[보고서]두충의 간기능개선 소재 개발

이금화

[국가R&D연구보고서] 두충의 간기능개선 소재 개발
Development of a functional material to protect non-alcohlic fatty liver disease from Eucommia ulmoides leaf 원문보기

보고서 정보
주관연구기관	전북대학교병원
연구책임자	이금화
참여연구자	박선아 , 이화영
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2019-02
과제시작연도	2018
주관부처	농촌진흥청 Rural Development Administration(RDA)
과제관리전문기관	농촌진흥청 Rural Development Administration
등록번호	TRKO201900016134
과제고유번호	1395056887
사업명	농업첨단핵심기술개발사업(R&D)
DB 구축일자	2019-10-26
키워드	두충잎.간기능.추출물.기전.기능성.NAFLD.ER stress.Eucommia ulmoides leaf.autophagy.mTOR.
DOI	https://doi.org/10.23000/TRKO201900016134

초록 ▼

❍ 두충잎 열수추출물의 표준화 분석
❍ 내피세포활성으로 인한 항산화 역할규명
❍ 두충잎 추출물의 고지방식이 동물모델에서 간기능 개선 효능 검증
- 두충잎 추출물의 지방간 동물모델에서 간독성억제효능 검증
- 두충잎 추출물의 중성지방축적 억제 기전 연구
- 두충잎 추출물의 소포체스트레스 억제효과 검증
- 두충잎 추출물의 간조직 내 산화적 스트레스 억제 효과 평가
- 두충잎 추출물의 lysosome활성 조절 규명
- Hep G2세포에서 palmitate 처리로 인한 두충잎 추출물의 보호효과 검증

Abstract ▼

Purpose&Contents
Development of a functional material to protect non-alcohlic fatty liver disease from Eucommia ulmoides leaf

Results
The recent discovery that the impairment of autophagic flux in non-alcoholic fatty liver disease (NAFLD) might be a strong determining factor in steatosis suggests the potential of therapeutic control of autophagic flux with natural agents in restoring NAFLD.
We investigated the potential of Eucommia ulmoides leaf extract (EUL) to control dyslipidemia in NAFLD. EUL supplementation (200 mg/kg) promoted recovery from high fat diet (HFD)-induced lipid dysmetabolism. This hepatoprotective efficacy was accompanied by suppression of endoplasmic reticulum (ER) stress, enhancing lysosomal functions and thereby increasing autophagic flux. We found a strong indication that inhibition of the mTOR-ER stress pathway was related to the enhanced autophagic flux. However, the direct antioxidative effect of EUL on cytoprotection cannot be ruled out as a significant contributing factor in NAFLD. Our findings will aid in further elucidating the mechanism of the anti-steatosis activity of EUL and highlight the therapeutic potential of EUL in the treatment of NAFLD

Expected Contribution
In an HFD-induced NAFLD model, EUL exerts positive effects on hepatocyte damage caused by HFD by reducing oxidative stress and restoring lipid metabolism, leading to lysosomal activation and the alleviation of ER stress. The results of this study may lead to the development of novel strategies to prevent complications of NAFLD

(출처 : Summary 5p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국 문 요 약 문 ... 4
Summary ... 5
목차 ... 6
제 1 장 연구 개발 과제의 개요 ... 7
제1절 연구개발의 개요 ... 7
제2절 연구 개발의 필요성 ... 7
제3절 연구 개발 범위 ... 9
제 2 장 연구 수행 내용 및 결과 ... 15
제1절 두충잎 추출물의 표준화분석 ... 15
제2절 두충의 간기능소재개발 –내피세포활성으로 인한 혈행 개선 효능 ... 17
제3절 두충잎추출물의 고지방식이 동물모델에서 간기능 개선 효능 검증 ... 23
제4절 두충잎추출물의 자연노화로 인한 간기능저하 모델에서 간 기능개선 효능 검증 ... 31
제5절 고찰 ... 36
제 3 장 목표달성도 및 관련분야 기여도 ... 38
제1절 : 목표대비 달성도 ... 38
제2절 : 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 38
제 4 장 연구 결과의 활용 계획 ... 39
제 5 장 연구 개발 결과의 보안 등급 ... 39
제 6 장 국가과학기술지식정보서비스에 등록한 연구시설·장비 현황 ... 39
제 7 장 연구개발과제의 대표적 연구실적 ... 40
제 8 장 기타사항 ... 41
제 9 장 참고문헌 ... 42
끝페이지 ... 45

표/그림 (28)

표 연도별 건강기능식품 생산실적 (‘13.12.31.기준, 출처: 식약처)
표 열수 추출물의 추출공정과 수득량
표 분석 compound
표 주요성분분석
표 Chromatogram of Standards
표 Chromatogram of EUL extract
표 HPLC analysis of EUL extracts. A) Chemical structure of aucubin. Chromatograms for aucubin were analyzed for standard solution B) and for EUL extracts C). EUL, Eucommia ulmoides leaf
표 EUL regulates the effects of ox-LDL on NO production. HUVECs were treated with 100, 250, or 500 μg/mL EUL, and then incubated with 20 μg/mL ox-LDL for another 24 h. A) NO release was measured as described in the Materials and Methods. B) Intracellular NO formation was measured via image analysis of NO-sensitive fluorochrome, DAF-2DA. All data are presented as mean ± SE from 3 independent experiments. *P < 0.05 vs. ox-LDL. EUL, Eucommia ulmoides leaf; HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein; DAF-2DA, diaminofluorescein–2 diacetate
표 EUL regulates ox-LDL-induced ROS production. HUVECs were treated with 100, 250, or 500 μg/mL EUL, and then were incubated with 20 μg/mL ox-LDL for another 24h. A) DPPH free radical scavenging activity of EUL in vitro B) Endothelial O2−production was assessed as described in the Materials and Methods. C) In DHE-staining analysis (another method of O2 − analysis), fluorescence was measured as described in the Materials and Methods. The fluorescence distribution of DHE oxidation was quantified as arbitrary units (right). D) HUVECs were exposed to 20 μg/mL ox-LDL in the presence or absence of 500 μg/mL EUL for 24 h. Representative chromatograms and retention times of 2-EOH and ethidium from 20 μg/mL ox-LDL in the presence or absence of 500 μg/mL EUL. The concentration of 2-hydroxyethidium (2-EOH) was determined by an LC–MS detector system. Data are expressed relative to the control (n=3, *P < 0.05 vs. ox-LDL). EUL, Eucommia ulmoides leaf; HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein; DHE, dihydroethidium
표 EUL maintains eNOS activity. A) HUVECs were treated with 100, 250, or 500 μg/mL EUL, then incubated with 20 μg/mL ox-LDL for 24 h. B) Cells were exposed to 20μg/mL ox-LDL in the presence or absence of 500 μg/mL EUL for 0, 6, 12, or 24 h. Immunoblotting was performed with anti-p-Thr495 eNOS, total eNOS or β-actin antibody. Data are expressed relative to the control (n=3, *P < 0.05 vs. ox-LDL) EUL, Eucommia ulmoides leaf; HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein
표 Aucubin regulates oxLDL-induced ROS production, affecting levels of NO production. HUVECs were treated with 5, 10, or 25 μg/mL aucubin, and then incubated with 20 μg/mL ox-LDL for another 24 h. A) NO release was analyzed as described in the Materials and Methods. B) Intracellular NO formation was measured via image analysis of the NOsensitive fluorochrome, DAF-2DA. C) Endothelial O2 − production was assessed as described in the Materials and Methods. D) In DHE-staining analysis (another form of O2−analysis), fluorescence was measured as described in the Materials and Methods. The fluorescence distribution of DHE oxidation was quantified as arbitrary units. E) HUVECs were exposed to 20 μg/mL ox-LDL in the presence or absence of 25 μg/mL aucubin for 24 h. Representative chromatograms and retention times of 2-EOH and ethidium from 20 μg/mL ox-LDL in the presence or absence of 25 μg/mL aucubin. The concentration of 2-hydroxyethidium (2-EOH) was determined by an LC–MS detector system. All data are presented as mean ± E from 3 independent experiments. *P < 0.05 vs. ox-LDL for 24 h. EUL, Eucommia ulmoides leaf; HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein; DHE, dihydroethidium;
표 Aucubin regulates oxLDL-induced eNOS expression.A) HUVECs were treated with 5, 10, or 25 μg/mL aucubin, and then incubated with 20 μg/mL ox-LDL for 24 h. B) HUVECs were exposed to 20 μg/mL ox-LDL in the presence or absence of 25 μg/mL aucubin for 0, 6, 12, or 24 h. Immunoblotting was performed with anti-p-Thr495 eNOS, total eNOS or β-actin antibody. All data are presented as mean ± SE from 3 independent experiments.*P < 0.05 vs. ox-LDL for 24 h, **P < 0.05 vs. ox-LDL for 24 h
표 Quantification of O2- production in the presence of L-NAME in Lucigenin-enhanced chemiluminescence assay
표 Body weight and fasting glucose level in HFD-induced hepatic functional damage. Results are means±SD from 3 separate experiments
표 EUL extract protect against HFD-induced hepatic functional damage. (A) AST, ALT, and GGT (B) were measured in HFD-treated rats. (B) H&E) staining assays was performed using the liver (100x magnification). Values are means ± SD. (n = 6, *P < 0.05 vs. NCD-group, **P < 0.05 vs. HFD-group)
표 EUL prevents HFD-induced hepatic steatosis. Rats were given a normal diet normal diet or a high-fat diet with or without 100mg/kg and 200mg/kg EUL extract for 6 wk, and serum and liver were harvested. (A). Biochemical analysis of serum samples. Levels of TG, TC and LDL-c were measured in the serum of rat in different experimental groups. (C) Liver weight, and (D) Liver weight/Body weight and (E) liver TG content were measured 6 wk after initial mulberry administration (n = 6).(E) Liver tissues retrieved 6 wk after initial mulberry administration were subjected to Oil red O staining, Scale bars, 200 μm. Values are means ± SD. (n = 6, *P < 0.05 vs. NCD-group, **P < 0.05 vs. HFD-group)
표 EUL inhibits ER stress in hyperlipidemic rats (A) Experimental scheme. Rat fed with HFD for 6 weeks were given 100mg/kg or 200mg/kg EUL extract for 8 weeks and sacrificed. Hepatic lysates were prepared from the NCD, HFD groups and HFD plus EUL group analyzed by western blotting assay. (A–C) Representative western blots with the indicated antibodies. (D) Representative EM images (upper panel) anti-p62 immunostaining (middle panel), and anti-p62 immunostaining images (lower panel)
표 EUL inhibits oxidative stress in hyperlipidemic rats (A) MDA levels in liver was assessed, respectively. GSH (B), SOD(C) , and GPX(D) activity were measured in HFD-treated rats. (E) Expression of oxidized protein in liver tissue lysates was determined by Oxy blot assay. Values are means ± SD. (n = 6, *P < 0.05 vs. NFD-group, #P < 0.05 vs. HFD-group)
표 EUL inhibits oxidative stress in hyperlipidemic rats DHE (A) and 4-HNE (B) were analyzed. Liver tissue was isolated and loaded with 5 μM dihydroethidium. Fluorescence images were acquired. Scale bars, 50 μm Quantification of fluorescence intensity for superoxide levels in liver. Values are means ± SD. (n = 6, *P < 0.05 vs. NFD-group, #P < 0.05 vs. HFD-group)
표 EUL regulates HFD-induced lysosomal BAX localization and lysosomal enzyme activation. (A) Cathepsin B activity in the liver lysate was measured. (B) Western blotting was carried out with antibody against BAX, cathepsin B, LAMP-1, or tubulin (C). Liver lysate were treated with NCD, HFD groups and HFD plus EUL group and the level of a-galactosidase, a-mannosidase, or acid phosphatase was measured (D). Values are means ± sem. (n = 6, *P < 0.05 vs. NFD-group, #P < 0.05 vs. HFD-group)
표 Effects of EUL on ER stress and lysosomal function in HepG2 cells. (A) HepG2 cells were treated the indicated concentrations of 50 μg/ml, 100 μg/ml, and 250 μg/ml EUL for 24 h for indicated periods, and protein level was etermined using the western blotting assay. (A–C) Representative western blots with the indicated antibodies. (D) HepG2 cells were incubated with the indicated concentrations of EUL extract for 24 h, and cathepsin B activity was determined. (E) HepG2 cells were treated with the indicated concentrations of 50 μg/ml, 100 μg/ml, and 250 μg/ml EUL for 24 h for indicated periods, and cytotoxicity was determined using the MTT assay. Values are means ± SD. n = 3, *P < 0.05 vs. control, #P < 0.05 vs. palmitate-treated
표 Effects of acitive compound on ER stress and lysosomal function in HepG2 cells. (A) HepG2 cells were treated the indicated concentrations of 250 μg/ml EUL, 25μg/ml aucubin, and 25μg/ml geniposide for 24 h for indicated periods, and protein level was etermined using the western blotting assay. (A–C) Representative western blots with the indicated antibodies. (D) HepG2 cells were incubated with the indicated concentrations of EUL, 25μg/ml aucubin, and 25μg/ml geniposide for 24 h, and cathepsin B activity was determined. (E) HepG2 cells were treated with the indicated concentrations of EUL, 25μg/ml aucubin, and 25μg/ml geniposide for 24 h for indicated periods, and cytotoxicity was determined using the MTT assay. Values are means ± SD. n = 3, *P < 0.05 vs. control, #P < 0.05 vs. palmitate-treated
표 Body weight and fasting glucose level in aging-induced hepatic functional damage. Results are means±SD from 3 separate experiments
표 EUL extract protect against aging-induced hepatic functional damage. (A) AST, ALT, and GGT (B) were measured in aging rats. (B) H&E) staining assays was performed using the liver (100x magnification). Values are means ± SD. (n = 6, *P < 0.05 vs. Young-group, **P < 0.05 vs. aging-group)
표 EUL prevents aging-induced hepatic steatosis. Rats were given a young rat or an aging rat with or without 100mg/kg and 200mg/kg EUL extract for 6 wk, and serum and liver were harvested. (A). Biochemical analysis of serum samples. Levels of TG, TC and LDL-c were measured in the serum of rat in different experimental groups. (C) Liver weight, and (D) Liver weight/Body weight and (E) liver TG content were measured 6 wk after initial mulberry administration (n = 6).(E) Liver tissues retrieved 6 wk after initial EUL administration were subjected to Oil red O staining, Scale bars, 200 μm. Values are means ± SD. (n = 6, *P < 0.05 vs. Young-group, **P < 0.05 vs. aging-group)
표 EUL inhibits oxidative stress in hyperlipidemic rats (A) MDA levels in liver was assessed, respectively. GSH (B), SOD(C) , and GPX (D) activity were measured in aging rats. (E) DHE was analyzed. Liver tissue was isolated and loaded with 5 μM dihydroethidium. Fluorescence images were acquired. Scale bars, 50 μm Quantification of fluorescence intensity for superoxide levels in liver. Values are means ± SD. (n = 6, *P < 0.05 vs. Young-group, #P < 0.05 vs. aging-group)
표 EUL inhibits ER stress in aging rats (A-B) Experimental scheme. Aging rat for 6 weeks were given 100mg/kg or 200mg/kg EUL extract for 8 weeks and sacrificed. Hepatic lysates were prepared from the Young, aging groups and aging plus EUL group analyzed by western blotting assay
표 EUL regulates aging-induced lysosomal BAX localization and lysosomal enzyme activation. (A) Immunoblotting was performed using antibodies against anti-LC-3, p62, and β-actin (B) Cathepsin B activity in the liver lysate was measured. (C) Western blotting was carried out with antibody against BAX, cathepsin B, LAMP-1, or tubulin (D). Liver lysate were treated with young, aging groups and aging plus EUL group and caspase-3 activity was measured (D). Values are means ± SD. (n = 6, *P < 0.05 vs. Young-group, #P < 0.05 vs. aging-group)

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[국가R&D연구보고서] 두충의 간기능개선 소재 개발
Development of a functional material to protect non-alcohlic fatty liver disease from Eucommia ulmoides leaf 원문보기