- 學位論文
刺菝葜(Smilax spinosa Mill.) 根部萃取物之抗氧化活性
Antioxidant Activity of Smilax spinosa Mill.
摘要
刺菝葜(Smilax spinosa Mill.)為Smilax屬多年生灌木，因具有抗菌、解毒及抗菌的功效，可應用於因氧化所產生的相關疾病、貧血及因產後與經期所引發的出血及發炎等症狀，在大西洋沿岸特別是尼加拉圭已被廣範應用為藥草。然而關於刺菝葜的抗氧化活性及化學組成的報告仍甚屬少數，因此需以科學方式驗證其藥理活性。本研究的主要目地是以刺菝葜根部經萃取後，所得到的甲醇(methanol, ME)萃取物、乙酸乙酯(ethyl acetate, EA)層、正丁醇(n-butanol)層及水層等可溶物，藉由1,1-Diphenyl-2-Picrylhydrazyl (DPPH) 自由基清除能力與天然的抗氧劑如抗壞血酸(ascorbic acid, 維他命C)及沒食子酸(gallic acid, GA)進行活性比較，並探討對二價鐵螯合之能力與2,2,2,2-(Ethane-1,2-diyldinitrilo)-tetraacetic 酸(EDTA)進行活性比較，同時建立EA層溶出物在不同濃度下對人類肝炎細胞(HepG2)之細胞毒性效果，及保護因H2O2引發對人類肝細胞FL83B之氧化逆境的細胞保護效果等模式，探討抗氧化能力之差異性。結果顯示，刺菝葜根部在ME萃取物、EA層、n-butanol層及水層等可溶物，均有高含量的總酚及總類黃酮，尤以EA層溶出物有最高之含量，分別為71.81± 0.36 mg gallic acid eq./g D.W，45.27± 31.27 mg quercetin eq./g D.W，而水層溶出物的含量則為最低。四種萃取物及可溶物之DPPH清除能力及二價鐵離子螯合能力在0.1 mg/mL濃度時均高於70%，但較EDTA之效果差。整體而言，EA層可溶物有最高的DPPH清除能力(93%於0.2 mg/mL)， EA層可溶物之EC50為0.043 mg/mL，低於維他命C 0.0675 mg/mL及沒食子酸0.068 mg/mL，顯示EA層溶出物具有最高的抗氧化活性。本研究再進一步以2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1)及3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 溴化物(MTT)等方式，探討EA層可溶物之細胞毒性及細胞生存性的細胞保護效果。結果顯示EA層可溶物在抑制人類肝炎細胞(HepG2)的細胞毒性效果隨著濃度增加，抑制HepG2細胞的增生及擴展之效果越佳，當濃度為1500 µg/mL時可將癌細胞100% 清除。EA層可溶物對人類健康的肝細胞(FL83B)生存的細胞保護效果，顯示當濃度為12.5 µg/mL時，達到77%的細胞存活率。總結本研究結果顯示刺菝葜根部萃取物的具有顯著的體外抗氧化活性，將來仍需更進一步的評估體內抗氧化活性，方能確保功能性及安全性。
並列摘要
Chainey root (Smilax spinosa Mill.) is popularly used in Nicaragua’s traditional medicine for its believed anti-bacterial, anti-toxin and antioxidant properties. Despite the widespread use of this plant in Nicaragua, especially on the Atlantic coast, literature contains few reports on the antioxidant activity and chemical composition of S. spinosa. Scientific evidence is required to verify its medicinal effects. Therefore, the aims of this study were to investigate the antioxidant properties of S. spinosa’s root methanolic (ME) extract and its ethyl acetate (EA), n-butanol and water soluble fractions. The total phenolic and flavonoid content, 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) free radical scavenging capacity and Iron (II) chelating activity were investigated. The cytotoxic effect of the EA fraction on human liver hepatoma (HepG2) cells and its cytoprotective properties against H2O2 induced oxidative stress on healthy human liver (FL83B) cells were established. Results demonstrated the antioxidant activity of S. spinosa root to be concentration dependant. Phenolics and flavonoids were found in the ME extract as well as in the EA, n-butanol and water soluble fractions. EA fraction revealed the highest gallic acid equivalence (GAE) of 71.81± 0.36 mg/g DW. Quercetin equivalent was also highest for the EA fraction (45.27± 31.27 mg/g). On the other hand, lowest phenolics and flavonoids were observed in the water soluble fraction. The DPPH scavenging capacity and Iron (II) chelating activity of all four fractions were above 70% at 0.1 mg/ mL. However, none of the fractions were better iron chelators than positive control EDTA. EA showed the highest DPPH scavenging percentage (93%) at 0.2 mg/mL. Among all test samples, the EC50 value of EA soluble fraction was the lowest (0.033 ± 0.002 mg/mL), indicating stronger antioxidant activity than those of positive controls ascorbic acid (0.067 ± 0.0002 mg/mL) and gallic acid (0.068 ± 0.0004 mg/mL). Because of its outstanding antioxidant activity, the EA fraction was selected for further assessment of cytotoxic and cytoprotective effects on the viability of human liver cells. Cytotoxic effect of S. spinosa root EA fraction on the proliferation of human liver hepatoma (HepG2) cells was assessed with the 2-(4-Iodophenyl)-3 - (4-nitrophenyl-5 (2, 4-disulfophenyl) - 2H-tetrazolium (WST-1) assay. S. spinosa EA fraction was capable of interrupting the development and proliferation of HepG2 cells at a concentration dependant manner. At 1500 µg/mL the scavenging of 100% cancerous cells was accomplish by the EA fraction. The Protective effect of the EA fraction on healthy human liver (FL83B) cells viability was assessed by treating the cells with EA for 1 hr prior to the addition of H2O2. The relative cell survival was determined by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. At a concentration of 12.5 µg/mL, the EA fraction enhanced a protective effect on the cells viability allowing 77% cells survival. Results provided a scientific support to the antioxidant properties of S. spinosa Mill. root extract in vitro. Further evaluation of its antioxidative properties in vivo is needed to ensure functionality and long term safety.