相思樹樹皮抽出物之抗氧化活性及護肝功效

Abstract

相思樹（Acacia confusa Merr.）為臺灣本土樹種，主要分佈於臺灣低海拔的山地與丘陵地，為傳統用藥之一。本研究針對樹皮乙醇抽出物及其各可溶部進行DPPH自由基清除試驗、總抗氧化力試驗、超氧自由基清除試驗、還原力測定、脂質過氧化抑制試驗及亞鐵離子螯合試驗等抗氧化活性評估，試驗結果顯示，樹皮乙醇抽出物各可溶部中，以乙酸乙酯可溶部清除DPPH自由基活性（EC50值 = 3.2 μg/mL）、總抗氧化力（2.0 mM Trolox）、清除超氧自由基活性（EC50值 = 4.7 μg/mL）、還原能力及螯合亞鐵離子能力（EC50值 = 253.0 μg/mL）最佳。因此，乃利用管柱層析與高效能液相層析進一步分離，共得25個化合物；並證實相思樹樹皮乙酸乙酯可溶部所分離的化合物包括3,4-Dihydroxybenzoic acid、3,4-Dihydroxybenzoic acid ethyl ester 、3,4,5-Trihydroxybenzoic acid（五倍子酸）及3,4,5-Trihydroxybenzoic acid ethyl ester等皆具極佳的抗氧化活性。因此，無論是相思樹樹皮或其化合物皆極具開發成為天然抗氧化劑之潛力，可避免自由基所帶來的各種疾病。 進一步利用四氯化碳誘導老鼠慢性肝損傷之動物試驗模式探討相思樹樹皮抽出物及五倍子酸（相思樹樹皮有效之抗氧化成分）之護肝功效。將六週齡大的SD大白鼠分成7組，每組六隻，包括（A）正常對照組、（B）CCl4處理組、（C）CCl4處理組並餵食水飛薊（50 mg/kg/day）、（D）CCl4處理組並餵食低劑量抽出物（50 mg/kg/day）、（E）CCl4處理組並餵食高劑量抽出物（250 mg/kg/day）、（F）CCl4處理組並餵食五倍子酸（50 mg/kg/day）及（G）餵食高劑量抽出物（250 mg/kg/day）。B、C、D、E及F組皮下注射0.75 mL/kg of BW之CCl4（40% CCl4/Olive oil, v/v），每星期一次，而A及G組則以等量之橄欖油取代，試驗為期九週。試驗結果顯示，相思樹樹皮抽出物及五倍子酸處理組皆能顯著降低肝損傷大白鼠血漿中AST及ALT活性，並有效降低CYP2E1表現量（p < 0.05）。且相思樹樹皮抽出物處理組能有效增加肝損傷大白鼠紅血球中超氧化物歧化酶活性、麩胱甘肽過氧化酶活性、GSH/GSSG（Glutathione/Oxidized glutathione）比值及肝臟中麩胱甘肽還原酶、麩胱甘肽過氧化酶及過氧化氫酶活性，並降低血漿中脂質過氧化物（p < 0.05）。此外，五倍子酸處理組則能有效增加肝損傷大白鼠紅血球及肝臟中的抗氧化酵素（超氧化物歧化酶、麩胱甘肽還原酶、麩胱甘肽過氧化酶及過氧化氫酶）活性及GSH/GSSG比值，並可降低血漿及肝臟中脂質過氧化物（p < 0.05）。綜合上述結果顯示，相思樹樹皮抽出物及五倍子酸皆具護肝功效，能有效抑制四氯化碳所引發的氧化壓力。

Acacia confusa Merr. (Leguminosae), a species indigenous to Taiwan, is widely distributed on the hills and lowlands of Taiwan, and it is traditionally used as a medicinal plant. The antioxidant activities of A. confusa bark extract and its soluble fractions were examined using various antioxidant assays, such as DPPH, TEAC, NBT, reducing power, lipid peroxidation, and metal chelating ability. Results showed that among all the fractions from ethanolic extracts of bark, the EtOAc soluble fraction exhibited the best antioxidant performance with DPPH free-radical scavenging activity (EC50 value = 3.2 μg/mL), trolox equivalent antioxidant capacity (2.0 mM Trolox), superoxide radical scavenging activity (EC50 value = 4.7 μg/mL), reducing power, and ferrous ion-chelating ability (EC50 value = 253.0 μg/mL). Furthermore, following by CC and HPLC, 25 pure phenolic compounds were isolated from ethanolic extracts of A. confusa bark. This study also demonstrated that phenolic compounds, such as 3,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid ethyl ester, 3,4,5-trihydroxybenzoic acid, and 3,4,5-trihydroxybenzoic acid ethyl ester obtained from EtOAc soluble fraction of A. confusa bark exhibited excellent antioxidant activities. Therefore, bark extracts and the derived phytochemicals from A. confusa have a great potential to prevent disease caused by the overproduction of radicals and also it might be used as a potential source of natural antioxidant. The hepatoprotective effects of A. confusa bark extract and gallic acid, an active constituent obtained from A. confusa bark, were evaluated against carbon tetrachloride (CCl4)-induced hepatotoxicity in male Sprague-Dawley rats. Rats were randomly assigned to seven groups; olive oil (control group), CCl4 injection (CCl4 group), dietary supplementation with silymarin at a dose of 50 mg/kg plus CCl4 injection (silymarin + CCl4 group), dietary supplementation with crude extract at a relatively low dose of 50 mg/kg plus CCl4 injection (low dose of 50 mg/kg crude extract + CCl4 group), dietary supplementation with crude extract at a relatively high dose of 250 mg/kg plus CCl4 injection (high dose of 250 mg/kg crude extract + CCl4 group) , dietary supplementation with gallic acid at a dose of 50 mg/kg plus CCl4 injection (gallic acid + CCl4 group), and dietary supplementation with crude extract at a relatively high dose of 250 mg/kg (high dose of 250 mg/kg crude extract group). Rats in CCl4 group, silymarin + CCl4 group, low dose of 50 mg/kg crude extract + CCl4 group, high dose of 250 mg/kg crude extract + CCl4 group, and gallic acid + CCl4 group were subcutaneously injected with 0.75 mL/kg of 40% CCl4 dissolved in olive oil once a week, while rats in control group and high dose of 250 mg/kg crude extract group were only injected with 0.75 mL/kg of olive oil. The experimental period was 9 weeks. Results showed the rats treated with crude extract and gallic acid showed significantly decreased plasma AST and ALT activities and CYP2E1 levels than those in CCl4 group (p < 0.05). Additionally, treatment with A. confusa bark extract significantly increased SOD activity, GPX activity and GSH/GSSG ratio in the erythrocytes, increased GRD, GPX and CAT activities in the hepatocytes, and decreased lipid peroxides in the plasma on CCl4-induced hepatotoxicity in rats (p < 0.05). Moreover, the antioxidant enzyme (SOD, GRD, GPX, and CAT) activities and GSH/GSSG ratio were increased by gallic acid in the erythrocytes and hepatocytes compared with those administered with CCl4 only (p < 0.05). Besides, gallic acid also reduced significantly lipid peroxides in the hepatocytes and plasma compared with CCl4 only (p < 0.05). These results suggested that A. confusa bark extract and gallic acid showed the hepatoprotective effect under CCl4-induced oxidative stress.