| 摘要: | 本研究探討野甘草 (Scoparia dulcis L.) 70%乙醇萃取物之抗氧化、鎮痛、抗發炎及保肝之活性，並以HPLC分析及定量活性成分之含量。
首先以Trolox當量的總抗氧化能力測定、DPPH自由基清除試驗、FRAP抗氧化能力試驗及總還原力試驗來評估野甘草70%乙醇萃取物及分劃層之抗氧化活性，並測定其總多酚及總黃酮含量；以醋酸扭體及福馬林舔足試驗，探討野甘草70%乙醇萃取物及betulinic acid之鎮痛作用；以λ-角叉菜膠誘發小鼠足蹠浮腫試驗，探討野甘草70%乙醇萃取物及betulinic acid抗發炎作用；檢測其腫脹足蹠組織之COX-2、NO、IL-1β、TNF-α與MDA含量，以及測定肝臟抗氧化酵素SOD、GSH-Px及GSH-Rd活性，藉以探討野甘草70%乙醇萃取物及betulinic acid之抗發炎作用機轉；以四氯化碳誘導小鼠急性肝損傷為動物模型，探討野甘草70%乙醇萃取物之保肝作用；最後分析野甘草70%乙醇萃取物中成分，並定量活性成分luteolin、apigenin及betulinic acid之含量。
結果顯示，野甘草70%乙醇萃取物具有體外抗氧化的活性，而各分劃層中以乙酸乙酯層抗氧化活性最佳，其總多酚類含量及總黃酮含量也是最高；鎮痛實驗結果顯示，野甘草70%乙醇萃取物及betulinic acid對於醋酸誘導的扭體反應及福馬林誘導的後期舔足時間具有明顯的抑制效果；抗發炎實驗結果則顯示野甘草70%乙醇萃取物及betulinic acid可明顯抑制λ-角叉菜膠誘發小鼠足蹠的腫脹；此外，野甘草70%乙醇萃取物及betulinic acid可以抑制足蹠組織發炎介質 (COX-2與NO) 含量及細胞激素 (IL-1β與TNF-α) 的濃度，減少脂質過氧化物MDA生成量，以及提升肝臟抗氧化酵素 (SOD、GSH-Px及GSH-Rd) 的活性；保肝實驗結果顯示，口服給予野甘草70%乙醇萃取物的小鼠其血清中的AST及ALT值會明顯降低，病理組織切片也顯示野甘草70%乙醇萃取物可以減少CCl4造成的肝臟損傷，此外，野甘草70%乙醇萃取物可明顯減少肝臟組織中MDA含量及增加GSH含量，並提高肝臟抗氧化酵素 (SOD、GSH-Px、GSH-Rd及GST) 之活性。
HPLC活性成分分析方面，發現乙酸乙酯層含有黃酮類成分luteolin與apigenin，氯仿層含有apigenin及三萜類成分betulinic acid；
採集品野甘草乾燥原植物中luteolin、apigenin及betulinic acid之含量分別為181.61-229.56 μg/g、154.82-183.42 μg/g及678.78-785.60 μg/g，而市場品乾燥原植物中luteolin、apigenin及betulinic acid之含量分別為123.83-219.02 μg/g、117.18-181.53 μg/g及481.37-715.69 μg/g，且其穩定性、準確度、精密度 (同日間及異日間差異)、LOD及LOQ等試驗均在可接受的範圍內。
綜合以上結果顯示，野甘草70%乙醇萃取物具有抗氧化活性，各分劃層中以乙酸乙酯層具有最佳抗氧化活性；野甘草70%乙醇萃取物及betulinic acid具有鎮痛及抗發炎作用，其抗發炎作用機轉可能為抑制IL-1β及TNF-α等細胞激素，進而抑制COX-2及NO的生成；提升肝臟中SOD、GSH-Px及GSH-Rd等抗氧化酵素活性，以清除自由基，進而減低足蹠組織中脂質過氧化的傷害，藉此而達到抗發炎的效果；此外，野甘草70%乙醇萃取物亦可減輕CCl4所造成的急性肝損傷，其保肝作用機轉可能是經由防止肝臟SOD、GSH-Px、GSH-Rd及GST等抗氧化酵素活性下降，提升GSH含量，進而減少脂質過氧化的傷害而達到保護肝臟的效果。
The aims of this study were intended to investigate the antioxidant, analgesic, anti-inflammatory and hepatoprotective activities of the 70% ethanol extract from Scoparia dulcis (SDE). The active constituents and their contents of SDE were analyzed by high performance liquid chromatography (HPLC).
The antioxidant activities of the SDE and its fractions were determined by trolox equivalent antioxidant capacity, DPPH free radical scavenging activity, ferric-reducing antioxidant power (FRAP) assay and reducing power assay. The total polyphenol and flavonoid contents were also evaluated. The analgesic effect of SDE and beulinic acid were determined by acetic acid-induced writhing response and formalin test. The anti-inflammatory activity of SDE and betulinic acid were evaluated based on λ-carrageenan-induced paw edema in mice. The anti-inflammatory mechanisms of SDE and betulinic acid were examined by detecting the levels of COX-2, NO, TNF-α, IL-1β and MDA in the edema paw tissue and the activities of SOD, GSH-Px and GSH-Rd in the liver. The hepatoprotective effect of SDE was evaluated based on carbon tetrachloride (CCl4)-induced acute liver injury in mice. Three active constituents, luteolin, apigenin and betulinic acid, were analyzed by HPLC and the contents were quantified.
The results revealed that SDE possessed antioxidant activity in vitro. Among all fractions, the ethyl acetate fraction exhibited the best antioxidant potency and the highest total polyphenol and total flavonoid contents. In the analgesic test, SDE and betulinic acid significantly decreased the acetic acid-induced writhing response and licking time in the late phase of the formalin test. In the anti-inflammatory model, the results showed that SDE and betulinic acid reduced the paw edema after λ-carrageenan administration. Moreover, SDE and betulinic acid affected the levels of COX-2, NO, TNF-α and IL1-β in the λ-carrageenan-induced edema paws. The MDA level in the edema paws was decreased and the activities of SOD, GPx and GSH-Rd in liver tissues were increased. In the hepatoprotective test, mice treated orally with SDE showed significant inhibition on serum ALT and AST. Histological analysis also showed that SDE reduced the extent of liver lesions induced by CCl4. Moreover, SDE decreased the MDA level, elevated the content of GSH and enhanced the activities of antioxidant enzymes including SOD, GPx, GSH-Rd and GST in the liver.
The active constituents were detected by HPLC. The results showed that two flavonoids (luteolin and apigenin) were found in the ethyl acetate fraction, and apigenin and betulinic acid (a triterpenoid) were found in the chloroform fraction. The contents of luteolin, apigenin and betulinic acid in collection samples of Scoparia dulcis were 181.61-229.56, 154.82-183.42 and 678.78-785.60 μg/g dry plant, respectively. The contents of luteolin, apigenin and betulinic acid in marketing samples of Scoparia dulcis were 123.83-219.02, 117.18-181.53 and 481.37-715.69 μg/g dry plant, respectively. The validation parameters, such as stability, accuracy, precision (intraday and interday), LOD and LOQ, were found to be highly satisfactory.
These results indicated that the SDE possessed antioxidant activity. The ethyl acetate fraction exhibited the best antioxidant potency and the highest total polyphenol and total flavonoid contents. SDE and betulinic acid had analgesic and anti-inflammatory activities. The anti-inflammatory mechanism of SDE and betulinic acid appear to be related to the reduction of the levels cytokines (IL-1β and TNF-α) that resulted in the inhibition of COX-2 and NO. SDE and betulinic acid also enhanced the activities of SOD, GSH-Px and GSH-Rd in the liver to scavenge the free radicals and reduce the damage of lipid peroxidation in inflammatory paw tissue. Otherwise, SDE could alleviate CCl4-induced acute liver injury in mice. The hepatoprotective mechanism of SDE was likely associated to prevent the reduction of the activities of SOD, GPx, GSH-Rd and GST and increase the content of GSH that resulted in inhibition the damage of lipid peroxidation caused by CCl4. |
