數種植物萃取物及 2,4,6-Trihydroxybenzoic acid 對活性雙羰基化合物之捕捉作用

Abstract

近年來許多研究指出，活性雙羰基物質 (reactive dicarbonyl species) 在體內累積將使細胞損傷，並於長時間存在下使蛋白質糖化，不可逆的形成糖化終產物 (advanced glycation endproduct，AGE)，在糖尿病及其併發症上扮演重要角色。Methylglyoxal (MG) 是活性雙羰基物質之一，在糖尿病患者血漿中發現有較高之含量，約為正常人的五倍。因此若將體內 MG 清除，則可降低其對細胞的傷害及抑制 AGE 的生成。本研究主要目的為探討一些含有豐富多酚物質的植物如茶、歐洲藍莓 (bilberry)、黑加侖 (black currant)、桑葉及葡萄籽與多酚化合物 2,4,6-trihydroxybenzoic acid (THBA) 以樣品與 MG 於模擬生理環境下 (37℃，pH 7.4) 對於 MG 之捕捉功效。 實驗第一部份為各種植物萃取物之 MG 捕捉功效探討。紅茶、包種茶、綠茶茶葉以沸水萃取後與 MG 反應，結果顯示以綠茶之 MG 捕捉效果最好，原因可能與其兒茶素之種類與含量有關。Bilberry 及黑加侖萃取物皆含大量花青素，在不同酸鹼度環境下與 MG 進行反應，發現於偏鹼性環境中，兩種萃取物都有很好的 MG 捕捉效果，正控制組 EGCG 使 MG 減少量約 82%，而 bilberry 與黑加侖萃取物分別為 76% 與 73%，且於萃取物之花青素當量濃度為 MG 濃度的 2 或 3 倍時，MG 減少程度甚至超越正控制組 EGCG；另外，bilberry 與黑加侖萃取物所含之花青素種類不同，因此 MG 捕捉能力也有差異。葡萄籽萃取物主要成份為原花青素 (proanthocyanidins)，可使 MG 量降低約 58%；而桑葉萃取物在同樣條件下之 MG 捕捉能力不佳，MG 減少量僅 23%，效果低於兒茶素類、花青素類萃取物及原花青素類萃取物。 第二部份的實驗為 THBA 與 MG 之反應情形探討。THBA 為一種簡單的多酚類化合物，其苯環上未鍵結的兩個碳上之電子密度很高。THBA 能有效降低 MG 含量，1.45 mM THBA 與同濃度 MG 反應，使 MG 減少 50% 所需時間 (T1/2) 為28.367 分鐘；以 3 mM 的濃度反應T1/2 為 27.170 分鐘，於反應時間 60 分鐘後，反應接近完成。於同樣條件下，反應 60 分鐘內，使 3 mM MG 減少 50% 所需 THBA 濃度 (EC50) 為 1.033 mM；1.45 mM 之 EC50 為 0.678 mM，且 THBA 與 MG 濃度較接近時，捕捉 MG 的能力較強。另外，THBA 於鹼性越高的環境下與 MG 反應，捕捉效果越強。THBA-MG 之結合產物以質譜儀分析，顯示在MG 量少的情況下產生鍵結一分子 MG 的產物；於 MG 量多的情況下則鍵結一分子或兩分子 MG 的產物皆被發現。

It has been demonstrated that reactive dicarbonyl species (RCS) irreversibly modified proteins over time and yielded the advanced glycation endproducts (AGEs), which are closely associated with several pathophysiological conditions, such as cataracts, diabetes complications, and Alzheimer’s disease. Methylglyoxal (MG) is one of the RCS. Higher levels of MG were observed in diabetic patients’ plasma than those in healthy people’s plasma. Thus decreasing the levels of MG will be a useful approach to prevent the formation of AGEs. The aims of the present study are to evaluate MG trapping ability of some plant extracts which were rich in phenolic compounds such as tea, bilberries, black currants, grape seeds and mulberry leaves, and one of the phenolic compounds, 2,4,6-trihydroxybenzoic acid (THBA) in simulated physiological conditions (pH 7.4 and 37 °C). The first part of current research was to evaluate the MG trapping abilities of selected plant extracts. Black, paochung and green tea leaves were extracted by boiling water, the MG trapping abilities were determined in the order of green tea, paochung tea and black tea. The result was highly correlated to their total phenolic contents. Bilberry and black currant extracts are rich in anthocyanins. When they reacted with MG at high pH conditions, both of them exhibited good MG trapping abilities. Their abilities were even better than the positive control, EGCG, when the anthocyanin equivalents were 2-3 times than MG concentration. The MG trapping ability difference between the two extracts might come from the different anthocyanin compositions of bilberry and black currant. The major compounds in grape seed extract are proanthocyanidins, the result showed that it also exhibited a good MG trapping ability. Mulberry leaves are rich in flavonoids such as quercetin, but its ability in MG trapping was not as good as those extracts rich in catechins, anthocyanins and proanthocyanidins. The second part of current research is to study the reaction between THBA and MG. THBA is a phenolic acid, which could be hydrolyzed from tannins of seaweeds or degraded from anthocyanins of tart cherries, and the structure is similar to phloretin, an antioxidant compound in apple. Our results showed that THBA is able to trap MG efficiently. The time for 50% MG reduction (t1/2) at 3 mM and 1.45 mM were determined to be 27.170 minutes and 28.367 minutes, respectively. THBA concentrations that can reduce 50% MG (EC50) were 0.835 mM THBA at 3 mM MG and 0.724 mM THBA at 1.45 mM MG.. To determine the structure of reaction adducts, THBA was react with MG in the ratio of 1:3 and 1:20 for 3 hours. The reaction mixtures were separated by HPLC, and reaction adducts collected for ESI mass spectroscopic analysis. At the ratio of 1:3, the molecular weight of adduct was shown to be 242 indicating one MG molecule was added onto one THBA molecule. At the ratio of 1:15, we got two compounds with molecular weight of 242 and 314, indicating that there were two compounds formed in the reaction, which might be one MG molecule added onto one molecule of THBA, and two MG molecules added onto one molecule of THBA.