AMP-activated protein kinase (AMPK), which is a heteromeric enzyme complex that works as a fuel guage to regulate the cellular and whole-body energy homeostasis, may be a key player in metabolic control. Thus, the AMPK regulators can be used as drug leads for metabolic syndrome such as obesity and t...
AMP-activated protein kinase (AMPK), which is a heteromeric enzyme complex that works as a fuel guage to regulate the cellular and whole-body energy homeostasis, may be a key player in metabolic control. Thus, the AMPK regulators can be used as drug leads for metabolic syndrome such as obesity and type-2 diabetes. During our screening for AMPK activator from natural products, Hedyotis diffusa showed potent activity. Dried H. diffusa was refluxed with 95% ethanol for 5 h. The ethanol extract was evaporated and partitioned with dichloromethane, ethylacetate and n-butanol. Eight active compounds were isolated by silica gel column chromatography from the dichloromethane and ethylacetate soluble fractions. Their structures were identified as digitolutein, 2-hydroxy-3-methylanthraquinone, ursolic acid, (E)-6-O-p-coumaroyl scandoside methyl ester / (Z) form 4 : 1 mixture, asperuloside, geniposide, daphylloside, and (E)-6-O-feruloyl scandoside methyl ester / (Z) form 4 : 1 mixture on the basis of the 13C-NMR, 1H-NMR and mass spectroscopic method. Among them, three compounds induced activation of AMPK and its down-stream, acetyl-CoA carboxylase (ACC) in skeletal muscle L6 cell at 30 and 60 μg/ml. These results suggest that the ethanol extract of H. diffusa and the compounds isolated are expected to be useful for regulating AMPK and ACC. Especially, ursolic acid significantly activated AMPK and ACC in L6 cells in a dose dependent manner. In artificially AMPK-activated HEK293 cell lysate, ursolic acid weakly inhibited AMPK dephosphorylation at 40 μM, suggesting that the effect was from inhibition of AMPK dephosphorylation. In addition, ursolic acid did not activate AMPK in HeLa cells lacking of LKB1. Also, co-treatment of STO-609, an inhibitor of CaMKK activity, with ursolic acid reduced the phosphorylaytion of AMPK, indicating that ursolic acid activates AMPK through LKB1 rather than CaMKK. To clarify whether ursolic acid affect on the distribution of LKB1 in the cytosol, recombinant pEGFP:LKB1 plasmid DNA was transfected to the HeLa cell which is lack of LKB1. As a result, in ursolic acid treated cells, LKB1 stayed in the nucleus as in the control group. Therefore, increase in the AMPK phosphorylation by ursolic acid was not concerned with the movement of LKB1 to the cytosol. For quality standardization, four compounds set up index components from H. diffusa and develop HPLC-UVD analysis method. Index components were analyzed within 20 minutes. And then, for effectiveness verification of analysis method, fulfilled validation. It showed a high linearity in the calibration curve as coefficient of correlation (R2) of 0.9991-0.9999, and LOD was 0.03-0.07 μg/ml, LOQ was 0.099-0.231 μg/ml. The intra-day and inter-day precision were 0.23-2.00% and 0.25-1.16%, intra-day and inter-day accuracy were 94.60-108.44% and 94.73-110.23%. As a result of this study, It can help to anti-obesity medicine development and quality management of H.diffusa.
AMP-activated protein kinase (AMPK), which is a heteromeric enzyme complex that works as a fuel guage to regulate the cellular and whole-body energy homeostasis, may be a key player in metabolic control. Thus, the AMPK regulators can be used as drug leads for metabolic syndrome such as obesity and type-2 diabetes. During our screening for AMPK activator from natural products, Hedyotis diffusa showed potent activity. Dried H. diffusa was refluxed with 95% ethanol for 5 h. The ethanol extract was evaporated and partitioned with dichloromethane, ethylacetate and n-butanol. Eight active compounds were isolated by silica gel column chromatography from the dichloromethane and ethylacetate soluble fractions. Their structures were identified as digitolutein, 2-hydroxy-3-methylanthraquinone, ursolic acid, (E)-6-O-p-coumaroyl scandoside methyl ester / (Z) form 4 : 1 mixture, asperuloside, geniposide, daphylloside, and (E)-6-O-feruloyl scandoside methyl ester / (Z) form 4 : 1 mixture on the basis of the 13C-NMR, 1H-NMR and mass spectroscopic method. Among them, three compounds induced activation of AMPK and its down-stream, acetyl-CoA carboxylase (ACC) in skeletal muscle L6 cell at 30 and 60 μg/ml. These results suggest that the ethanol extract of H. diffusa and the compounds isolated are expected to be useful for regulating AMPK and ACC. Especially, ursolic acid significantly activated AMPK and ACC in L6 cells in a dose dependent manner. In artificially AMPK-activated HEK293 cell lysate, ursolic acid weakly inhibited AMPK dephosphorylation at 40 μM, suggesting that the effect was from inhibition of AMPK dephosphorylation. In addition, ursolic acid did not activate AMPK in HeLa cells lacking of LKB1. Also, co-treatment of STO-609, an inhibitor of CaMKK activity, with ursolic acid reduced the phosphorylaytion of AMPK, indicating that ursolic acid activates AMPK through LKB1 rather than CaMKK. To clarify whether ursolic acid affect on the distribution of LKB1 in the cytosol, recombinant pEGFP:LKB1 plasmid DNA was transfected to the HeLa cell which is lack of LKB1. As a result, in ursolic acid treated cells, LKB1 stayed in the nucleus as in the control group. Therefore, increase in the AMPK phosphorylation by ursolic acid was not concerned with the movement of LKB1 to the cytosol. For quality standardization, four compounds set up index components from H. diffusa and develop HPLC-UVD analysis method. Index components were analyzed within 20 minutes. And then, for effectiveness verification of analysis method, fulfilled validation. It showed a high linearity in the calibration curve as coefficient of correlation (R2) of 0.9991-0.9999, and LOD was 0.03-0.07 μg/ml, LOQ was 0.099-0.231 μg/ml. The intra-day and inter-day precision were 0.23-2.00% and 0.25-1.16%, intra-day and inter-day accuracy were 94.60-108.44% and 94.73-110.23%. As a result of this study, It can help to anti-obesity medicine development and quality management of H.diffusa.
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#Hedyotis diffusa 비만 AMPK
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