KR20210004133A - Compositions for preventing or treating fatty liver disease comprising Allomyrina dichotoma larva extract - Google Patents
Compositions for preventing or treating fatty liver disease comprising Allomyrina dichotoma larva extract Download PDFInfo
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- KR20210004133A KR20210004133A KR1020190080050A KR20190080050A KR20210004133A KR 20210004133 A KR20210004133 A KR 20210004133A KR 1020190080050 A KR1020190080050 A KR 1020190080050A KR 20190080050 A KR20190080050 A KR 20190080050A KR 20210004133 A KR20210004133 A KR 20210004133A
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- adle
- fatty liver
- hfd
- extract
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Abstract
Description
본 발명은 지방간 예방 및 치료용 조성물에 관한 것으로서, 더 상세하게는 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는 지방간 예방 및 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing and treating fatty liver, and more particularly, to a composition for preventing and treating fatty liver comprising an active ingredient of a beetle larva extract or powder.
비-알콜성 지방간 질환(Non-alcoholic fatty liver disease, NAFLD)은 선진국에서 가장 흔한 간 질환으로 그 빈도가 지속적으로 증가하고 있다(Loomba, R. et al., Nat Rev Gastroenterol Hepatol, 10, 686-690. 2013). 비-알콜성 지방간 질환의 발생률은 제2형 당뇨병에서 70-75%로 보고되고 간경화 및 암과 같은 심각한 간 질환에 영향을 미치는 것으로 알려져 있다(Mantovani, A. et al., A meta-analysis. Diabetes Care, 41, 372-382. 2018). 또한 비정상적인 지방 섭취는 간세포의 세포질에서 중성지방(triglyceride, TG), 콜레스테롤 및 지방 소립(lipid droplet)과 같은 간 지질(hepatic lipid) 축적에 기여하고 지방 축적에 의한 지방독성(lipotoxicity)은 제2형 당뇨병의 발병 기전에서 간장 인슐린 저항성을 유도하였다(Neuschwander-Tetri, B.A. Hepatology, 52, 774-788. 2010). 현재 제2형 당뇨병에 사용되는 약물에는 고지혈증(hyperlipidemia)과 고인슐린혈증(hyperinsulinemia) 치료를 위한 고 비용이 소요되며 대부분의 항 당뇨병 약물에는 부작용이 있다. 또한 제2형 당뇨병의 발생에 대한 비-알코올성 지방간 질환의 영향은 아직 명확하지 않고 제2형 당뇨병의 발생에 대한 비-알코올성 지방간 질환의 영향은 아직 명확하지 않다. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in developed countries and its frequency continues to increase (Loomba, R. et al., Nat Rev Gastroenterol Hepatol, 10, 686- 690. 2013). The incidence of non-alcoholic fatty liver disease is reported to be 70-75% in
한편, 최근 유엔식량농업기구(FAO)에 따르면 식용 곤충은 지방, 단백질, 무기질, 비타민 및 기타 영양소가 풍부하여 앞으로 식이 보충제로서의 가능성이 있음을 보고하였다(Chung, M.Y.H. et al., J. Life Sci. 23, 664-668. 2013). 많은 나라에서 다양한 종류의 곤충이 이미 전통 음식이나 민간요법으로 사용되었지만 인간에 대한 안정성과 긍정적인 효과에 대한 과학적 증거가 부족하여 소비량은 제한적이다. 장수풍뎅이(Allomyrina dichotoma)는 전통적 약제로 항-간질 세포증, 항-종양제 및 당뇨병 치료에 널리 사용되고 있는데 종래 연구에서 장수풍뎅이 유충의 추출물에 항-신생물제(anti-neoplastic)가 포함되어 있는 것으로 밝혀졌으며, 약리 활성 성분은 주로 유충에서 발견되었다(Sagisaka, A. et al., Insect Mol Biol, 10, 293-302. 2001). Meanwhile, according to the Food and Agriculture Organization of the United Nations (FAO) recently, edible insects are rich in fat, protein, minerals, vitamins and other nutrients, so it has the potential as a dietary supplement in the future (Chung, MYH et al., J. Life Sci. 23, 664-668. 2013). In many countries various types of insects have already been used as traditional foods or folk remedies, but consumption is limited due to the lack of scientific evidence of their safety and positive effects on humans. Longevity beetle ( Allomyrina dichotoma ) is a traditional drug and is widely used in anti-epileptic cytopathy, anti-tumor agent, and diabetes treatment.In conventional studies, the extract of the beetle larvae contains anti-neoplastic agents. It was found, and the pharmacologically active ingredient was mainly found in larvae (Sagisaka, A. et al., Insect Mol Biol , 10, 293-302. 2001).
그러나 장수풍뎅이 유충 추출물(ADLE)이 지방간을 예방하거나 고지방식이(HFD)에 의해 유도된 제2형 당뇨병에서 고혈당을 개선할 수 있는지에 대한 연구는 여전히 미개척 분야라 할 수 있다. However, research into whether or not beetle larva extract (ADLE) can prevent fatty liver or improve hyperglycemia in
본 발명은 상기와 같은 문제점을 포함하여 여러 문제점들을 해결하기 위한 것으로서, 간 지방생성을 효율적으로 억제하여 간 지방증을 완화하고 고혈당을 개선시키는 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는 지방간 예방 및 치료용 조성물을 제공하는 것을 목적으로 한다. 그러나 이러한 과제는 예시적인 것으로, 이에 의해 본 발명의 범위가 한정되는 것은 아니다.The present invention is to solve a number of problems including the above problems, and prevent fatty liver comprising an active ingredient of a longevity beetle larva extract or powder that effectively suppresses liver adipogenesis to relieve hepatic steatosis and improve high blood sugar It is an object of the present invention to provide a therapeutic composition. However, these problems are exemplary, and the scope of the present invention is not limited thereby.
본 발명의 일 관점에 따르면, 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는, 지방간 예방 및 치료용 조성물이 제공된다. According to one aspect of the present invention, there is provided a composition for preventing and treating fatty liver, comprising an active ingredient of a beetle larva extract or powder.
본 발명의 다른 일 관점에 따르면, 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는, 지방간 개선용 건강기능식품이 제공된다. According to another aspect of the present invention, there is provided a health functional food for improving fatty liver, comprising an active ingredient of a longevity beetle larva extract or powder.
상기한 바와 같이 본 발명의 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는 지방간 예방 및 치료용 조성물은 간 지방생성의 억제를 통해 고혈당을 효과적으로 개선시키고 지질 축적을 유의하게 억제하였으므로 지방간 및 제2형 당뇨병 치료를 위한 치료제로 활용할 수 있다. 물론 이러한 효과에 의해 본 발명의 범위가 한정되는 것은 아니다.As described above, the composition for preventing and treating fatty liver comprising the active ingredient of the extract or powder of the beetle larva of the present invention effectively improved hyperglycemia through inhibition of liver adipogenesis and significantly inhibited lipid accumulation, so fatty liver and
도 1은 고지방식이(HFD) 투여 마우스에 본 발명의 장수풍뎅이 유충 추출물(ADLE)을 6주간 투여 후 체중, 공복 혈당, 식이섭취량 및 FER에 대한 변화를 분석한 그래프로 (a)는 ADLE 투여에 따른 6주간 체중의 변화, (b)는 각 실험군에 대한 체중의 변화(각 주당 체중에서 기저 본체 중량을 뺀 것), (c)는 식이섭취량을 (d)는 식이 효율비(FER)를 (E)는 공복 혈당수준을 나타내었다. 분석 결과는 평균±SD (n=6-8)로 계산하였다.
***p<0.001 vs. NFD 군. #p<0.05, ##p<0.01, 및 ###p<0.001 vs. HFD 군. NFD: 정상지방식이, HFD: 고지방식이, HFD + ADLE: 고지방식이 + ADLE (장수풍뎅이 유충 추출물, 100 mg/kg), HFD + Met(메트포르민, 100 mg/kg).
도 2는 HFD-급이 마우스에 본 발명의 ADLE를 투여하여 글루코스 대사 및 인슐린 감수성을 분석한 것으로 (a)는 ADLE 주입 후 혈당의 변화를 분석한 그래프, (b)는 복강 내 포도당 내성 검사(GTT), (c)는 포도당 주입 후 혈당의 변화를 분석한 그래프, (d)는 인슐린 주입 후 혈당 변화에 대한 복강 내 인슐린 내성 검사(ITT) 결과이다. 값은 군당 6-8 마리의 마우스의 평균 ± 표준 편차이다.
***p＜0.001 vs. NFD group. #p＜0.05 vs. HFD 군. NFD: 정상지방식이, HFD: 고지방식이, HFD + ADLE: 고지방식이 + ADLE (장수풍뎅이 유충 추출물, 100 mg/kg), HFD + Met(메트포르민, 100 mg/kg).
도 3은 고지방 식이 마우스(HFD)에 본 발명의 ADLE를 6주간 투여하여 혈청 화학(serum chemistry)에 대한 효과를 분석한 것으로 (a)는 혈청 지질 프로파일, (b)는 AST 및 ALT 결과를 분석한 그래프이다. 결과는 평균 ± SD. (n= 6-8)으로 표시하였다.
**p＜0.01, ***p < 0.001 vs. NFD 군. #p < 0.05, ##p＜0.01, 및 ###p < 0.001 vs. HFD 군. NFD: 정상지방식이, HFD: 고지방식이, HFD + ADLE: 고지방식이 + ADLE (장수풍뎅이 유충 추출물, 100 mg/kg), HFD + Met(메트포르민, 100 mg/kg).
도 4는 HFD-식이 마우스의 간 지방증(hepatic steatosis)에 있어 ADLE의 효과를 분석한 것으로 (a)는 오일 O 염색법(원배율 x 400)으로 염색된 간 조직의 섹션을 나타내는 현미경 사진이고 (b)는 간 트리 글리세 라이드(TG, mg/g 단백질) 및 총 콜레스테롤(TC, mg/g 단백질) 함량을 분석한 그래프이다. 결과는 평균 ± SD. (n = 6-8)으로 표시하였다.
*p＜0.05, **p < 0.01 vs. NFD 군. #p < 0.05 및 ##p＜0.01 vs. HFD 군. NFD: 정상지방식이, HFD: 고지방식이, HFD + ADLE: 고지방식이 + ADLE (장수풍뎅이 유충 추출물, 100 mg/kg), HFD + Met(메트포르민, 100 mg/kg).
도 5는 HepG2 세포에서 세포 독성, 세포 내 지질 축적 및 지방 생성과 관련된 마커에 대한 ADLE의 효과를 분석한 것으로 (a)는 HepG2 세포에서 세포 독성에 대한 ADLE의 효과를 분석한 그래프이고 (b)는 트리글리세리드 측정 키트를 이용하여 HepG2 세포의 세포 내 지질 축적을 분석한 그래프이며 (c)는 실시간 PCR로 유전자의 mRNA 수준을 분석한 그래프이다. 상대적인 양은 참조 유전자에 상응하는 시클로필린(cyclophilin)으로 표준화되었다. (d 및 e)는 단백질의 발현 수준을 분석한 웨스턴 블랏 분석의 겔 사진이다. Quantity one 소프트웨어로 각 밴드의 강도를 측정하였고, β-액틴에 대해 상대적인 양을 계산했다. 실험 b, c 및 d에서 사용된 ADLE의 최종 농도는 0.5 mg/mL이다. 데이터는 평균 ± SD (n = 3-5)로 표시하였다.
*p < 0.05, **p < 0.001, 및 ***p < 0.001 vs. 무처리 대조군(CON). #p < 0.05, ##p < 0.001, 및 ###p < 0.001 vs. 0.5 mM 팔미트산염만 처리(PAL).
도 6은 HFD-식이 마우스 간에서 지방생성과 관련된 마커에 대한 ADLE의 효과를 분석한 것으로 (a)SREBP-1c, FA 합성 효소(FAS) 및 ACC의 mRNA 발현수준을 분석한 그래프이다. (b 및 c)는 SREBP-1, FA 합성효소(FAS) 및 ACC의 단백질 발현을 분석한 겔 사진 및 그래프이다. 밴드의 강도는 농도 측정 분석(densitometric analysis)에 의해 정량화하였고 상응하는 β-액틴으로 표준화하였다. 정량적 RT-PCR을 측정된 유전자 발현에 사용하였고 유전자 발현은 시클로필린의 수준으로 정상화되었다. 결과는 평균 ± SD(n = 6 ~ 8)로 계산되었다.
**p < 0.01 및 *** p< 0.001 vs. NFD 군. #p < 0.05, ## p < 0.01, 및 ### p < 0.001 vs. HFD 군. NFD: 정상지방식이, HFD: 고지방식이, HFD + ADLE: 고지방식이 + ADLE (장수풍뎅이 유충 추출물, 100 mg/kg), HFD + Met(메트포르민, 100 mg/kg).
도 7은 HFD-식이 마우스 간에서 AMPK 인산화에 대한 ADLE의 효과를 분석한 것으로 (a)는 총-AMPK 및 phospho-AMPK의 간 발현에 대한 웨스텃 블랏 분석 겔 사진이다. β- 액틴은 대조군으로 사용하였다. (b)는 p-AMPK/t-AMPK 비를 분석한 그래프이다. 도는 3가지 독립적인 실험에서 하나의 대표적인 블랏을 나타내며 phospho-AMPK/총-AMPK 비율의 농도 값은 막대 그래프에 표시된다. 값은 평균±SD (n=6-8)로 표시된다.
***p < 0.001 vs. NFD 군. ##p < 0.01 vs. HFD 군. NFD: 정상지방식이, HFD: 고지방식이, HFD + ADLE: 고지방식이 + ADLE(장수풍뎅이 유충 추출물, 100 mg/kg), HFD + Met(메트포르민, 100 mg/kg).
도 8은 본 발명의 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는 지방간 예방 및 치료용 조성물에 대한 개발 과정을 개략적으로 나타내고 있는 개요도이다. Figure 1 is a graph analyzing changes in body weight, fasting blood sugar, dietary intake and FER after administration of the longevity beetle larva extract (ADLE) of the present invention to mice administered on a high fat diet (HFD) for 6 weeks. Change in body weight for 6 weeks according to, (b) is the change in body weight for each experimental group (the weight per week minus the base body weight), (c) is the dietary intake and (d) is the diet efficiency ratio (FER). (E) shows the fasting blood sugar level. Analysis results were calculated as mean±SD (n=6-8).
*** p<0.001 vs. NFD group. # p<0.05, ## p<0.01, and ### p<0.001 vs. HFD group. NFD: a normal fat diet, HFD: a high fat diet, HFD + ADLE: a high fat diet + ADLE (Betworm larva extract, 100 mg/kg), HFD + Met (metformin, 100 mg/kg).
Figure 2 is an analysis of glucose metabolism and insulin sensitivity by administering ADLE of the present invention to HFD-fed mice, (a) is a graph analyzing changes in blood sugar after ADLE injection, (b) is an intraperitoneal glucose tolerance test ( GTT), (c) are graphs analyzing changes in blood sugar after glucose injection, and (d) are results of intraperitoneal insulin resistance test (ITT) for changes in blood sugar after insulin injection. Values are the mean ± standard deviation of 6-8 mice per group.
*** p<0.001 vs. NFD group. # p<0.05 vs. HFD group. NFD: a normal fat diet, HFD: a high fat diet, HFD + ADLE: a high fat diet + ADLE (Betworm larva extract, 100 mg/kg), HFD + Met (metformin, 100 mg/kg).
3 is an analysis of the effect on serum chemistry by administering ADLE of the present invention to high-fat diet mice (HFD) for 6 weeks, (a) is a serum lipid profile, (b) is an analysis of AST and ALT results. It is a graph. Results are mean ± SD. (n=6-8).
** p <0.01, *** p <0.001 vs. NFD group. # p <0.05, ## p <0.01, and ### p <0.001 vs. HFD group. NFD: a normal fat diet, HFD: a high fat diet, HFD + ADLE: a high fat diet + ADLE (Betworm larva extract, 100 mg/kg), HFD + Met (metformin, 100 mg/kg).
Figure 4 is an analysis of the effect of ADLE on hepatic steatosis of HFD-fed mice (a) is a micrograph showing a section of liver tissue stained with oil O staining (original magnification x 400) (b ) Is a graph analyzing liver triglyceride (TG, mg/g protein) and total cholesterol (TC, mg/g protein) content. Results are mean ± SD. (n = 6-8).
* p <0.05, ** p <0.01 vs. NFD group. # P <0.05 and ## p <0.01 vs. HFD group. NFD: a normal fat diet, HFD: a high fat diet, HFD + ADLE: a high fat diet + ADLE (Betworm larva extract, 100 mg/kg), HFD + Met (metformin, 100 mg/kg).
5 is an analysis of the effect of ADLE on the markers related to cytotoxicity, intracellular lipid accumulation, and adipogenesis in HepG2 cells. (a) is a graph analyzing the effect of ADLE on cytotoxicity in HepG2 cells (b) Is a graph analyzing intracellular lipid accumulation in HepG2 cells using a triglyceride measurement kit, and (c) is a graph analyzing the mRNA level of a gene by real-time PCR. Relative amounts were normalized to cyclophilin, which corresponds to the reference gene. (d and e) are gel photographs of Western blot analysis analyzing the expression level of the protein. The intensity of each band was measured with Quantity one software and the relative amount was calculated for β-actin. The final concentration of ADLE used in experiments b, c and d is 0.5 mg/mL. Data are expressed as mean ± SD (n = 3-5).
* p <0.05, ** p <0.001, and *** p <0.001 vs. No treatment control (CON). # p <0.05, ## p <0.001, and ### p <0.001 vs. 0.5 mM palmitate only (PAL).
6 is an analysis of the effect of ADLE on a marker related to adipogenesis in the liver of HFD-fed mice, and is a graph showing the mRNA expression level of (a) SREBP-1c, FA synthase (FAS), and ACC. (b and c) are gel photographs and graphs analyzing protein expression of SREBP-1, FA synthase (FAS), and ACC. The intensity of the band was quantified by densitometric analysis and normalized to the corresponding β-actin. Quantitative RT-PCR was used for the measured gene expression and gene expression was normalized to the level of cyclophilin. Results were calculated as mean ± SD (n = 6-8).
** p <0.01 and *** p <0.001 vs. NFD group. # p <0.05, ## p <0.01, and ### p <0.001 vs. HFD group. NFD: a normal fat diet, HFD: a high fat diet, HFD + ADLE: a high fat diet + ADLE (Betworm larva extract, 100 mg/kg), HFD + Met (metformin, 100 mg/kg).
7 is an analysis of the effect of ADLE on AMPK phosphorylation in the liver of HFD-fed mice. (a) is a photograph of a Westet blot analysis gel for liver expression of total-AMPK and phospho-AMPK. β-actin was used as a control. (b) is a graph analyzing the ratio of p-AMPK/t-AMPK. Figure shows one representative blot from three independent experiments, and the concentration values of the phospho-AMPK/total-AMPK ratio are shown in the bar graph. Values are expressed as mean±SD (n=6-8).
*** p <0.001 vs. NFD group. ## p <0.01 vs. HFD group. NFD: a normal fat diet, HFD: a high fat diet, HFD + ADLE: a high fat diet + ADLE (longevity beetle larva extract, 100 mg/kg), HFD + Met (metformin, 100 mg/kg).
Figure 8 is a schematic diagram showing the development process for a composition for the prevention and treatment of fatty liver comprising the active ingredient of the beetle larva extract or powder of the present invention.
용어의 정의:Definition of Terms:
본 문서에서 사용되는 "장수풍뎅이(Allomyrina dichotoma)는"는 분류학상 곤충강(Insecta) 딱정벌레목(Coleoptera) 장수풍뎅이과(Dynastidae)에 속하며 1년에 한 번 생활사를 완결하는 1화생 곤충으로 3번의 탈피를 하여 번데기가 된 후 6-7월경에 성충으로 우화한다. 성충을 지낸 후 8월말에서 9월에 걸쳐 교미를 하고 산란하며, 약 2주 후에 1령 유충으로 부화한다. 장수풍뎅이 유충은 딱정벌레류의 유충을 말하며, 흔히 민간요법으로 이용되어온 것은 장수풍뎅이 유충이며, 간경화, 간암 등의 간 질환에 효능이 있는 것으로 알려져 있고, 이외에도 허약체질의 보양효과와 각종 성인병 치료에 효과가 있는 것으로 알려져 있다." Allomyrina dichotoma " used in this document belongs to the family Insecta, Coleoptera, Dynastidae, taxonomically, and is a one-figure insect that completes its life cycle once a year. It turns into a pupa and then emerges as an adult in June-July. After being an adult, they mate and spawn from the end of August to September, and hatch into 1-instar larvae after about 2 weeks. Longevity beetle larvae refer to the larvae of beetles, and it is the longevity beetle larva that has been commonly used as a folk remedy, and is known to be effective in liver diseases such as cirrhosis and liver cancer. It is known to have.
발명의 상세한 설명:Detailed description of the invention:
본 발명의 일 관점에 따르면, 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는, 지방간 예방 및 치료용 조성물이 제공된다.According to one aspect of the present invention, there is provided a composition for preventing and treating fatty liver, comprising an active ingredient of a beetle larva extract or powder.
상기 지방간 예방 및 치료용 조성물에 있어서, 상기 추출물은 물, C1 내지 C4의 저급알코올 또는 이들의 혼합물로 추출한 것일 수 있고 상기 저급알코올은 메탄올, 에탄올 또는 프로판올일 수 있으며 상기 지방간은 비알콜성 지방간일 수 있다. In the composition for preventing and treating fatty liver, the extract may be extracted with water, C1 to C4 lower alcohol or a mixture thereof, and the lower alcohol may be methanol, ethanol or propanol, and the fatty liver is non-alcoholic fatty liver. I can.
상기 지방간 예방 및 치료용 조성물에 있어서, 건조된 장수풍뎅이 유충을 분쇄기로 분쇄하여 분말로 제조하는 분말 제조단계; 상기 분말에 에탄올 및 물을 1:9 내지 9:1의 비율로 첨가 후 50 내지 70℃에서 2 내지 4시간 동안 추출하는 추출물 제조단계; 상기 추출물을 여과 후 농축 및 동결건조시킨 후 탈 이온수에 용해시켜 수득하는 추출물 수득단계를 포함하는 제조방법에 의해 제조될 수 있다. In the composition for preventing and treating fatty liver, a powder manufacturing step of pulverizing the dried longevity beetle larva with a grinder to prepare a powder; Extract preparation step of extracting for 2 to 4 hours at 50 to 70 ℃ after adding ethanol and water in a ratio of 1:9 to 9:1 to the powder; It can be prepared by a manufacturing method comprising the step of obtaining an extract obtained by filtration, concentration, lyophilization, and dissolution in deionized water.
본 발명의 다른 일 관점에 따르면, 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는, 지방간 개선용 건강기능식품이 제공된다.According to another aspect of the present invention, there is provided a health functional food for improving fatty liver, comprising an active ingredient of a longevity beetle larva extract or powder.
상기 지방간 개선용 건강기능식품에 있어서, 탕제, 드링크제, 산제, 환제, 캡슐, 정제 또는 젤리의 제형으로 제공될 수 있다. In the health functional food for improving fatty liver, it may be provided in the form of a bath, a drink, a powder, a pill, a capsule, a tablet, or a jelly.
본 발명의 약학적 조성물의 유효량은 환자의 환부의 종류, 적용부위, 처리회수, 처리시간, 제형, 환자의 상태, 보조제의 종류 등에 따라 변할 수 있다. 사용량은 특별히 한정되지 않지만, 0.01μg/kg/day 내지 10 mg/kg/day일일 수 있다. 상기 1일량은 1일에 1회, 또는 적당한 간격을 두고 하루에 2~3회에 나눠 투여해도 되고, 수일(數日) 간격으로 간헐(間歇)투여해도 된다. The effective amount of the pharmaceutical composition of the present invention may vary depending on the type of the affected part of the patient, the application site, the number of treatments, the treatment time, the formulation, the condition of the patient, the type of adjuvant and the like. The amount used is not particularly limited, but may be 0.01 μg/kg/day to 10 mg/kg/day. The daily dose may be administered once a day, divided into 2-3 times a day at appropriate intervals, or intermittently administered at intervals of several days.
본 발명의 약학적 조성물은, 조성물 총 중량에 대하여 0.1-100 중량%로 함유될 수 있고 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 또한, 약학적 조성물의 제조에는 고체 또는 액체의 제제용 첨가물을 사용할 수 있다. 제제용 첨가물은 유기 또는 무기 중 어느 것이어도 된다. 부형제로서는 예를 들면 유당, 자당, 백당, 포도당, 옥수수 전분(cornstarch), 전분, 탈크, 소르비트, 결정 셀룰로오스, 덱스트린, 카올린, 탄산칼슘 및 이산화규소 등을 들 수 있다. 결합제로서는 예를 들면 폴리비닐알코올, 폴리비닐에테르, 에틸셀룰로오스, 메틸셀룰로오스, 아라비아고무, 트래거캔스(tragacanth),젤라틴, 셀락(shellac), 히드록시프로필셀룰로오스, 히드록시프로필메틸셀룰로오스, 구연산칼슘, 덱스트린 및 펙틴(pectin) 등을 들 수 있다. 활택제로서는 예를 들면 스테아린산마그네슘, 탈크, 폴리에틸렌글리콜, 실리카, 경화식물유 등을 들 수 있다. 착색제로서는 통상 의약품에 첨가하는 것이 허가되어 있는 것이라면 모두 사용할 수 있다. 이들의 정제, 과립제에는 당의(糖衣), 젤라틴코팅, 기타 필요에 따라 적절히 코팅할 수 있다. 또한, 필요에 따라 방부제, 항산화제 등을 첨가할 수 있다.The pharmaceutical composition of the present invention may be contained in an amount of 0.1-100% by weight based on the total weight of the composition, and may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. In addition, solid or liquid additives for preparation may be used in the preparation of pharmaceutical compositions. The additive for formulation may be either organic or inorganic. Examples of excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. As a binder, for example, polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, And dextrin and pectin. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant that is permitted to be added to pharmaceuticals can be used. These tablets and granules can be appropriately coated with a sugar coat, gelatin coating, or other necessary. In addition, preservatives, antioxidants, and the like may be added as necessary.
본 발명의 약학적 조성물은 당 업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며(예: 문헌 [Remington's Pharmaceutical Science, 최신판;Mack Publishing Company, Easton PA), 제제의 형태는 특별히 한정되는 것은 아니다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌[Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042(Chapter 87: Blaug, Seymour)에 기술되어 있다. The pharmaceutical composition of the present invention can be prepared in any formulation conventionally prepared in the art (for example, Remington's Pharmaceutical Science, the latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited. . These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a formula generally known for all pharmaceutical chemistry.
본 발명의 약학적 조성물은 경구 또는 비경구로 투여되는 것이 가능하며, 바람직하게는 비경구 투여로 정맥내 주입, 피하 주입, 뇌실내 주입(intracerebroventricular injection), 뇌척수액내 주입(intracerebrospinal fluid injection), 근육내 주입 및 복강 주입 등으로 투여할 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenteral administration by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, intramuscular It can be administered by injection or intraperitoneal injection.
본 발명의 건강기능식품은 캡슐, 정제, 분말, 과립, 액상, 환, 편상, 페이스트상, 시럽, 겔, 젤리 또는 바(bar) 형태의 제제인 것을 특징으로 하는 알코올성 지방간 개선 또는 예방용 건강기능식품. 상기 건강기능식품은 건강기능식품에 적합한 다양한 제형, 예컨대, 탕제, 드링크제, 산제, 환제, 캡슐, 정제(코팅정, 당의정, 설하정 등), 젤리 등의 제형이 사용될 수 있다.The health functional food of the present invention is a health function for improving or preventing alcoholic fatty liver, characterized in that it is a formulation in the form of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jelly or bars. food. The health functional food may be used in a variety of formulations suitable for health functional food, such as a bath, drink, powder, pills, capsules, tablets (coated tablets, dragees, sublingual tablets, etc.), jelly, and the like.
본 발명의 건강기능식품은 건강기능식품에 적합한 다양한 제형, 예컨대, 탕제, 드링크제, 산제, 환제, 캡슐, 정제(코팅정, 당의정, 설하정 등), 젤리 등의 제형이 사용될 수 있다.The health functional food of the present invention can be used in a variety of formulations suitable for health functional foods, for example, formulations such as baths, drinks, powders, pills, capsules, tablets (coated tablets, dragees, sublingual tablets, etc.), jelly.
본 발명에서 "건강기능식품"이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다. In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and the nutrients for the structure and function of the human body It means ingestion for the purpose of controlling or obtaining useful effects for health purposes such as physiological effects.
장수풍뎅이 유충(Allomyrina dichotoma larva)은 미래의 식량 자원으로서 영양 가치가 있으며 여러 가지 약리학적 기능을 가지고 있으나 간장 인슐린 저항성(hepatic insulin resistance) 효과에 대해서는 여전히 연구가 미미하다. 이에 본 발명자들은 고지방 식이로 유도된 C57BL/6J 마우스 모델을 이용하여 장수풍뎅이 유충 추출물(ADLE)의 간독성 관련 인슐린 저항 효과 및 팔미틴산염을 처리한 HepG2 세포에서의 분자 기전을 조사했다. 그 결과, ADLE는 공복 혈당, 인슐린 저항성, 혈청 및 간 조직의 지질 프로필 감소, 축적된 간 지방증(liver steatosis)을 완화시켰다. 또한 간 조직에서 AMPK 인산화의 활성화를 통해 지방생성 관련 아세틸 CoA 카르복실라아제(ACC), FAS 및 SREBP-1c 발현을 감소시켰으며 지방생성 관련 mRNA 및 단백질의 하향조절을 통해 HepG2 세포에서 지질 축적을 유의하게 억제하는 것으로 나타났다. 상기 결과는 본 발명의 ADLE가 AMPK 신호 전달 경로의 활성화에 의한 간 지방생성(hepatic lipogenesis)의 억제를 통해 고혈당을 효과적으로 개선시키고 천연 의료용 화합물을 함유함으로써 제2형 당뇨병 치료를 위한 유망한 치료제가 될 수 있음을 시사하는 것이다(도 8).The beetle larvae ( Allomyrina dichotoma larva) have nutritional value as a food resource of the future and have several pharmacological functions, but the effects of hepatic insulin resistance are still insignificant. Accordingly, the present inventors investigated the hepatotoxic-related insulin resistance effect of the beetle larva extract (ADLE) and the molecular mechanism in HepG2 cells treated with palmitate using a C57BL/6J mouse model induced by a high fat diet. As a result, ADLE alleviated fasting blood sugar, insulin resistance, decreased serum and lipid profile of liver tissue, and accumulated liver steatosis. In addition, it reduced the expression of adipogenesis-related acetyl CoA carboxylase (ACC), FAS, and SREBP-1c through activation of AMPK phosphorylation in liver tissue, and lipid accumulation in HepG2 cells through down-regulation of adipogenesis-related mRNA and protein. It has been shown to significantly inhibit. The above results show that the ADLE of the present invention effectively improves hyperglycemia through inhibition of hepatic lipogenesis by activation of the AMPK signaling pathway and can be a promising therapeutic agent for the treatment of
이하, 실시예를 통하여 본 발명을 더 상세히 설명한다. 그러나 본 발명은 이하에서 개시되는 실시예에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있는 것으로, 이하의 실시예는 본 발명의 개시가 완전하도록 하며, 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이다. Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and the following embodiments make the disclosure of the present invention complete, and the scope of the invention to those of ordinary skill in the art. It is provided to fully inform you.
실시예 1: 장수풍뎅이 유충 추출물의 준비 Example 1: Preparation of beetle larva extract
본 발명에서 사용한 장수풍뎅이 유충(Allomyrina dichotoma larva)은 예천 버그랜드(경상북도 예천군)에서 구입하였고 건조된 장수풍뎅이 유충을 분쇄기로 분쇄하여 분말로 제조하였다. 그 후 상기 분말에 에탄올/물(70:30, v/v)을 20 mL/g의 비율로 첨가하여 60℃에서 3시간 동안 3회 추출한 후, 여과지(Toyo no.2, Advantec)를 통해 2회 여과하였고 상기 여과액을 진공회전증발기(T <40℃)에서 농축 및 동결건조시킨 후, 탈 이온수(deionized water)에 용해시켰다.The longevity beetle larvae ( Allomyrina dichotoma larva) used in the present invention was purchased from Yecheon Bugland (Yecheon-gun, Gyeongsangbuk-do), and the dried longevity beetle larvae were pulverized with a grinder to prepare a powder. Thereafter, ethanol/water (70:30, v/v) was added to the powder at a ratio of 20 mL/g, extracted three times at 60° C. for 3 hours, and then 2 through a filter paper (Toyo no.2, Advantec). The filtrate was filtered twice, and the filtrate was concentrated and lyophilized in a vacuum rotary evaporator (T <40° C.), and then dissolved in deionized water.
실시예 2: 세포 배양 Example 2: Cell culture
세포 배양을 위해 인간 간모세포종(hepatoblastoma) 세포주인 HepG2 세포를 5% CO2 및 37℃ 대기 조건에서 10% 우태아혈청(Gibco) 및 페니실린/스트렙토마이신(Welgene, Daegu, Korea)을 포함하는 Dulbecco's modified Eagle's medium (Gibco, Paisley, UK)에서 배양하였다. Dulbecco's modified containing 10% fetal bovine serum (Gibco) and penicillin/streptomycin (Welgene, Daegu, Korea) in 5% CO 2 and 37°C atmospheric conditions with HepG2 cells for cell culture. It was cultured in Eagle's medium (Gibco, Paisley, UK).
실시예 3: 팔미트산염의 제조 및 MTT 분석 Example 3: Preparation of palmitate and MTT analysis
팔미트산염의 20 mM 저장 용액을 만들기 위해 팔미트산 나트륨(Sigma, St. Louis, MO, USA)을 1:3 부피비로 5% 소혈청알부민(Sigma)과 혼합하였다. 그 후, HepG2 세포를 24시간 동안 ADLE의 존재 또는 부재하에 0.5 mM 팔미트산에 노출시켰고 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide(MTT; thiazolyl blue)(Duchefa Biochemie BV, Haarlem, Netherlands)를 사용하여 비색계(colorimetry)로 세포 생존력을 측정 하였다. 그 후, 불용성 포르마잔 결정을 2-프로판올에 용해시키고 마이크로 플레이트 분광광도계를 사용하여 540 nm에서 검출 하였다.Sodium palmitate (Sigma, St. Louis, MO, USA) was mixed with 5% bovine serum albumin (Sigma) in a volume ratio of 1:3 to make a 20 mM stock solution of palmitate. Thereafter, HepG2 cells were exposed to 0.5 mM palmitic acid in the presence or absence of ADLE for 24 hours, and 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue) (Duchefa Biochemie BV, Haarlem, Netherlands) was used to measure cell viability by colorimetry. Then, the insoluble formazan crystal was dissolved in 2-propanol and detected at 540 nm using a microplate spectrophotometer.
실시예 4: 동물 모델 Example 4: Animal model
본 발명에서 사용한 4주령된 C57BL/6J 수컷 마우스는 한국생명공학연구원(KRIBB, 대전, 한국)으로 부터 제공받았다. 실험동물의 모든 절차는 을지대학교 기관 동물 관리 및 사용위원회(EUIACUC-18-7)의 승인을 받았다. 상기 마우스는 일주일 동안 표준 식단과 물을 자유식이로 일주일동안 급이하였다. 상기 마우스는 사료와 물을 자유롭게 섭취하면서 12:12 시간 명암 주기로 주위 온도(23℃)에서 유지되었고 1주일간 적응시킨 후 6주간 고지방식(HFD, 60% 지방, D12492, Research Diets, New Brunswick, NJ, USA)를 급이하였다(n = 36). 그러나 대조군 마우스는 같은 기간 동안 정상 지방식이(normal-fat diet, NFD)로 급이하였다. HFD 급이 6주 후에 혈당치와 체중을 검사하였고 혈당치가 250 mg/dl을 초과하는 마우스를 실험에 사용했다. 당뇨병 마우스는 본 발명의 ADLE(100 mg/kg/day) 또는 비히클(증류수)로 6주간 매일 경구 투여하였고 양성 대조군으로 메트포민(metformin)을 6주간 경구 투여(100 mg/kg/day) 하였다. 그 후, 체중과 식이섭취량을 매주 기록하였고 6주간의 급이 기간 동안 2주 마다 혈당을 측정하였으며 식이 효율비(FER)는 실험 기간(g) 중 실험 기간(g)/총 식이섭취량을 적용하여 증가된 공식 FER= 체중에 따라 계산하였다.A 4-week-old C57BL/6J male mouse used in the present invention was provided by the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, Korea). All procedures for experimental animals were approved by Eulji University Institutional Animal Care and Use Committee (EUIACUC-18-7). The mice were fed a standard diet and water for a week on a free diet for a week. The mice were kept at ambient temperature (23°C) with a 12:12 hour light/dark cycle while freely ingesting food and water, and after being acclimated for 1 week, a high fat diet (HFD, 60% fat, D12492, Research Diets, New Brunswick, NJ) , USA) was fed (n = 36). However, control mice were fed on a normal-fat diet (NFD) for the same period. Blood glucose levels and body weight were examined 6 weeks after HFD feeding, and mice with blood glucose levels exceeding 250 mg/dl were used in the experiment. Diabetic mice were orally administered with ADLE (100 mg/kg/day) or vehicle (distilled water) of the present invention for 6 weeks and metformin as a positive control group for 6 weeks (100 mg/kg/day). Thereafter, body weight and dietary intake were recorded weekly, and blood glucose was measured every two weeks during the six-week feeding period.The dietary efficiency ratio (FER) was applied to the experimental period (g)/total dietary intake during the experimental period (g). Calculated according to the increased formula FER = body weight.
실시예 5: IPGTT 및 IPITTExample 5: IPGTT and IPITT
본 발명자들은 복강 내의 당부하시험(IPGTT) 및 복강 내의 인슐린 내성시험(IPITT)을 수행하였다. 구체적으로 18시간 동안 마우스를 금식한 후, 꼬리 정맥(0 분)으로부터 원터치 글루코미터(Johnson & Johnson, New Brunswick, NJ, USA)를 이용하여 전혈에서 혈당을 측정하였다. 그 후 PBS에 녹인 인슐린 용액을 복강 내 주입(2g/kg)하였고 혈당을 30, 60 및 120분에 검출하였다. 또한 ITT 검사를 위해서 마우스를 4시간 동안 단식시킨 후 꼬리 정맥을 통해 글루코미터(glucometer)로 전혈에서 측정하였고 PBS에 녹인 인슐린 용액을 복강 내 주입 (2 단위 / kg)하였으며 혈당을 30 , 60 및 120분에 검출하였다. 상기 측정 후 0 내지 120분 사이의 곡선하면적(AUC) 사다리꼴 모델(trapezoid model)을 사용하여 포도당 제거 활성(glucose clearance activity)을 정량적으로 평가하였다. 임의의 2개의 시점 사이의 AUC는 (순차 판독 사이의 시간차) × (1차 시점의 포도당 수준 + 2차 시점의 포도당 수준)/2)로 계산되었다.The present inventors performed an intraperitoneal glucose tolerance test (IPGTT) and an intraperitoneal insulin resistance test (IPITT). Specifically, after fasting mice for 18 hours, blood glucose was measured in whole blood from the tail vein (0 min) using a one-touch glucometer (Johnson & Johnson, New Brunswick, NJ, USA). Then, the insulin solution dissolved in PBS was injected intraperitoneally (2g/kg), and blood glucose was detected at 30, 60 and 120 minutes. In addition, for the ITT test, mice were fasted for 4 hours and then measured in whole blood with a glucometer through the tail vein, and an insulin solution dissolved in PBS was intraperitoneally injected (2 units/kg), and blood glucose levels were 30, 60, and 120. It was detected in minutes. After the measurement, the glucose clearance activity was quantitatively evaluated using an AUC trapezoid model between 0 and 120 minutes. The AUC between any two time points was calculated as (time difference between sequential readings) × (glucose level at the first time point + glucose level at the second time point)/2).
실시예 6: 혈액 내 생화학 분석 Example 6: Biochemical analysis in blood
혈액 내 생화학 분석을 위한 혈청 지질 농도는 상용화 키트를 이용하여 제조사의 지침(Asan Pharmaceutical Co., Seoul, Korea)에 따라 혈청 내 총 콜레스테롤(TC), 중성 지방(TG) 및 고밀도 콜레스테롤(HDL) 수준을 측정하였다. 또한 저밀도 콜레스테롤(LDL)의 농도는 종래의 공식(Friedewald, W.T. et al., Clin Chem, 18, 499-502. 1972)을 이용하여 계산하였다(총 콜레스테롤-HDL 콜레스테롤-(트리글리 세라이드/5)). 간 기능 지표로 알려진 aspartate aminotransferase(AST)와 alanine transferase(ALT)는 제조사의 지침(Asan Pharm. Kit)에 따라 측정하였고 단위는 IU/L 단위로 나타내었으며 모든 분석은 자외선 분광계 (TECAN Group Ltd, Shanghai, China)로 측정하였다.Serum lipid concentration for biochemical analysis in blood is determined by using a commercial kit, according to the manufacturer's instructions (Asan Pharmaceutical Co., Seoul, Korea), according to the serum total cholesterol (TC), triglyceride (TG) and high-density cholesterol (HDL) levels. Was measured. In addition, the concentration of low-density cholesterol (LDL) was calculated using a conventional formula (Friedewald, WT et al., Clin Chem, 18, 499-502. 1972) (total cholesterol-HDL cholesterol-(triglyceride/5)). . Aspartate aminotransferase (AST) and alanine transferase (ALT), known as liver function indicators, were measured according to the manufacturer's instructions (Asan Pharm. Kit), and the units were expressed in IU/L, and all analyzes were performed using an ultraviolet spectrometer (TECAN Group Ltd, Shanghai). , China).
실시예 7: 간 TG 및 TC의 평가 Example 7: Evaluation of liver TG and TC
간 TG 및 TC의 평가를 위해 간에서의 지질 함량은 종래의 방법(Folch, J. et al., J Biol Chem, 226, 497-509. 1957)을 사용하여 측정하였다. 구체적으로 마우스 간(20-40 mg)을 차가운 PBS에서 균질화시킨 후 메탄올/클로로포름(1:2)으로 0.2 mL의 균질액을 추출하였고 2,500 xg에서 10분간 원심분리 하였다. 그 후, 유기상(organic phase)의 분액을 수집하여, 질소하에 건조시켰고, Triton X-100/에탄올 혼합물(1:1, v/v)에 재현탁시켰다. 정량 키트(아산 제약)를 사용하여 간 TG 및 TC 함량을 측정하였고 데이터는 간 추출물의 단백질 농도의 차이에 대해 표준화되었다.For the evaluation of liver TG and TC, the lipid content in the liver was measured using a conventional method (Folch, J. et al., J Biol Chem , 226, 497-509. 1957). Specifically, after homogenizing mouse liver (20-40 mg) in cold PBS, 0.2 mL of homogenate was extracted with methanol/chloroform (1:2), and centrifuged at 2,500 xg for 10 minutes. Then, an aliquot of the organic phase was collected, dried under nitrogen, and resuspended in a Triton X-100/ethanol mixture (1:1, v/v). Liver TG and TC content was measured using a quantification kit (Asan Pharmaceutical), and the data were normalized for the difference in protein concentration of liver extract.
실시예 8: 웨스턴 블랏 분석 Example 8: Western blot analysis
웨스턴 블랏 분석을 위해 간 조직 및 세포 용해물을 프로테아제 억제제 칵테일(Sigma) 및 페닐 메탄슬포닐 플루오라이드(Sigma, PMSF)의 존재하에 포유동물 단백질 추출 완충액(Sigma Chemical Co., St. Louis, MO USA)에서 균질화시켰다. 그 후 상기 용해물을 4℃에서 12,000 rpm으로 20분간 원심분리 하였고 제조사의 지침에 따라 단백질 분석용 염료 시약 농축액(Bio-Rad Laboratories, Hercules, CA, USA)를 사용하여 상등액의 단백질 함량을 측정하였다. 또한 동일한 농도의 단백질을 SDS-폴리아크릴아미드 겔 전기영동으로 분리하였고 니트로 셀룰로오스 블로팅 멤브레인(Amersharm, GE Healthcare Life Science, Germany)으로 이송하였다. 항-SREBP-1(1:1000, Abcam), 항-ACC(1:1000, Cell Signaling Technology), 항-FAS (1:1000, Cell signaling technology), 항-AMPKα(1:1000, Cell signaling technology), 항-phospho-AMPKα(Thr172)(1:1000, Cell signaling technology) 및 항-β-actin(1:2500, Abcam)을 이용하여 면역블로팅(immunoblotting)을 수행하였다. 그 후, ELC 키트(Millipore, USA)를 사용하여 강화된 화학발광(chemiluminescence) 방법으로 단백질 밴드를 시각화 하였고 상기 밴드를 Quantity 1 version 4.6.7 소프트웨어(Bio-Rad)를 이용하여 정량화하였다.For Western blot analysis, liver tissue and cell lysates were prepared in mammalian protein extraction buffer (Sigma Chemical Co., St. Louis, MO USA) in the presence of protease inhibitor cocktail (Sigma) and phenyl methanesulfonyl fluoride (Sigma, PMSF). ). Thereafter, the lysate was centrifuged at 4° C. at 12,000 rpm for 20 minutes, and the protein content of the supernatant was measured using a dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA) for protein analysis according to the manufacturer's instructions. . In addition, proteins of the same concentration were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose blotting membrane (Amersharm, GE Healthcare Life Science, Germany). Anti-SREBP-1 (1:1000, Abcam), anti-ACC (1:1000, Cell Signaling Technology), anti-FAS (1:1000, Cell signaling technology), anti-AMPKα (1:1000, Cell signaling technology ), anti-phospho-AMPKα (Thr172) (1:1000, Cell signaling technology) and anti-β-actin (1:2500, Abcam) were used to perform immunoblotting. Then, the protein band was visualized by an enhanced chemiluminescence method using an ELC kit (Millipore, USA), and the band was quantified using
실시예 9: 정량 실시간 PCR Example 9: Quantitative real-time PCR
qRT-PCR을 위해 Trizol 시약(Invitrogen, Grand Island, NY, USA)을 사용하여 마우스 간으로부터 총 RNA를 추출하였고 PrimescriptTM 1 'strand cDNA 합성 키트 (Takara Bio Inc., Shiga, Japan)를 이용하여 cDNA를 합성하였다. 실시간 PCR 반응은 SYBR Premix Ex Taq II, ROX plus(Takara Bio Inc., Shiga, Japan)를 사용하여 제조사의 지침에 따라 Applied biosystem Inc.(Forster City, CA)의 ABI 실시간 PCR 시스템에서 수행되었다. 증폭 조건은 90℃에서 10분, 95 ℃에서 15초, 60℃에서 1분의 조건으로 총 40 사이클로 수행되었다. 또한 기준 유전자(reference gene)로 시클로필린(Cyclophilin)을 사용하였으며, 모든 결과를 시클로필린 mRNA의 존재량으로 표준화하였고 mRNA의 상대적인 양은 2△△Ct 방법으로 계산하였다. 상기 증폭에 사용된 프라이머의 염기서열을 하기 표 1에 표시하였다. For qRT-PCR, total RNA was extracted from mouse liver using Trizol reagent (Invitrogen, Grand Island, NY, USA), and cDNA was prepared using PrimescriptTM 1'strand cDNA synthesis kit (Takara Bio Inc., Shiga, Japan). Synthesized. Real-time PCR reactions were performed in an ABI real-time PCR system of Applied biosystem Inc. (Forster City, CA) according to the manufacturer's instructions using SYBR Premix Ex Taq II, ROX plus (Takara Bio Inc., Shiga, Japan). The amplification conditions were performed at 90°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute for a total of 40 cycles. In addition, cyclophilin was used as a reference gene, and all results were normalized to the amount of cyclophilin mRNA, and the relative amount of mRNA was calculated by the 2 △△Ct method. The nucleotide sequence of the primers used for the amplification is shown in Table 1 below.
실시예 10: 오일 레드 O 염색 분석 Example 10: Oil Red O staining analysis
조직학적 검사를 위해 마우스 간 일부를 절개하여 4% 완충된 포름알데히드 용액(pH 7.4)에서 24시간 동안 고정시켰다. 동결 절단(Cryosections, 10 μm)을 위해 10분 동안 오일 레드 O 용액(Cayman Chemical, Ann Arbor, MI, USA)과 함께 배양하였다. 그 후, 상기 조직을 Olympus BX61 현미경(Olympus Co., Tokyo, Japan)에 의한 Olympus DP70 디지털 카메라(Olympus Co.)로 검출하였다. For histological examination, a portion of mouse liver was dissected and fixed in 4% buffered formaldehyde solution (pH 7.4) for 24 hours. Incubated with Oil Red O solution (Cayman Chemical, Ann Arbor, MI, USA) for 10 minutes for cryosections (10 μm). Thereafter, the tissue was detected with an Olympus DP70 digital camera (Olympus Co.) by an Olympus BX61 microscope (Olympus Co., Tokyo, Japan).
실험예 1: 체중, 식이섭취량 및 공복 혈당의 변화 Experimental Example 1: Changes in body weight, dietary intake and fasting blood sugar
본 발명자들은 고지방식이(HFD) 유도에 따른 마우스의 체중, 식이섭취량 및 공복 혈당의 변화를 관찰하였다. 먼저, C57BL/6J 마우스에 제2형 당뇨병을 유발하기 위해 고지방식이를 6주간 급이하였고 초기 및 최종 시점(6주)에서 체중, 식이섭취량 및 혈당치의 변화를 측정하였다. The present inventors observed changes in body weight, dietary intake, and fasting blood sugar of mice according to the induction of a high fat diet (HFD). First, in order to induce
그 결과, 투여 6주 후에 HFD 군의 체중은 정상지방식이(NFD)과 비교하여 2.3 배로 유의하게 증가하였으며(p <0.001), HFD 군에서는 식이섭취가 감소했고 공복 혈당도 NFD 군보다 HFD 군에서 증가하였다(표 2 참조). 제2형 당뇨병 유도 C57BL/6J 마우스의 체중, 식이섭취 및 혈당 변화를 하기 표 2에 요약하였다.As a result, 6 weeks after administration, the body weight of the HFD group significantly increased by 2.3 times compared to that of the normal fat diet (NFD) (p <0.001), and the dietary intake decreased in the HFD group, and the fasting blood glucose was also higher in the HFD group than in the NFD group. Increased (see Table 2). Changes in body weight, dietary intake and blood glucose of
(g/day)Dietary intake
(g/day)
(mg/dL)Blood sugar
(mg/dL)
*값은 평균 ± SD이다. NFD: 정상지방식이, HFD: 고지방식이*Values are mean ± SD. NFD: normal fat diet, HFD: high fat diet
*** t- 검정에 의한 p <0.001의 실험군 간의 유의한 차이.*** Significant difference between experimental groups with p <0.001 by t-test.
실험예 2: ADLE의 투여에 따른 체중 및 혈당의 변화 Experimental Example 2: Changes in body weight and blood sugar according to ADLE administration
본 발명의 ADLE의 투여가 마우스의 당뇨병 표현형(diabetic phenotype)을 감소시키는지를 조사하기 위해 ADLE 100 mg/kg을 제2형 당뇨병 유도 마우스에 6주간 투여하였고 메트포르민(MET, 100 mg/kg)을 양성 대조군으로 사용하였다.To investigate whether the administration of ADLE of the present invention reduces the diabetic phenotype of mice,
그 결과, HFD 군의 체중은 NFD 군보다 유의하게 높았으며(p <0.001), ADLE 및 메트포르민 투여는 HFD 군에 비해 체중을 감소한 것으로 나타났다(도 1a). 그러나 체중의 변화를 관찰한 결과, ADLE이 아닌 MET을 투여한 HFD 군이 HFD 군에 비해 체중이 유의하게 감소한 것으로 나타났다(도 1b). 식이섭취량에서는 NFD군보다 HFD군에서 유의적 감소를 보이나 ADLE, Met투여에 의한 섭취량의 유의적 차이는 없었으며(도 1c) HFD에 의해 증가된 식이 효율비(FER)는 ADLE 또는 MET의 투여에 의해 변화되지 않았다(도 1d). 그러나 ADLE 투여는 HFD 군에 비해 공복혈당 수준을 유의하게 감소시켰으며(p <0.05), MET 투여군과 비슷한 효과를 나타내었다(도 1e).As a result, the body weight of the HFD group was significantly higher than that of the NFD group (p <0.001), and ADLE and metformin administration decreased the body weight compared to the HFD group (Fig. 1a). However, as a result of observing the change in body weight, it was found that the HFD group administered MET, not ADLE, significantly decreased the body weight compared to the HFD group (Fig. 1b). In terms of dietary intake, there was a significant decrease in the HFD group than in the NFD group, but there was no significant difference in the intake by ADLE and Met administration (Fig. 1c), and the dietary efficiency ratio (FER) increased by HFD was determined by the administration of ADLE or MET. Did not change by (Fig. 1D). However, ADLE administration significantly reduced the fasting blood glucose level compared to the HFD group (p <0.05), and showed similar effects to the MET administration group (Fig. 1e).
실험예 3: 포도당 및 인슐린 내성 분석 Experimental Example 3: Glucose and insulin resistance analysis
본 발명자들은 ADLE 투여가 당뇨병 유도 마우스에서 내당능(glucose resistance)과 인슐린 감수성을 감소시키는지를 조사하기 위해, ADLE 투여 여부에 관계없이 6주 후 HFD 군에서 포도당 내성 검사(GTT) 및 인슐린 내성 검사(ITT)를 수행하였다.In order to investigate whether ADLE administration reduces glucose resistance and insulin sensitivity in diabetes-induced mice, the present inventors investigated glucose tolerance test (GTT) and insulin resistance test (ITT) in HFD group after 6 weeks regardless of ADLE administration. ) Was performed.
그 결과, 포도당 내성은 NFD 군과 비교하여 HFD 군에서 손상되었으나, ADLE 투여군에서는 개선되었다. 또한 HFD 군의 AUC가 NFD 군보다 높았으며(p <0.001), ADLE의 투여로 유의하게 감소한 것으로 나타났다(p <0.05, 도 2a 및 2b). 아울러, HFD에 의한 인슐린 내성의 증가는 ADLE 투여에 의해 감소되었으며 HFD 군와 비교하여 ITT 값의 AUC에서 유의 한 감소가 관찰되었다(p <0.05, 도 2c 및 2d).As a result, glucose tolerance was impaired in the HFD group compared to the NFD group, but improved in the ADLE administration group. In addition, the AUC of the HFD group was higher than that of the NFD group (p <0.001), and it was found to be significantly reduced by the administration of ADLE (p <0.05, FIGS. 2A and 2B). In addition, the increase in insulin resistance caused by HFD was reduced by ADLE administration, and a significant decrease was observed in the AUC of the ITT value compared to the HFD group (p <0.05, FIGS. 2C and 2D).
실험예 4: 지질 프로파일 및 AST/ALT 분석 Experimental Example 4: Lipid Profile and AST/ALT Analysis
본 발명자들은 당뇨병 유도 마우스(HFD)의 혈청 지질 프로파일에 대한 ADLE의 효과를 조사하기 위해 ADLE 추출물을 포함 또는 포함하지 않은 HFD 군에서 총 콜레스테롤(TC), TG, 고밀도 지단백질(HDL) 및 저밀도 지단백질(LDL)의 수준을 비교했다.In order to investigate the effect of ADLE on the serum lipid profile of diabetes-inducing mice (HFD), the present inventors investigated the total cholesterol (TC), TG, high-density lipoprotein (HDL) and low-density lipoprotein in the HFD group with or without ADLE extract. LDL) levels were compared.
그 결과, TC, HDL 및 LDL의 혈청 농도는 NFD 군보다 HFD 군에서 유의하게 증가 하였지만(p <0.01 또는 p <0.001), HFD 군 및 ADLE 투여 HFD군 간에 유의한 차이는 관찰되지 않았다. 그러나 HFD 군에서 TG의 증가는 ADLE 투여로 유의하게 감소하였고(p <0.05), 그 효과는 MET 투여군과 유사하게 나타났다(도 3a). 또한 간 기능에 대한 ADLE의 효과를 시험하기 위해, AST 및 ALT의 효소 활성을 측정한 결과 HFD에 의한 증가된 AST 수준은 ADLE 및 MET(각각 20.16% 및 29.83%) 투여에 따라 현저히 감소되었고 혈청 ALT의 수준은 HFD 군이 NFD 군에 비해 166% 까지 유의하게 증가하였으며 ADLE 및 MET(각각 46.27% 및 13.09%) 투여에 따라 유의하게 감소되었다(도 3b).As a result, serum concentrations of TC, HDL, and LDL were significantly increased in the HFD group than in the NFD group (p <0.01 or p <0.001), but no significant difference was observed between the HFD group and the ADLE-administered HFD group. However, the increase in TG in the HFD group was significantly reduced by ADLE administration (p <0.05), and the effect was similar to that of the MET administration group (Fig. 3a). In addition, to test the effect of ADLE on liver function, as a result of measuring the enzymatic activity of AST and ALT, the increased AST level by HFD was significantly decreased with administration of ADLE and MET (20.16% and 29.83%, respectively), and serum ALT was significantly reduced and serum ALT. The level of was significantly increased by 166% in the HFD group compared to the NFD group, and significantly decreased with ADLE and MET (46.27% and 13.09%, respectively) administration (Fig. 3b).
실험예 5: 간에서의 지질 축적 분석 Experimental Example 5: Analysis of lipid accumulation in liver
당뇨병 마우스에서 ADLE 투여에 의해 TG 및 GOT의 혈청 농도가 유의하게 감소되었으므로 본 발명자들은 간 절편에서 오일 레드 O 염색을 이용하여 간 지방 축적을 조사하였다.Since the serum concentrations of TG and GOT were significantly reduced by ADLE administration in diabetic mice, the present inventors investigated liver fat accumulation using Oil Red O staining in liver sections.
그 결과, 간의 오일 레드 O 염색 세포(적색 반점)는 NFD 군에 비해 HFD 군에서 증가하였고, ADLE 및 MET 투여에 의해 염색 세포가 현저하게 감소되었음을 확인하였다(도 4a). 또한 각 실험군의 간 용해물에서 TG 및 TC 함량을 확인한 결과, HFD 군의 TG 및 TC 농도는 NFD 군에 비해 유의하게 증가하였고, ADLE 투여에 의해 억제되는 것으로 나타났다(p <0.05, 도 4b).As a result, it was confirmed that the liver oil red O stained cells (red spots) increased in the HFD group compared to the NFD group, and stained cells were significantly reduced by administration of ADLE and MET (FIG. 4A). In addition, as a result of confirming the TG and TC contents in the liver lysates of each experimental group, the TG and TC concentrations of the HFD group were significantly increased compared to the NFD group, and were found to be inhibited by ADLE administration (p <0.05, FIG. 4b).
실험예 6: 항-지방생성 효과 Experimental Example 6: Anti-adipogenic effect
본 발명의 ADLE의 항-지방생성 효과와 ADLE의 분자 메커니즘을 조사하기 위해 팔미트산염(palmitate, 500 μM)으로 유도된 HepG2 세포에서 ADLE(0.1 ~ 1.0 mg/ml)를 처리하여 지방생성(lipogenic) 발현을 관찰하였다. In order to investigate the anti-adipogenic effect of ADLE and the molecular mechanism of ADLE of the present invention, ADLE (0.1 ~ 1.0 mg/ml) was treated in HepG2 cells induced with palmitate (500 μM) ) The expression was observed.
그 결과, HepG2 세포에서 ADLE의 세포 독성을 확인 하였을 때, 추출물을 0.1 ~ 0.5 mg/ml로 24시간 동안 처리한 경우 세포 생존율에 변화가 없었지만 1 mg/ml으로 처리한 경우 유의한 감소를 나타내었다(도 5a). 또한 0.5 mg/ml ADLE를 사용하여 세포 내 TG 함량을 관찰했을 때, 본 발명의 ADLE의 처리는 HepG2 세포에서 팔미트산염에 의해 유발된 TG 수준을 완화시켰다(p <0.05, 도 5b).As a result, when confirming the cytotoxicity of ADLE in HepG2 cells, there was no change in cell viability when the extract was treated at 0.1 ~ 0.5 mg/ml for 24 hours, but when treated with 1 mg/ml showed a significant decrease. (Fig. 5a). In addition, when 0.5 mg/ml ADLE was used to observe the intracellular TG content, the treatment of ADLE of the present invention alleviated the TG level induced by palmitate in HepG2 cells (p <0.05, FIG. 5B).
또한, ADLE에 의해 지방생성 유전자(lipogenic genes)가 감소된 TG 수준에 관여하는지 여부를 시험하기 위해, 스테롤 조절 요소 결합 단백질(sterol regulatory element-binding protein, SREBP), 지방산 합성 효소(fatty acid synthase, FAS) 및 아세틸-CoA 카르복시라제(acetyl-CoA carboxylase, ACC)의 mRNA 및 단백질 수준을 조사한 결과, 팔미트산염에 의해 증가된 SREBP-1c, FAS 및 ACC의 mRNA 발현은 ALDE에 의해 감소되었고(도 5c) 단백질 발현 수준도 팔미트산염에 의해 상향 조절되었으며 ADLE로 처리한 후 HepG2 세포에서 유의하게 감소하였다(도 5d). 마지막으로, AMPK(AMP-activated protein kinase)의 탈인산화(dephosphorylation)가 간 지질(hepatic lipid) 대사의 조절에 중요한 역할을 한다는 보고에 따라 상기 실험 조건에서 AMPK의 인산화를 조사한 결과, AMPK의 기본 발현은 변화하지 않았지만 Thr 172(pAMPK)에서 AMPK의 인산화는 팔미트산염에 의해 감소되었으며 ADLE 동시 처리에 의해 개선되었다. 또한 팔미트산염을 처리한 HepG2 세포에서 감소된 pAMPK/AMPK 비율은 ADLE 처리에 의해 억제되었다(도 5e).In addition, to test whether lipogenic genes are involved in reduced TG levels by ADLE, sterol regulatory element-binding protein (SREBP), fatty acid synthase, As a result of examining the mRNA and protein levels of FAS) and acetyl-CoA carboxylase (ACC), the mRNA expression of SREBP-1c, FAS and ACC increased by palmitate was decreased by ALDE (Fig. 5c) The protein expression level was also upregulated by palmitate and significantly decreased in HepG2 cells after treatment with ADLE (Fig. 5D). Finally, according to the report that dephosphorylation of AMPK (AMP-activated protein kinase) plays an important role in the regulation of hepatic lipid metabolism, phosphorylation of AMPK was investigated under the above experimental conditions. As a result, the basic expression of AMPK Was not changed, but phosphorylation of AMPK at Thr 172 (pAMPK) was reduced by palmitate and was improved by simultaneous ADLE treatment. In addition, the decreased pAMPK/AMPK ratio in HepG2 cells treated with palmitate was inhibited by ADLE treatment (Fig. 5e).
실험예 7: AMPK의 지방생성 유전자와 인산화 Experimental Example 7: Adipogenesis Gene and Phosphorylation of AMPK
본 발명자들은 AMPK의 지방생성 유전자 및 인산화가 ADLE로 처리된 HepG2 세포의 감소된 지질 축적에 관여한다는 것을 발견하여 HFD 유도 당뇨병 마우스의 간 용해물을 이용하여 상기 메카니즘을 확인했다.The present inventors found that the adipogenic gene and phosphorylation of AMPK are involved in decreased lipid accumulation in HepG2 cells treated with ADLE, and thus the mechanism was confirmed using liver lysates of HFD-induced diabetic mice.
그 결과, 지질생성 유전자의 mRNA 및 단백질 발현양은 NFD 군에 비해 HFD 군의 간에서 증가하였고 ADLE 처리에 따라 유의하게 억제되었다(도 6a 및 6b). 또한 HFD 군의 pAMPK/tAMPK 비율은 대조군과 비교하여 유의하게 감소하였고(p <0.001), ADLE 처리에 의해 회복되었다(p <0.01)(도 7a 및 7b). As a result, the mRNA and protein expression levels of the lipid-generating gene were increased in the liver of the HFD group compared to the NFD group, and were significantly suppressed by ADLE treatment (FIGS. 6A and 6B ). In addition, the ratio of pAMPK/tAMPK in the HFD group was significantly decreased compared to the control group (p <0.001), and recovered by ADLE treatment (p <0.01) (Figs. 7A and 7B).
결론적으로, 본 발명의 일 실시 예에 따른 장수풍뎅이 유충 추출물 또는 분말의 유효성분을 포함하는 지방간 예방 및 치료용 조성물은 지방생성 관련 유전자의 발현을 감소시키고 지방생성 관련 mRNA 및 단백질의 하향조절을 통해 HepG2 세포에서 지질 축적을 유의하게 억제하였으며 AMPK 신호 전달 경로의 활성화에 의한 간 지방생성의 억제를 통해 고혈당을 효과적으로 개선시켰으므로 지방간 및 제2형 당뇨병 치료를 위한 치료제로 활용가능하다(도 8). In conclusion, the composition for preventing and treating fatty liver comprising an active ingredient of a beetle larva extract or powder according to an embodiment of the present invention reduces the expression of adipogenesis-related genes and down-regulates adipogenesis-related mRNA and protein. Since it significantly inhibited lipid accumulation in HepG2 cells and effectively improved hyperglycemia through inhibition of liver adipogenesis by activation of the AMPK signaling pathway, it can be used as a therapeutic agent for the treatment of fatty liver and
본 발명은 상술한 실시예 및 실험예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예 및 실험예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구범위의 기술적 사상에 의하여 정해져야 할 것이다.The present invention has been described with reference to the above-described Examples and Experimental Examples, but these are only exemplary, and those of ordinary skill in the art understand that various modifications and equivalent other Examples and Experimental Examples are possible therefrom. I will understand. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.
<110> EULJI UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION <120> Compositions for preventing or treating fatty liver disease comprising Allomyrina dichotoma larva extract <130> PD19-5811 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SREBP-1c F <400> 1 cttctggaga catcgcaaac 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SREBP-1c R <400> 2 ggtagacaac agccgcatc 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACC F <400> 3 aggaagatgg cgtccgctct g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACC R <400> 4 ggtgagatgt gctgggtcat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS F <400> 5 cttgggtgct gactacaacc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS R <400> 6 gccctcccgt acactcactc 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cyclophilin F <400> 7 tggagagcac caagacagac a 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cyclophilin R <400> 8 tgccggagtc gacaatgat 19 <110> EULJI UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION <120> Compositions for preventing or treating fatty liver disease comprising Allomyrina dichotoma larva extract <130> PD19-5811 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SREBP-1c F <400> 1 cttctggaga catcgcaaac 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SREBP-1c R <400> 2 ggtagacaac agccgcatc 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ACC F <400> 3 aggaagatgg cgtccgctct g 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACC R <400> 4 ggtgagatgt gctgggtcat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS F <400> 5 cttgggtgct gactacaacc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS R <400> 6 gccctcccgt acactcactc 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cyclophilin F <400> 7 tggagagcac caagacagac a 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cyclophilin R <400> 8 tgccggagtc gacaatgat 19
Claims (7)
상기 추출물은 물, C1 내지 C4의 저급알코올 또는 이들의 혼합물로 추출한 것인, 조성물.The method of claim 1,
The extract will be extracted with water, C1 to C4 lower alcohol or a mixture thereof.
상기 저급알코올은 메탄올, 에탄올 또는 프로판올인, 조성물.The method of claim 2,
The lower alcohol is methanol, ethanol or propanol, composition.
상기 지방간은 비알콜성 지방간인, 조성물.The method of claim 1,
The fatty liver is non-alcoholic fatty liver, composition.
건조된 장수풍뎅이 유충을 분쇄기로 분쇄하여 분말로 제조하는 분말 제조단계;
상기 분말에 에탄올 및 물을 1:9 내지 9:1의 비율로 첨가 후 50 내지 70℃에서 2 내지 4시간 동안 추출하는 추출물 제조단계;
상기 추출물을 여과 후 농축 및 동결건조시킨 후 탈 이온수에 용해시켜 수득하는 추출물 수득단계를 포함하는 제조방법에 의해 제조되는, 조성물.The method of claim 1,
Powder manufacturing step of pulverizing the dried long-sleeved beetle larvae into powder by grinding with a grinder;
Extract preparation step of extracting for 2 to 4 hours at 50 to 70 ℃ after adding ethanol and water in a ratio of 1:9 to 9:1 to the powder;
A composition prepared by a manufacturing method comprising the step of obtaining an extract obtained by filtering the extract, concentrating and lyophilizing it, and dissolving it in deionized water.
탕제, 드링크제, 산제, 환제, 캡슐, 정제 또는 젤리의 제형으로 제공되는, 건강기능식품.
The method of claim 6,
A health functional food provided in the form of a bath, drink, powder, pill, capsule, tablet or jelly.
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| CN115812849A (en) * | 2022-12-09 | 2023-03-21 | 中国热带农业科学院环境与植物保护研究所 | Complete feed for improving utilization rate of hermetia illucens powder and preparation method thereof |
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