| Summary: | 碩士 === 國立臺灣大學 === 食品科技研究所 === 96 === Diabetes mellitus is a metabolism disorder characterized by chronic hyperglycemia due to inefficient action of insulin or insufficient production of insulin. One of the mechanisms proposed for diabetic complication is the formation of advanced glycation end products, known as AGE. AGE could bind with cellular receptors, increase oxidative stress and activate pathways that release proinflammatory compounds and cytokines, form irreversible AGE-proteins and finally modify proteins and enzymes and deactivate their functions. Lotus leaf and adlay have numerous biological activities and are potential supplements for diabetic patients. Therefore, the objective of this study was to screen antiglycative agents from lotus leaf and adlay seed using in vitro experiments and examine the effects of AGE on glucose uptake of adipocytes. Based on in vitro BSA-Glucose assay, which simulates the in vivo AGE formation, ethanolic extract of adlay testa demonstrated a high inhibitory effect against AGE formation, especially in butanol subfractions C and D (ATE-Bu-C, ATE-Bu-D). Butanol subfractions 4, 5 and 6 from methanolic extract of lotus leaf had the highest inhibitory effects against AGE formation (LLMB 4, LLMB 5, LLMB 6). Amongst all the compounds isolated from these subfractions up to date, quercetin, catechin, caffeic acid and chlorogenic acid demonstrated high capabilities to inhibit AGE formation. Therefore, ATE-Bu-C, ATE-Bu-D, LLMB 4, LLMB 5, LLMB 6, catechin, quercetin, chlorogenic acid and caffeic acid were selected for the next stage of this study. From hemoglobin-δ-gluconolactone assay, which simulates the in vivo early stage of AGE formation, chlorogenic acid and catechin were the better early glycation inhibitors with inhibitory percentage of 25% and 23%, which were comparable to the inhibitory effect of the positive control aminoguanidine (23%). Effective subfractions and compounds were then examined on their abilities to inhibit MGO via MGO-BSA assay and MGO trapping capacity, which simulate the middle stage of AGE formation. Catechin (2.5 mM) demonstrated a 85% MGO (1.5 mM) trapping ability. Although caffeic acid had the highest inhibition against MGO-mediated protein modification, catechin demonstrated the strongest MGO trapping abilities with an inhibitory percentage of 85% at 2.5 mM. Chlorogenic acid and caffeic acid did not exhibit any MGO trapping abilities. ATE-Bu-C and ATE-Bu-D did not demonstrate much MGO trapping capability whereas LLMB 4, LLMB 5 and LLMB 6 demonstrated 21 to 28% MGO inhibition abilities. Based on HPLC analysis, catechin concentrations were determined to be 0.032 mM and 0.039 mM in LLMB 4 and LLMB 5. Hence, MGO trapping abilities of LLMB 4 and LLMB 5 were at least partially came from catechin. MGO had been demonstrated to be cytotoxic to cells and its cytotoxicity was examined in 3T3-L1 as well. Concentrations of MGO below 2 mM did not appear to have any detectable cytotoxic effects on the viability of 3T3-L1. Therefore, 1 mM and 2 mM of MGO were used to evaluate MGO’s effects on the glucose uptake of 3T3-L1. The glucose uptake ability of 3T3-L1 progressively decreased to 52% and 40% of the control group respectively after 36 hours of 1 mM and 2 mM MGO treatment. This result suggested that AGEs are involved in the development of impaired insulin sensitivity in adipocytes. In conclusion, adlay inhibits the early and middle stage of glycation. The primary effective subfractions were ATE-Bu-C and ATE-Bu-D and the major effective pure compounds were chlorogenic acid and caffeic acid. Lotus leaf also inhibits the early and middle stage of glycation and it traps MGO as well. The primary effective subfractions were LLMB 4, LLMB 5 and LLMB 6 and the major effective compounds were quercetin and catechin.
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