HPLC-DPPH法评价雪松松针抗氧化活性成分

Evaluation of antioxidant activity compounds extracted from the pine needle of Cedrus deodara by HPLC-DPPH screening method

目的评价雪松松针抗氧化活性成分。方法以维生素C(VC)和没食子酸为阳性对照,采用高效液相色谱(HPLC)法测定1,1-二苯基-2-苦肼基(DPPH)溶液加入雪松松针抗氧化活性成分后DPPH峰面积的衰减情况,并通过计算DPPH自由基的清除率和半数抑制浓度(IC_(50)),来评价雪松松针3种有效部位及8个单体成分的抗氧化活性。采用Agilent Eclipse Plus-C_(18)(250 mm×4.6 mm,5μm)为色谱柱,乙腈-水(62∶38,V/V)为流动相;检测波长为517 nm;流速为1.0 mL/min;柱温为25℃;进样量为20μL。结果雪松松针各有效部位清除DPPH自由基活性的能力大小依次为总黄酮>总多酚>总木脂素,IC_(50)分别为76.10、100.50、115.40μg/mL。与阳性对照比较,除山柰酚-3-O-β-D-葡萄糖苷外,7个单体化合物清除DPPH自由基活性的能力大小依次为二氢杨梅素>金丝桃苷>原儿茶酸>没食子酸>阿魏酸>杨梅素>山柰酚-3-O-(6″-O-E-阿魏酰基)-β-D-吡喃葡萄糖苷>VC>异鼠李素,IC50分别为16.50、19.40、23.73、27.24、32.10、32.20、47.60、93.20、297.20μg/mL。结论建立了HPLC-DPPH法用于评价雪松松针抗氧化活性;雪松松针具有较强的抗氧化活性,其中有效部位总黄酮、总多酚及单体成分二氢杨梅素、金丝桃苷等是具有开发前景的纯天然抗氧化剂。

OBJECTIVE To evaluate the antioxidant activity compounds extracted from the pine needle of Cedrus deodara.METHODS Using vitamin C(VC)and gallic acid as positive control,HPLC method was employed to determine the changes of the DPPH peak area after antioxidant compounds from pine needle of C.deodara were treated with 1,1-diphenyl-2-picrylhydrazyl radical(DPPH).Then,the antioxidant activities of 3 active parts and 8 monomer compositions from pine needle of C.deodara were evaluated by calculating the inhibition rate of DPPH free radicals and the half inhibitory concentration(IC_(50)).HPLC conditions were using Agilent Eclipse Plus-C_(18) column(250 mm×4.6 mm,5μm)with acetonitrile-water(62∶38,V/V)as the mobile phase.The detection wavelength was 517 nm;the flow rate was 1.0 mL/min;the column temperature was 25℃;the injection volume was 20μL.RESULTS The order of DPPH free radical inhibition activity of active parts from pine needle of C.deodara was total flavonoids>total polyphenols>total lignans,with IC50 of 76.10,100.50 and 115.40μg/mL,respectively.Compared with positive control,except for kaempferol-3-O-β-D-glucoside,the sequence of seven monomer compounds for DPPH free radical inhibition activity was dihydromyricetin>hypericin>protocatechuic acid>gallic acid>ferulic acid>myricetin>kaempferol-3-O-(6″-O-E-feruloyl)-β-D-glucopyranoside>VC>isohamnin,with IC_(50) of 16.50,19.40,23.73,27.24,32.10,32.20,47.60,93.20 and 297.20μg/mL,respectively.CONCLUSIONS HPLC-DPPH method is established to evaluate the antioxidant activity of pine needle of C.deodara.The pine needle of C.deodara has strong antioxidant activity,among which the effective compounds of total flavonoids,total polyphenols,monomer dihydromyricetin and hypericin are natural antioxidants with development prospects.