薏苡籽實與荷葉之抗糖基化效應

Effects of fractions from adlay seed and lotus leaf extracts on antiglycation

張文思(Fiona Wen-Szu Chang)

指導教授 ： 江文章

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02029

摘要

糖尿病為一新陳代謝異常相關之疾病，主要為胰島素利用率不佳或胰島素分泌不足，所造成的慢性高血糖。糖化終產物(advanced glycation end products, AGEs)生成的機制與糖尿病併發症有關。糖化終產物會與細胞上的受體結合，增加氧化壓力並活化釋放前發炎物質與細胞激素之訊息傳導路徑、形成不可逆的AGE蛋白質並改變蛋白質與酵素之結構使其失去活性。荷葉與薏仁因其豐富的生理活性，可望作為糖尿病病患平日之保健食品。因此，本實驗之目的在篩選荷葉及薏苡籽實中之有效抗糖基化成分並檢視AGEs是否會對脂肪細胞之葡萄糖代謝能力造成不良影響。以BSA-Glucose assay模擬體內最終形成的AGE之篩選結果顯示，薏苡種皮乙醇萃取物之正丁醇區分物的次區分物C與D (ATE-Bu-C, ATE-Bu-D)有最高的抗蛋白糖基化活性。在荷葉甲醇萃取物(LLM)方面，以正丁醇區分物的次區分物4、 5與6 (LLMB 4, LLMB 5, LLMB 6)之抗蛋白糖基化效果最為顯著。從所有選定之次區分物內已分出來的純化合物裡，以槲皮素、兒茶素、咖啡酸及綠原酸有最高的抗蛋白糖基化活性。因此，ATE-Bu-C、ATE-Bu-D、LLMB 4、LLMB 5、LLMB 6、槲皮素、兒茶素、咖啡酸及綠原酸被選定進入下階段的實驗。以Hemoglobin-δ-gluconolactone assay模擬體內早期AGE形成試驗結果顯示，綠原酸及兒茶素具有25%及23%抑制早期蛋白糖基化反應之功效，且它們的功效與正控制aminoguanidine的效果是相當的(23%)。繼續評估選定之有效次區分物及純化合物是否具有抑制MGO (Methylglyoxal)反應或與MGO直接螯合之能力來模擬抑制體內中期AGE形成的效果。結果指出，咖啡酸擁有最佳之抑制MGO糖化蛋白質之能力，但兒茶素(2.5 mM)有最佳之與MGO直接結合的能力(85%)。然而，咖啡酸與綠原酸皆不具有與MGO直接形成adduct的能力。ATE-Bu-C及ATE-Bu-D並沒有任何與MGO形成adduct的能力，但LLMB 4、LLMB 5及LLMB 6約有21至28%的抑制率。經HPLC定量結果顯示，LLMB 4與LLMB 5各含0.032 mM與0.039 mM之兒茶素。據此推測，LLMB 4與LLMB 5螯合MGO之能力最少有部分與其兒茶素含量有關。MGO之生成會對細胞造成毒性，因此本實驗評估MGO對3T3-L1脂肪細胞之細胞株之毒性。結果顯示，MGO在濃度2 mM以下無細胞毒性，因此以1 mM及2 mM的MGO探討其對3T3-L1葡萄糖攝取之能力影響。經過36小時的1 mM及2 mM MGO處理，3T3-L1葡萄糖攝取之能力降至控制組的52%及40%。 從這個結果推測，AGEs的確與胰島素抗性具有相關性。綜上所述，薏仁可抑制早期與中期之AGE形成，其主要作用次區分物為ATE-Bu- C與ATE-Bu-D，其主要作用成分應來自於綠原酸及咖啡酸；荷葉亦可抑制早期與中期之AGE形成並與MGO形成聚合物，其主要作用次區分物為 LLMB 4、LLMB 5與LLMB 6，其主要作用成分應來自
於槲皮素及兒茶素。

關鍵字

薏苡籽實 ； 荷葉 ； 3T3-L1脂肪細胞 ； 糖尿病 ； 糖化終產物 ； 葡萄糖攝取

並列摘要

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.

並列關鍵字

adlay seed ； lotus leaf ； 3T3-L1 adipocytes ； diabetes mellitus ； advanced glycation end products ； glucose uptake

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