 (+FA(oleic acid))후 oil red O로 세포 내 축적된 중성지방을 측정한 결과 처리하지 않은 대조군 (Control) 보다 지방적이 염색되어 더 붉은 색을 띄는 것을 관찰할 수 있었다. 반면 양성대조군인 WY-14643으로 처리된 세포의 경우(+FA +WY)는 지방적이 현저히 감소되어 있었으며, GSC-ON188 (+FA +ON188)을 처리한 세포에서도 지방적이 현저히 감소된 것을 발견할 수 있었다. 따라서 GSC-ON188은 지방산 산화 유전자의 발현증가를 통해 세포내 지방적의 감소에 기여한다.In order to investigate whether the increased expression of fatty acid oxidase actually inhibits intracellular fat accumulation, AML-12 hepatocytes were treated with oleic acid fatty acid. In the case of olive acid treatment (+ FA (oleic acid)), olive oil accumulated in the cells was observed as reddish color due to fatty staining rather than control (control). On the other hand, in the cells treated with positive control WY-14643 (+ FA + WY), the lipid was markedly decreased and the lipid was significantly decreased even in the GSC-ON188 (+ FA + ON188) treated cells there was. Therefore, GSC-ON188 contributes to the decrease of intracellular lipid by increasing expression of fatty acid oxidized gene.
4. 고지방식이 섭식 마우스에서 서목태 동충하초 발효추출물에 의한 지방간 억제 효능4. Effect of Fermented Extract of Cordyceps militaris on the Fatty Liver
간세포에서 관찰된 지방산 산화증가를 확인하기 위해 8주령 수컷 C57BL6 마우스(쥐)에 고지방식이(HFD)를 2주간 섭식시킨 후 고지방식이에 GSC-ON188를 50,100, 200 mg/kg/day로 혼합하여 4주간 섭식시켰다. 이때 대조군으로 일반식이군 (NCD)과 양성대조군으로 PPARα활성화제인 fenofibrate 20 mg/kg/day (를 섭취시킨 마우스 군을 사용하여 비교하였다. 섭식시킨 4주간 일반식이군(NCD)은 현저히 낮은 체중을 보였으나 고지방식이군(HFD)은 시간이 지남에 따라 체중이 급격히 증가하였다. GSC-ON188 50, 100 mg/kg/day 군은 고지방식이군과 체중에 큰 차이를 보이지 않았으나 200 mg/kg/day을 복용한 마우스는 유의적인 체중의 감소를 나타내었다(도 5의 A). To investigate the increase in fatty acid oxidation observed in hepatocytes, male C57BL6 mice (male rats) were fed high-fat diet (HFD) for 2 weeks and then mixed with GSC-ON188 at 50, 100 and 200 mg / kg / day And fed for 4 weeks. (NCD) and a positive control group (PPARα activator, fenofibrate 20 mg / kg / day) were used in the control group. The 4-week feeding diets (NCD) showed significantly lower body weight (100 mg / kg / day) showed no significant difference in body weight between the high fat diet group and the high fat diet group, but 200 mg / kg / day Showed a significant decrease in body weight (Fig. 5A).
그리고 fenofibrate 복용 마우스는 현저한 체중의 감소를 나타내었는데 이는 먹이섭취량의 감소로 인한 것으로 판단된다(도 5의 B). 먹이 섭취에 있어서 다른 마우스군들은 큰 차이를 보이지 않았다(도 5의 B).The mice receiving fenofibrate showed a significant decrease in body weight, presumably due to a decrease in food intake (Fig. 5B). There was no significant difference in other groups of mice on food intake (Fig. 5B).
마우스의 간조직을 분리하여 Hematoxylin & Eosin으로 염색하였을 때 고지방식이군은 일반식이군과 달리 간조직에 지방적을 축적하였으며 간세포 내에도 지방이 축적된 병리적 형태를 보였다. 그러나 대조군인 일반식이와 fenofibrate 복용군은 일반적 간조직의 병리적 형태를 보였다(도 6의 A). When mouse liver tissue was isolated and stained with Hematoxylin & Eosin, high - fat diet group showed fatty accumulation in liver tissue unlike normal diet group, and hepatic cell showed pathological form accumulating fat. However, the control group, the general diet and the fenofibrate group, showed a general hepatic pathology (Fig. 6A).
GSC-ON188을 복용한 마우스의 간조직은 50 mg/kg/day 용량에서는 일부 지방적이 조직 내에 축적된 형태를 보였으나 고지방식이군보다 그 크기는 작았다. GSC-ON188 복용량이 높아지면서 일반식이군과 유사한 간조직의 형태를 보였으며 지방적이 사라진 형태를 관찰하였다(도 6의 A).The liver tissue of GSC-ON188-treated mice showed accumulation in some lipid tissues at the dose of 50 mg / kg / day, but the size was smaller than that of the high-fat diet. As the dose of GSC-ON188 increased, the liver tissue morphology similar to that of the normal diabetic group was observed, and the disappearance of the lipid was observed (Fig. 6A).
한편 복부지방조직을 분리하여 Hematoxylin & Eosin으로 염색하였을 때 고지방식이군은 일반식이군보다 지방세포의 크기가 현저히 큰 것을 관찰할 수 있었으며 양성 대조군인 fenofibrate 복용군은 지방세포의 크기가 현저히 작아져 있는 것을 관찰할 수 있었다. GSC-ON188을 복용한 마우스는 지방세포의 크기가 용량이 높아짐에 따라 현저히 작아지는 것으로 보아 지방세포 내 지방의 축적이 GSC-ON188에 의해 현저히 억제되는 것으로 판단된다. 따라서 서목태 동충하초 발효추출물은 지방간의 억제와 지방세포의 분화 및 성장을 억제하는 것으로 판단된다.On the other hand, when the abdominal adipose tissue was separated and stained with Hematoxylin & Eosin, it was observed that the fat cells of the high fat diet group were significantly larger than that of the normal diet group, and the fat cells of the positive control group, fenofibrate, . GSC-ON188 mice were significantly reduced in size as the adipocyte size increased, suggesting that accumulation of adipocyte fat was significantly inhibited by GSC-ON188. Therefore, it is concluded that the extract of Fermented Cordyceps sinensis extract inhibits fatty liver and differentiation and growth of adipocytes.
이상에서와 같이, 본 발명의 상세한 설명에서 구체적인 실시예에 관해 설명하였으나, 본 발명의 기술이 당업자에 의하여 용이하게 변형 실시될 가능성이 자명하며, 이러한 변형된 실시예들은 본 발명의 특허청구범위에 기재된 기술사상에 포함된다할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. And will be included in the described technical idea.
Claims (8)
b) 상기 발효된 서목태 동충하초 발효 추출물을 열수로 추출하는 단계로 이루어진 것을 특징으로 하는 서목태 동충하초 발효 추출물의 제조 방법.a) Pseudomonas aeruginosa was extracted with distilled water at 105 ° C for 2 hours, and the extract was inoculated with lactic acid bacteria (Pediococcus pentosaceus ON188), fermented for 24 hours, heated at 100 ° C for 10 minutes, and sonicated for 3 minutes step; And
and b) extracting the fermented extract from the fermented Cordyceps mellitus fermentation extract with hot water.
상기 단계 a)의 발효가 24 ~ 48시간 동안 수행되는 것을 특징으로 하는 서목태 동충하초 발효 추출물의 제조 방법.The method according to claim 1,
Wherein the fermentation of step (a) is carried out for 24 to 48 hours.
상기 단계 b)의 추출이 6 ~ 24시간 동안 수행되는 것을 특징으로 하는 서목태 동충하초 발효 추출물의 제조 방법.The method according to claim 1,
Wherein the extraction of step b) is carried out for 6 to 24 hours.
[Claim 6] The pharmaceutical composition according to claim 5, wherein the fermented extract of Cordyceps mellitus does not exhibit cytotoxicity.
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