龍眼花Proanthocyanidin A2萃出物與純物質Proanthocyanidin A2在大鼠體內抗氧化活性之探討

Abstract

本研究室於先前研究中發現龍眼花具有優異之抗氧化效果，並已鑑定其活性成分為 proanthocyanidin A2 (PA2)，但對於 PA2 在動物體內的抗氧化活性尚未明瞭，故本研究以 tert-butyl hydroperoxide (t-BHP) 誘導氧化壓力為動物模式，探討純物質 PA2 與富含 PA2 的龍眼花萃出物 (PE) 於大鼠體內的抗氧化活性，並且比較兩者的抗氧化效果。 首先龍眼花 (9.12 kg) 以 80% acetone 萃取，再以 ethyl acetate 進行溶劑區分萃取，得到龍眼花乙酸乙酯區分物 (LF-A-EA)，經由管柱層析與再結晶純化，得到 8.10g 的PA2 (純度98.53 ± 1.12%) 及 7.31g 的 PE (含PA2 56.30 ± 1.37%)，經過換算得知本次研究之龍眼花每公斤中約含有 1.33 g 的 proanthocyanidin A2。 動物試驗先以管餵方式給予動物去離子水、正控制組 Silibinin 或龍眼花材料 2 週後，再以 t-BHP 誘導氧化壓力，共分 5 組進行試驗：Control 組 (去離子水 1mL/kg BW)、t-BHP 組 (去離子水1mL/kg BW)、Silibinin 組 (100 mg/kg BW)、PA2 組 (100 mg/kg BW) 及 PE 組 (175 mg/kg BW)，其中 PA2 組與 PE 組的樣品劑量是於 PA2 含量相同的前提下餵食大鼠，兩組劑量都含有 98.53 mg/kg BW 的 PA2。連續管餵樣品14 天後，於第 14 天以腹腔注射 t-BHP (0.2 mmol/kg BW) 或生理食鹽水 (Control 組)，並在注射後 18 小時犧牲，收集血液、尿液與肝臟進行抗氧化分析。結果顯示 PA2 與 PE 可降低因 t-BHP 處理而增加之 aspartate aminotransferase (AST)、alanine aminotransferase (ALT) 及 lactate dehydrogenase (LDH) 活性；在脂質過氧化分析方面，相較於 t-BHP 組，PA2 與 PE 可顯著減少血漿及肝臟中 malondialdehyde (MDA) 之生成，尿液中的 8-isoprostagladin F2α (8-iso-PGF2α) 含量也顯著減少，表示 PA2 與 PE 能降低體內脂質過氧化的發生；在抗氧化物質與抗氧化酵素分析方面，PA2 與 PE 能增加因 t-BHP 所減少之抗氧化酵素 glutathione peroxidase (GPx)、glutathione reducatse (GRd)、glutathione S-transferase (GST)、superoxide dismutase (SOD) 及 catalase (CAT) 之活性，也能提升抗氧化物質 glutathione (GSH) 之濃度；另外也發現給予動物 PA2 與 PE 後，抗氧化酵素活性與 GSH 含量皆與 Control 組無顯著差異。肝臟組織病理觀察中，顯示 PA2 與 PE 具有保護肝臟組織之效果，防止 t-BHP 所造成的肝損傷，如漿膜炎、細胞壞死及單核炎症細胞浸潤。 綜合以上實驗結果，顯示 PA2 與 PE 於動物體內皆具有良好的抗氧化活性，且抗氧化活性沒有顯著差異，表示 PE 中的其他成分並無增加抗氧化的效果，PA2 為主要的活性成分。PA2 對 t-BHP 誘導的氧化傷害有保護作用，其保護機制可能經由減少脂質過氧化、維持體內抗氧化酵素的活性及維持抗氧化物質的含量，達到提升抗氧化防禦系統之能力，使得生物體免於 t-BHP 的氧化傷害。

Our laboratory has found that longan flowers contained components with excellent antioxidant activities, and proanthocyanidin A2 (PA2) was identified as the most active component. However, the relationship between in vivo antioxidant activity and longan flower active compounds is not clear yet. Therefore, the aim of this study is to investigate the potential protective effects of PA2 and PA2 rich extract on tert-butyl hydroperoxide (t-BHP) induced oxidative damage in rats. Longan flower (9.12kg) was extracted with 80% acetone and then partitioned with ethyl acetate liquid-liquid. The ethyl acetate fraction (LF-A-EA) was separated by column chromatography and purified by recrystallization. Purified PA2 (8.10g; purity 98.53 ± 1.12%) and PA2 rich extract (7.31g; contains 56.3 ± 1.37% of PA2) were obtained. The content of proanthocyanidin A2 in longan flower was estimated to be 1.33 g/kg of dry weight. SD rats were tube-fed with either deionized water, positive control (Silibinin) or longan flower sample for 14 days before inducing oxidative damage with t-BHP. The test animals were divided into five groups: Control group (ddH2O 1mL/kg BW); t-BHP group (ddH2O 1mL/kg BW); Silibinin group (100 mg/kg BW); PA2 group (100 mg/kg BW); and PA2 rich extract group (175 mg/kg BW). The PA2 group or PA2 rich extract group had the same content of PA2 (98.53 mg/kg BW). After daily supplementation by gavage to rats for 14 days, t-BHP was injected intraperitoneally at a dosage of 0.2 mmol/kg BW, and 18hr later the rats were sacrificed. The control group was injected with saline solution. Blood, urine and liver samples were collected from each group for antioxidant analysis. Results showed that pretreatment with PA2 or PA2 rich extract by gavage for 14 days before a single dose of t-BHP (0.2 mmol/kg) lowered serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), as well as reduced the formation of malondialdehyde (MDA) in plasma and liver, also decreased 8-isoprostagladin F2α (8-iso-PGF2α) content in urine. Moreover, the PA2 group and PA2 rich extract group increased hepatic glutathione peroxidase (GPx), glutathione reducatse (GRd), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) activities, as well as glutathione (GSH) level as compared with the t-BHP group. The GSH level and the activities of antioxidant enzymes were observed to move closer to the values of the control group. In addition, histopathological evaluation of the rat livers revealed that PA2 or PA2 rich extract reduced the incidence of liver lesions induced by t-BHP including serositis with necrotic cells and mononuclear cell infiltration. Based on the results described above, we speculate that PA2 or PA2 rich extract may play a role in the prevention of oxidative damage in vivo. However, results of statistical analysis demonstrated that there were no significant differences between the PA2 and PA2 rich extract group, that means the components in PA2 rich extract can not increase the antioxidant activities of PA2. Therefore, it can be speculated that supplementation with PA2 protects against t-BHP induced liver injury by attenuating lipid peroxidation, maintaining the GSH level and the activities of antioxidant enzymes which enhance antioxidant defense.