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Nutrigenomics approach to understanding the role of dietary phenolic acids in sulfate conjugation
|關鍵字:||血小板;Platelet;轉硫酶;酚酸化合物;抗氧化性;相乘效果;細胞保護;訊息傳遞;抗氧化基因調控序列;human phenolsulfotransferases;phenolic acids;antioxidant activity;Synergistic effect;Cytoprotective;Signal transd||出版社:||食品科學系||摘要:||
硫化代謝作用(sulfation)是屬於人體中PhaseⅡ之共軛作用之一，可幫助體內藥物及環境中化學物質失去其毒性或是調控內源性物質之生合成及一些神經傳導方面之反應，通常都將硫化共軛反應稱之為去毒化作用。而硫化代謝作用主要是藉由人體中之轉硫酶(sulfotransferase, ST)，轉移體內3’-phosphoadenosine-5’-phosphate sulfate (PAPS)上的硫酸根至受質上，以增加反應受質之水溶性，並使其易於排出體外。因此體內之轉硫酶便扮演著重要的角色來催化轉硫作用的運作進行，研究指出不同的轉硫酶依其所作用之基質不同所以表現出的功能特性亦具有差異。酚酸化合物是一群具有許多優良效應之食品活性成分，由結構上可區分為苯甲酸及肉桂酸等兩大類化合物。研究指出酚酸化合物具有良好之藥理活性，可被應用來當作抗氧化、抗突變及抗癌性試劑。因此本研究主要探討膳食中多酚化合物對人類轉硫酶作用之影響。內容主要分為六部分：(一) 酚酸化合物對轉硫酶活性之影響及其抗氧化力之相關性、(二) 酚酸化合物相乘作用對轉硫酶活性影響之探討、(三) 探討蔬果對人體轉硫酶活性之影響與其抗氧化能力、總多酚間之相關性、(四) 酚酸化合物誘發細胞內轉硫酶基因表現之探討、(五) 酚酸化合物對動物體內硫化作用及抗氧化狀態之影響探討、(六) 酚酸化合物誘發轉硫酶基因表現後之細胞保護效應探討。
結果顯示：(一) p-hydroxybenzoic acid、gallic acid、gentisic acid、ferulic acid、及p-coumaric等酚酸化合物具有顯著性提升人體血小板內轉硫酶之活性。以ORACROO.及TEAC評估後得知這些酚酸化合物具有不錯之抗氧化能力，線性回歸分析證實酚酸化合物抗氧化能力之強弱與影響轉硫酶活性之間的確具有顯著之關連性。(二) 另外，系統性的組合兩種或三種具有促進轉硫酶活性酚酸化合物於複合作用下對轉硫酶活性之影響。亦發現酚酸化合物在相互作用後對人體血小板轉硫酶活性提升上具有顯著性的相乘效果(synergistic effect)。(三) 值得注意的是蘆筍、花椰菜、芹菜、甘藍菜、及茄子等蔬果亦具有顯著性提升細胞內轉硫酶之活性，配合高效能液相層析結果發現這些蔬果含有大量之酚酸化合物，推測蔬菜中所含有之酚酸化合物可能為影響轉硫酶活性之主要因子。進一步以細胞模式探討酚酸化合物對轉硫酶之調控機制，結果發現酚酸化合物具有誘發細胞內轉硫酶之活性，其中又以gallic acid及ferulic acid效果最佳，(四) 以RT-PCR及西方墨點法來分析胞內轉硫酶基因及蛋白質表現之變化，推測酚酸化合物提升胞內轉硫酶活性乃源於調節細胞中轉硫酶基因及蛋白質所導致。 (五) 利用動物實驗模式確認酚酸化合物於活體內對硫化代謝作用及抗氧化狀態之影響，結果發現酚酸化合物能顯著性的誘發大鼠肝臟內P-form及M-form轉硫酶mRNA表現，此結果與轉硫酶活性之提升具有相同誘發趨勢。此外，酚酸化合物亦能有效提升大鼠血漿中之總抗氧化能力。生物利用率之結果顯示，gallic acid及p-coumaric acid可有效被活體吸收，於餵食2小時之後，在血漿中可偵測到最大之吸收量，其Cmax值分別為665±23及550±33 nmol/L。酚酸化合物誘發活體內轉硫酶活性與其總抗氧化力之間有著顯著之相關性，推測酚酸化合物可有效改善活體內之硫化共軛代謝作用及其抗氧化狀態。(六) Nrf2 可啟動與antioxidant response element 相關的許多抗氧化基因表現，本研究結果也證實酚酸化合物可活化細胞中Nrf2，使其由細胞質進入核內，且此活化過程需要JNK及p38 MAPK等訊息傳遞蛋白質參與。推論酚酸化合物可能經由NK及p38 MAPK活化Nrf2進入核內，進一步啟動轉硫酶(PST)基因表現，造成細胞中轉硫酶蛋白質的生成，對細胞產生抗氧化傷害等保護作用。
Sulfation (sulfonation) is one of the major phase II conjugative reactions involved in the biotransformation of various endogenous compounds, drugs, and xenobiotics as well as in steroid biosynthesis, catecholamine metabolism, and thyroid hormone homeostasis. Phenolic acids such as hydroxybenzoic acids and hydroxycinnamic acids are antioxidant compounds in fruits and vegetables. Research on phenolic acids is of current interest due to the important biological and pharmacological properties attributed to their antioxidant properties. Therefore, the objective of this study was to investigate the effect of dietary polyphenolic compounds on human sulfotransferase. There are six topics included in this study: (1) Effect of phenolic Acid on human phenolsulfotransferases in relation to their antioxidant activity (2) Synergistic effect of antioxidant phenolic acids on human phenolsulfotransferase activity (3) Effect of vegetables on human phenolsulfotransferases in relation to their antioxidant activity and total phenolics (4) Induction of phenolsulfotransferase expression by antioxidant phenolic acids in human hepatoma HepG2 cells (5) Modulation of hepatic phase II phenolsulfotransferase and antioxidant status by phenolic acids in rats. (6) Cytoprotective effects of P-form phenolsulfotransferase induction by phenolic acid
Our studies revealed that (1) p-hydroxybenzoic acid, gallic acid, gentisic acid, ferulic acid, and p-coumaric acid all could increase the activities of both PST-P and PST-M. These phenolic acids also possessed antioxidant capacity in the ORAC and TEAC assays. (2) Furthermore, in both two-compound and three-compound combinations with each of other phenolic acids, gallic acid and gentistic acid exhibit the potential synergistic effects in the promotion of PSTs activities. The overall effects of phenolic acids on the activities of PST-P and PST-M are highly correlated to their ORAC values, suggesting that antioxidant phenolic acids might alter sulfate conjugation. (3) Moreover, the PST-P activity was significantly induced by asparagus, broccoli, cauliflower, celery, and eggplant, whereas PST-M activity was induced by asparagus, broccoli, carrot, eggplant, and potato at a concentration of 100 g/ml. The major polyphenols in broccoli, the most potential inducer in both forms of PSTs activities, was antioxidant phenolic acids. HPLC retention times and standard spiked indicated the presence of gallic acid, p-hydroxybenzoic acid, p-coumaric acid, gentisic acid, and ferulic acid in broccoli. These results imply that vegetables have a capability of inducing PST activity, and the PST induction may be possibly ascribed to antioxidant phenolic acids in vegetable extracts. (4) Furthermore, human hepatoma cell line HepG2 was used as a model to investigate the effect of antioxidant phenolic acids on enzymatic activity and expression of one of the major phase II sulfateconjugation enzymes, PST-P. The results showed that gallic acid, gentisic acid, p-hydroxybenzoic acid, and p-coumaric acid were found to increase the PST-P activity in a dose-dependent manner. A significant correlation between the expressions of PST-P mRNA and the corresponding PST-P activity was observed. The results demonstrated that certain antioxidant phenolic acids could induce PST-P activity in HepG2 cells by promoting PST-P mRNA and protein expression, suggesting a novel mechanism by which antioxidant phenolic acids may be implicated in phase II sulfate conjugation. (5) Used the animal models to investigate the the modulatory effect of phenolic acids on hepatic phase II phenolsulfotrnasferases and antioxidant status in vivo. According to the results, phenolic acids in dosage of 100 mg/body weight significantly increased PST-P and PST-M activities as compared with the that of the control rats (p<0.05). Reverse transcription polymerase chain reaction results indicated that the changes in PST-P and PST-M mRNA levels by phenolic acids were similar to those noted in the enzymes activity levels. The plasma obtained form phenolic acids-administrated rats were significantly increased the oxygen radical absorbance capacity (ORAC) values than that form control rats. In a bioavailability study, following oral administration of gallic acid and p-coumaric acid (100 mg/kg body weight), the phenolic acid were detected in the plasma and the Cmax values after 2.0 h administration were 665±23, and 550±33 nmol/L, respectively. There was a significant correlation between the activity of both forms of PSTs and the antioxidant capacity of ORAC value by phenolic acids, suggesting that phenolic acids might alter sulfate conjugation and antioxidant capacity in living systems. (6) Nrf2 can initiate transcription of many antioxidant response element (ARE) mediated antioxidant genes expression. In this study, it was found that antioxidant phenolic acids could induce translocation of cytoplasmic Nrf2 into nucleus and JNK/p38 MAPK signaling cascade was involved in this process. It was demonstrated that antioxidant phenolic acids could remarkably induce PST-P protein expression and this induction of PST-P by antioxidant phenolic acids could be inhibited by p38 MAPK inhibitor SB203580. Taken together, we speculate that antoxidant phenolic acids activate translocation of Nrf2 into nucleus through JNK/p38 MAPK. Nrf2 then binds to the regulatory transcription region of PST gene in the nucleus and initiates gene transcription that results in final expression of chemoprotective PST-P protein.
Our results provide better understanding of the effects of phenolic acids on human PST activities, as well as information regarding the intake of phenolic antioxidant for human health.
Keywords: Platelet, human phenolsulfotransferases, phenolic acids, antioxidant activity, Synergistic effect, Cytoprotective, Signal transduction, antioxidant responsive element
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