만성음주자의 경우 비대해진 Cytochrome P4502E1에 의해 알코올이 산화되며, 이 과정에서 강화된 NADPHP450 reducatse (CYPR)와 상호작용하게 되어 활성산소종이 발생하여, 간세포 손상의 원인이 된다. 본 연구에서는 알코올 유도 CYP2E1으로부터 발생된 활성산소종의 소거계검색을 위한 in vitro assay계를 확립하고 식용식물로부터 소거물질을 탐색하였다. Human hepatocyte인 HepG2 세포주에 ...
만성음주자의 경우 비대해진 Cytochrome P4502E1에 의해 알코올이 산화되며, 이 과정에서 강화된 NADPHP450 reducatse (CYPR)와 상호작용하게 되어 활성산소종이 발생하여, 간세포 손상의 원인이 된다. 본 연구에서는 알코올 유도 CYP2E1으로부터 발생된 활성산소종의 소거계검색을 위한 in vitro assay계를 확립하고 식용식물로부터 소거물질을 탐색하였다. Human hepatocyte인 HepG2 세포주에 viral transfection을 통하여CYP2E1과 CYPR을 동시발현시킨 세포주와 CYP2E1만을 발현시킨 세포주를 사용한 in vitro assay system을 확립하였다. 두 세포주의 CYP2E1 효소활성은 동일하였으며, CYP2E1 및 CYPR 동시발현 세포주의 경우, reductase 효소 활성이 control세포주 대비, 약 7배 증가하였다. 무엇보다도 알코올, 아세트아미노펜, N, N-dimethylnitrosoamine등의 약물에서 CYP2E1 및 CYPR 동시발현 세포주의 경우가 50%이상 증가된 감수성을 나타내었다. 확립된 in vitro assay계를 이용하여 30종의 식용작물을 대상으로 알코올 유도 CYP2E1에 의한 활성산소종 소거계를 검색한 결과, 녹차 (Camellia sinensis)의 메탄올 추출물이 50%이상의 활성을 나타내었다. 녹차의 주성분인 8종의 카테킨을 대상으로 실험한 결과, (-)EC, (-)EGCG, (-)CG, (-)ECG가 70%이상의 활성과 활성산소종 소거능을 보였으며, 알코올로 손상된 GSH level을 상승시킴으로써 알코올 유도 CYP2E1으로 인한 활성산소종으로부터의 간세포를 보호활성이 확인되었다. 알코올 투여 흰쥐를 대상으로 녹차 추출액을 공급하여 이에 의한 간 보호효과를 확인하고, P450 isozyme의 변화를 확인한 결과, 녹차에 의해 알코올로 상승된 CYP2E1의 감소를 보였으나, total P450양의 증가와 혈액에서의 ALT, AST의 상승, histology에서 steatosis와 inflamaion의 증가를 보임으로써, in vitro결과와 차이를 보였다.
또다른 알코올 유도 CYP2E1 억제물질 탐색용 in vitro assay계의 하나로, 높은 발현율과 yield 를 지닌CYP2E1, CYPR및 cytochrome b5가 발현된 E.coli membrane fraction을 사용하여, 350종의 식용생물의 추출물을 대량검색하였다. 높은 활성을 보인 보리 (Hordeum vulgare)의 메탄올 추출물을 대상으로 HP20 ion exchange column, LH20, HW40 chromatography를 거쳐 HPLC에서 정제물을 동정한 결과 분자량 284의 active compound인 M-Ma-I-IIIb을 얻었다. CYP2E1 억제에 뛰어난 활성을 보인 보리의 메탄올 추출물로 알코올 급여 흰쥐를 대상으로 동물실험을 한 결과, CYP2E1뿐 아니라, 전체 P450 content, CYP1A2, CYP2B등 여러 P450을 낮추는 결과를 보였고, high dose(0.3%)에서 알코올성 간손상을 완화시키는 결과를 보였다.
본 연구 결과는 향후 녹차와 보리에 함유되어있는 카테킨과 향후 동정가능한 억제활성물질을 통하여 녹차와 보리로부터 알코올 유도 CYP2E1에 의한 간손상을 예방 또는 억제할 수 있는 의약, 식품의약 및 기능성 식품용 천연소재의 실용화의 가능성과 실제 가능한 농도를 제시하였다.
만성음주자의 경우 비대해진 Cytochrome P4502E1에 의해 알코올이 산화되며, 이 과정에서 강화된 NADPH P450 reducatse (CYPR)와 상호작용하게 되어 활성산소종이 발생하여, 간세포 손상의 원인이 된다. 본 연구에서는 알코올 유도 CYP2E1으로부터 발생된 활성산소종의 소거계검색을 위한 in vitro assay계를 확립하고 식용식물로부터 소거물질을 탐색하였다. Human hepatocyte인 HepG2 세포주에 viral transfection을 통하여CYP2E1과 CYPR을 동시발현시킨 세포주와 CYP2E1만을 발현시킨 세포주를 사용한 in vitro assay system을 확립하였다. 두 세포주의 CYP2E1 효소활성은 동일하였으며, CYP2E1 및 CYPR 동시발현 세포주의 경우, reductase 효소 활성이 control세포주 대비, 약 7배 증가하였다. 무엇보다도 알코올, 아세트아미노펜, N, N-dimethylnitrosoamine등의 약물에서 CYP2E1 및 CYPR 동시발현 세포주의 경우가 50%이상 증가된 감수성을 나타내었다. 확립된 in vitro assay계를 이용하여 30종의 식용작물을 대상으로 알코올 유도 CYP2E1에 의한 활성산소종 소거계를 검색한 결과, 녹차 (Camellia sinensis)의 메탄올 추출물이 50%이상의 활성을 나타내었다. 녹차의 주성분인 8종의 카테킨을 대상으로 실험한 결과, (-)EC, (-)EGCG, (-)CG, (-)ECG가 70%이상의 활성과 활성산소종 소거능을 보였으며, 알코올로 손상된 GSH level을 상승시킴으로써 알코올 유도 CYP2E1으로 인한 활성산소종으로부터의 간세포를 보호활성이 확인되었다. 알코올 투여 흰쥐를 대상으로 녹차 추출액을 공급하여 이에 의한 간 보호효과를 확인하고, P450 isozyme의 변화를 확인한 결과, 녹차에 의해 알코올로 상승된 CYP2E1의 감소를 보였으나, total P450양의 증가와 혈액에서의 ALT, AST의 상승, histology에서 steatosis와 inflamaion의 증가를 보임으로써, in vitro결과와 차이를 보였다.
또다른 알코올 유도 CYP2E1 억제물질 탐색용 in vitro assay계의 하나로, 높은 발현율과 yield 를 지닌CYP2E1, CYPR및 cytochrome b5가 발현된 E.coli membrane fraction을 사용하여, 350종의 식용생물의 추출물을 대량검색하였다. 높은 활성을 보인 보리 (Hordeum vulgare)의 메탄올 추출물을 대상으로 HP20 ion exchange column, LH20, HW40 chromatography를 거쳐 HPLC에서 정제물을 동정한 결과 분자량 284의 active compound인 M-Ma-I-IIIb을 얻었다. CYP2E1 억제에 뛰어난 활성을 보인 보리의 메탄올 추출물로 알코올 급여 흰쥐를 대상으로 동물실험을 한 결과, CYP2E1뿐 아니라, 전체 P450 content, CYP1A2, CYP2B등 여러 P450을 낮추는 결과를 보였고, high dose(0.3%)에서 알코올성 간손상을 완화시키는 결과를 보였다.
본 연구 결과는 향후 녹차와 보리에 함유되어있는 카테킨과 향후 동정가능한 억제활성물질을 통하여 녹차와 보리로부터 알코올 유도 CYP2E1에 의한 간손상을 예방 또는 억제할 수 있는 의약, 식품의약 및 기능성 식품용 천연소재의 실용화의 가능성과 실제 가능한 농도를 제시하였다.
In order to establish the better model for the evaluation of toxicological properties by ethanol-induced human cytochrome P450 2E1(CYP2E1), HepG2 cell lines were constructed for the stable and constitutive expression of human CYP2E1 or both CYP2E1 and NADPH P450 reductase (CYPR), using the recombina...
In order to establish the better model for the evaluation of toxicological properties by ethanol-induced human cytochrome P450 2E1(CYP2E1), HepG2 cell lines were constructed for the stable and constitutive expression of human CYP2E1 or both CYP2E1 and NADPH P450 reductase (CYPR), using the recombinant retroviral expression vector. Exposure of the CYP2E1 expressing HepG2 cells to high concentration of ethanol (200 mM) for 5 days resulted in more than 50% increase in cytotoxicity, assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) production, and loss of the normal morphology, in comparison with HepG2 cells containing control vector. This model appears to be useful in efforts to establish a CYP2E1-dependent ethanol hepatotoxicity system and to screen the protective materials from edible plant extract. More than twenty extracts from western herbs were screened in transfected HepG2 cell overexpressing CYP2E1 to identify the natural products, which possess protective effect on CYP2E1. The extracts of Camellia sinensis showed the most potent protective effect, approximately 51 % increase in cell viability as compare to controls. Treatment of the cells with various catechins increased the cell viability by more than 2-fold. (-)-Epicatechin gallate and (-)-catechin gallate at the lowest concentration (5 M) attenuated cell death induced CYP2E1 by 60-65%. Prevention of the ethanol toxicity by catechin treatment was associated with the maintenance of intracellular GSH level and morphology. The effects of feeding the diet containing methanol extract of green tea (Camellia sinensis) on alcohol-induced damages were investigated in male ICR mice. Mice were divided into six groups, control, ethanol, 0.15%, 0.30% of green tea, and ethanol plus 0.15%, 0.30% of green tea group. For four weeks, 35% (v/v) ethanol was supplemented with Lieber Decarli diet. Green tea plus ethanol feeding groups showed significant weight loss and moderate hepatotoxicity, while pair group was normal. Ethanol group increased the total cytochrome P450 and isoenzyme CYP2E1, and CYP1A2 activity. Green tea (0.3%) group stimulated CYP1A, CYP2B and CYP2E1, but ethanol plus green tea group decreased those levels. The level of catalase activity and GSH level in the ethanol group was significantly lower than that of the control group while ethanol plus green tea group stimulated the catalase activity and GSH level. These results indicated that green tea extract modulate enzyme responsible for ethanol, which may provide a potent mechanism for synergic action of alcoholic liver damages by green tea extract.
More than three hundred extracts from edible plants were screened in relation to their inhibitory effects on CYP2E1 in E. coli membrane fraction To identify the natural products, which possess inhibitory effect on CYP2E1. The extracts of Hordeum vulgare showed the most potent inhibitory effect, approximately 20 % inhibition as compared to controls. In order to isolate the active compounds in barley extracts, crude methanol extract (C-M) was further separated with n-hexane (M-H), chloroform (M-C), and ethyl acetate (M-E), and then the final residue (M-M) was dried. The M-M fraction, which had highest in inhibitory effect on CYP2E1, was repeatedly subjected to HP20 ion exchange column, Sephadex LH20, Toyopearl HW40, and finally high performance liquid chromatography using an ODS column. The 50% (v/v) aqueous methanol eluted fraction from the HP20 column represented the high inhibitory effects (M-Ma). The active fraction I (M-Ma-I) from the subsequent LH20 column was concentrated and then applied to HW40 gel chromatography. I isolated one major substance (M-Ma-I-IIIb) from the active fraction III (M-Ma-I-III) from HW40 column using HPLC. By means of EI-mass spectrometry, the molecular mass of the isolated substance was 284 m/z. The effects of feeding the diet containing methanol extract of barely (Hordeum vulgare) on alcohol-induced damages were examined in male ICR mice. Mice were divided into six groups, control, ethanol, 0.15, 0.30% of barley, and ethanol plus 0.15 %, 0.30 % of barley group. For four weeks, 35% (v/v) ethanol was supplemented with Lieber-Decarli diet. Ethanol group increased the total cytochrome P450 and isoenzyme CYP2E1, CYP1A activity. These effects of ethanol treatment were attenuated by administration of barley extract. In conclusion, the administration of barley extract ameliorates the ethanol-induced enhancement in liver microsomal parameters dependent on CYP2E1 activity.
In order to establish the better model for the evaluation of toxicological properties by ethanol-induced human cytochrome P450 2E1(CYP2E1), HepG2 cell lines were constructed for the stable and constitutive expression of human CYP2E1 or both CYP2E1 and NADPH P450 reductase (CYPR), using the recombinant retroviral expression vector. Exposure of the CYP2E1 expressing HepG2 cells to high concentration of ethanol (200 mM) for 5 days resulted in more than 50% increase in cytotoxicity, assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) production, and loss of the normal morphology, in comparison with HepG2 cells containing control vector. This model appears to be useful in efforts to establish a CYP2E1-dependent ethanol hepatotoxicity system and to screen the protective materials from edible plant extract. More than twenty extracts from western herbs were screened in transfected HepG2 cell overexpressing CYP2E1 to identify the natural products, which possess protective effect on CYP2E1. The extracts of Camellia sinensis showed the most potent protective effect, approximately 51 % increase in cell viability as compare to controls. Treatment of the cells with various catechins increased the cell viability by more than 2-fold. (-)-Epicatechin gallate and (-)-catechin gallate at the lowest concentration (5 M) attenuated cell death induced CYP2E1 by 60-65%. Prevention of the ethanol toxicity by catechin treatment was associated with the maintenance of intracellular GSH level and morphology. The effects of feeding the diet containing methanol extract of green tea (Camellia sinensis) on alcohol-induced damages were investigated in male ICR mice. Mice were divided into six groups, control, ethanol, 0.15%, 0.30% of green tea, and ethanol plus 0.15%, 0.30% of green tea group. For four weeks, 35% (v/v) ethanol was supplemented with Lieber Decarli diet. Green tea plus ethanol feeding groups showed significant weight loss and moderate hepatotoxicity, while pair group was normal. Ethanol group increased the total cytochrome P450 and isoenzyme CYP2E1, and CYP1A2 activity. Green tea (0.3%) group stimulated CYP1A, CYP2B and CYP2E1, but ethanol plus green tea group decreased those levels. The level of catalase activity and GSH level in the ethanol group was significantly lower than that of the control group while ethanol plus green tea group stimulated the catalase activity and GSH level. These results indicated that green tea extract modulate enzyme responsible for ethanol, which may provide a potent mechanism for synergic action of alcoholic liver damages by green tea extract.
More than three hundred extracts from edible plants were screened in relation to their inhibitory effects on CYP2E1 in E. coli membrane fraction To identify the natural products, which possess inhibitory effect on CYP2E1. The extracts of Hordeum vulgare showed the most potent inhibitory effect, approximately 20 % inhibition as compared to controls. In order to isolate the active compounds in barley extracts, crude methanol extract (C-M) was further separated with n-hexane (M-H), chloroform (M-C), and ethyl acetate (M-E), and then the final residue (M-M) was dried. The M-M fraction, which had highest in inhibitory effect on CYP2E1, was repeatedly subjected to HP20 ion exchange column, Sephadex LH20, Toyopearl HW40, and finally high performance liquid chromatography using an ODS column. The 50% (v/v) aqueous methanol eluted fraction from the HP20 column represented the high inhibitory effects (M-Ma). The active fraction I (M-Ma-I) from the subsequent LH20 column was concentrated and then applied to HW40 gel chromatography. I isolated one major substance (M-Ma-I-IIIb) from the active fraction III (M-Ma-I-III) from HW40 column using HPLC. By means of EI-mass spectrometry, the molecular mass of the isolated substance was 284 m/z. The effects of feeding the diet containing methanol extract of barely (Hordeum vulgare) on alcohol-induced damages were examined in male ICR mice. Mice were divided into six groups, control, ethanol, 0.15, 0.30% of barley, and ethanol plus 0.15 %, 0.30 % of barley group. For four weeks, 35% (v/v) ethanol was supplemented with Lieber-Decarli diet. Ethanol group increased the total cytochrome P450 and isoenzyme CYP2E1, CYP1A activity. These effects of ethanol treatment were attenuated by administration of barley extract. In conclusion, the administration of barley extract ameliorates the ethanol-induced enhancement in liver microsomal parameters dependent on CYP2E1 activity.
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