Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50935
標題: Antioxidant and apoptosis-inducing properties of water extracts from Chinese medicinal plants in Human hepatoma HepG2 cells
中草藥水萃取物抗氧化及誘導肝癌細胞凋亡之研究
作者: 徐美如
Hsu, Mei-Ju
關鍵字: 中草藥
抗氧化
高效液相層析法
低密度脂蛋白
肝癌HepG2細胞
細胞凋亡
Chinese medicinal plants、antioxidative、high performance liquid chromatography (HPLC)、low-density lipoprotein (LDL)、hepat
出版社: 食品科學系
摘要: 本研究以砂仁、紅棗、山防風、山葡萄、荔枝草、雞血藤及白鶴靈芝等七種中草藥為材料,進行上述中草藥水萃取物其抗氧化性及誘導細胞凋亡之研究。結果顯示,在中草藥水萃取物清除DPPH自由基之效力上,以雞血藤最好(IC50為8.3 micro g/mL),紅棗(IC50為526.7 microg/mL)最差。總抗氧化力(TEAC)表現方面依次為雞血藤>荔枝草≧白鶴靈芝>山葡萄>山防風>砂仁>紅棗。清除過氧化自由基效力(ORAC)以雞血藤、白鶴靈芝、山防風及山葡萄表現較佳。測定中草藥水萃取物之總抗氧化物的含量,發現七種樣品中以雞血藤水萃取物的多酚類、類黃酮含量最高(分別為390.9和69.1 mg/g)。以HPLC分析定量七種中草藥水萃取物之主要成分,除了紅棗無酚酸及類黃酮化合物,其餘六種中草藥與標準品比對後,發現主要成分為p-hydroxybenzoic acid、gallic acid、protocatechuic acid、caffeic acid、vanillic acid、epicatechin (EC)、p-coumaric acid、gentisic acid。進一步將中草藥以酸、鹼水解處理後,經HPLC分析結果顯示山防風及荔枝草含有高量的caffeic acid、protocatechuic acid;砂仁、山葡萄與雞血藤則含有多量的gallic acid、protocatechuic acid、salicylic acid;紅棗經過水解處理後,主要酚酸成分為chlorogenic acid。探討七種中草藥水萃取物延緩LDL氧化修飾之影響結果顯示,當作用劑量為5 microg/mL時,其延長共軛雙烯生成之遲滯時間分別為雞血藤(483分鐘)>荔枝草(261分鐘)>山防風(230分鐘)>山葡萄(202分鐘)>白鶴靈芝(145分鐘)>砂仁(79分鐘)>紅棗(69分鐘)。TBARS試驗結果發現,雞血藤(5 microg/mL)具有最佳抑制MDA生成的效力,其次為荔枝草。同時,在電泳相對移動量(REM)的測定上,結果中草藥水萃取物仍以雞血藤的效力最好,與對照組ascorbic acid有相似之抑制效應。以人類肝腫瘤細胞株HepG2為模式,探討七種中草藥水萃取物是否有促進細胞凋亡之效應。結果顯示,荔枝草和雞血藤對於HepG2細胞呈現生長抑制效應,細胞增殖有明顯下降的情形,其IC50值分別為233.7及226.8 microg/mL,而對Clone 9則無抑制增生效果。以流式細胞儀分析HepG2細胞週期分布之變化,砂仁、山葡萄、荔枝草和雞血藤處理組可誘發sub-G1 phase arrest,且隨著樣品濃度增加而上升,其中效力最佳的雞血藤(500 microg/mL)反應72小時,其sub-G1 phase由0.72%顯著提高至19.42%(p<0.05)。在Annexin V/PI雙染試驗,結果顯示荔枝草和雞血藤具最佳效力,凋亡細胞數分別為5.48%及9.62%(Control為0.69%)。Western blot分析結果發現,山葡萄、荔枝草和雞血藤可能藉由增加p53的表現,抑制Bcl-2蛋白並促進caspase-3活化而引發下游PARP裂解,進而誘導HepG2細胞走向自體凋亡的途徑。
The objectives of this study were to investigate the antioxidative and anti-tumor effects of 7 Chinese medicinal plants extracts (Amomi fructus, Zizyphus jujuba Mill., Echinops grijsii, Ampelopsis brevipedunculata (Maxim.) Trautv., Salvia plebeia R. Brown, Millettia reticulata Benth, and Rhinacanthus nasutus L. Kurz). Among the 7 Chinese medicinal plants extracts, M. reticulata performed the highest scavenging effect on DPPH radical (IC50=8.3 microg/mL), and the worst one was Z. jujuba (IC50=526.7 microg/mL). The TEAC of the Chinese medicinal plants extracts was in an order of: M. reticulata>S. plebeia≧R. nasutus>A. brevipedunculata>E. grijsii>A. fructus>Z. jujuba. As to the ORAC, the data indicated that M. reticulata, R. nasutus, E. grijsii and A. brevipedunculata had high ORACROO. value among the 7 Chinese medicinal plants tested. M. reticulata contained the most abundance of polyphenols and isoflavones (390.9 and 69.1 mg/g, respectively) among the 7 Chinese medicinal plants extracts. Chinese medicinal plants extracts were analyzed by HPLC. We found that these Chinese medicinal plants except Z. jujuba consisted of p-hydroxybenzoic acid, gallic acid, protocatechuic acid, caffeic acid, vanillic acid, epicatechin (EC), p-coumaric acid, and gentisic acid. Furthermore, after the treatment of alkaline and acid hydrolyses, the results showed that the major compounds in E. grijsii and S. plebeia were protocatechuic acid and caffeic acid; A. fructus, A. brevipedunculata and M. reticulata were rich in gallic acid, protocatechuic acid and salicylic acid; Z. jujuba contained high quantity of chlorogenic acid. In the Cu2+/LDL oxidative modification system, the ability of the Chinese medicinal plants extracts (5 microg/mL) that retarded LDL oxidation through prolonging the lag phase of conjugated diene formation were: M. reticulata (483 min)>S. plebeia (261 min)>E. grijsii (230 min)>A. brevipedunculata (202 min)>R. nasutus (145 min)>A. fructus (79 min)>Z. jujuba (69 min). M. reticulata possessed the highest inhibitory effect on the formation of MDA, and the next was S. plebeia. Through the determination of the REM, the data indicated that M. reticulata performed the highest inhibitory effect on the oxidative modification of LDL induced by Cu2+. To evaluate the apoptosis-inducing activity of Chinese medicinal plants extracts on human hepatocellular carcinoma HepG2 cells, the results showed that S. plebeia and M. reticulata exhibited the most potent anti-proliferation effect on HepG2 cells, and the IC50 values were 233.7 and 226.8 microg/mL, respectively. Flow cytometric analysis showed that the significant arrest of cell cycle at the sub-G1 phase by A. fructus, A. brevipedunculata, S. plebeia and M. reticulata were in a time- and dose- dependent manner (p<0.05). Annexin V/PI determination on S. plebeia and M. reticulata treated HepG2 cells showed the increase of apoptotic bodies formation (5.48 and 9.62%, respectively and control was 0.69%). Western blot assay showed that A. brevipedunculata, S. plebeia and M. reticulata up-regulated the expression of p53 and down-regulated Bcl-2, followed by turning on the activation of caspase-3 and resulted in the proteolysis of PARP, finally executed apoptosis in HepG2 cells.
URI: http://hdl.handle.net/11455/50935
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