New Evaluation Methods For The Scavenging Effect Of Plant Polyphenols On DPPH Free Radical And Their Application

The traditional antioxidant activity evaluation methods were based onspectrophotometry, fluorophotometry and chemiluminescence method, which existsome drawbacks such as low sensitivity, high sample consumption, bad reliability,and spectral interference caused by plant ingredients. HPLC and electrochemicalmethod have shown the good application prospect on the evaluation of antioxidantactivity with higher sensitivity, better reliability and lower sample consumption; theycan also avoid the interference of coloring matter.In view of the shortcomings of the traditional antioxidant activity evaluationmethods, the spectrum, electrochemical and liquid chromatography characteristics ofthe scavenging reaction of plant polyphenols on DPPH radical were investigated inthis paper，and the novel spectrum, electrochemical and liquid chromatographymethods for the evaluation of DPPH scavenging activity of plant polyphenols weresuggested. The proposed methods were applied to the antioxidant activity research ofTibetan medicine terminalia.The main research contents and results are as follows:（1） HPLC analysis of plant polyphenols and the active ingredients forscavenging DPPH free radicalAB-8macroporous resin was selected to the further separation and purificationof terminalia polyphenols. The content of polyphenols compounds in terminalia wasdetermined by HPLC. An HPLC method for the simultaneous determination of fourplant polyphenols （gallic acid, vanillic acid, caffeic acid and p-coumaric acid） in theextract of terminalia was proposed. The optimum chromatographic separationconditions: XDB-C18（4.6×150mm i.d,5μm） column.The mobile phase: methanol（A） and1%ethylic acid （B） with a linear gradient program at flow rate of1.0mL·min^-1. The detection wave-length was280nm, and the injection volume was20μL. The HPLC analysis was carried out on the column temperature of25oC. Theresults showed that the contents of gallic acid, vanillic acid, caffeic acid andp-coumaric acid were1.22mg·g^-1,0.78mg·g^-1,0.13mg·g^-1,1.11mg·g^-1, respectively.The bioactive components for the scavenging DPPH radical in terminalia were identified by HPLC via addition DPPH free radical into purred polyphenols.12components were found to have the DPPH free radical scavenging activity interminalia, and4of them were identified as gallic acid, vanillic acid, caffeic acid andp-coumaric acid. Gallic acid has the highest scavenging activity, followed by caffeicacid, the scavenging activity of vanillic acid and p-coumaric acid was weaker thanthat of gallic acid and caffeic acid.（2） HPLC analysis method for the DPPH free radical scavenging of plantpolyphenolsAn HPLC method specific for the determination of the DPPH free radicalscavenging activities of plant polyphenols was suggested. The HPLC conditions:Agilent1200XDB-C18（4.6×150mm i.d,5μm） column; mobile phase: methanol andwater （50:50, v/v）; flow rate:1.0mL·min^-1; detection wavelength:517nm; columntemperature:25oC; injection volume:20μL. Detection6times everyday in3days,and detect the peak area and retention times of the DPPH blank solution （R.S.D.<4%）. DPPH free radical scavenging activities of allic acid, vanillic acid, caffeic acidand p-coumaric acid were researched by developed method, and detect calibrationequation of four samples scavenging DPPH free radical.The DPPH free radical scavenging activities of plant polyphenols wasdetermined by HPLC, the DPPH radical scavenging activity can be evaluated via thecomparison of their calibration equation and the concentration for50%radicalscavenging rate （IC50） calculated based on the calibration equation. The radicalscavenging activity was ranked as follows: gallic acid> caffeic acid> extracts ofterminalia> rutin> vanillic> p-coumaric acid.（3） The spectral characteristic of plant polyphenols and DPPH radical and thespectrum titration analysisThe spectral characteristic of DPPH free radical and four plant polyphenolswas investigated. The resules showed that detection wavelength was517nm;the stable time of DPPH was1h. The maximum scavenging time of four plantpolyphenols for DPPH was ranked as follows: gallic acid（10min）﹤caffeicacid（20min）﹤vanillic acid（30min）﹤p-coumaric acid（35min）. Gallic acidand caffeic acid can effectively and rapidly scavenge. It was found that the terminalia polyphenols extract had strong anti-oxidative activity, and gallicacid and caffeic acid are two main active ingredient of scavenging DPPH freeradical in terminalia.Based on the traditional spectrophotometry estimation method of DPPHscavenging effect, a spectrum titration method of plant polyphenols scavengingDPPH free radical was investigated. We determinated the spectrum titration curve ofgallic acid, vanillic acid, caffeic acid and p-coumaric acid for different concentrationof DPPH scavenging effect. And got the stoichiome tricrelationship of four plantpolyphenols and DPPH was1:3,1:2,1:0.0015,1:0.0004, respectively.The error analysis indicated that the error of the spectrum titration was related toDPPH concentration and the ability of plant polyphenols scavenging DPPH.Thehigher clear ability, the smaller RSD is. The RSD of high concentration （0.007mg/m）and middle concentration （0.004mg/mL） less than2%for gallic acid and caffeic acid.But the error of low concentration （0.002mg/mL） increased obviously for four plantpolyphenols.（4） The electrochemical oxidation mechanism of plant polyphenols andmicro-electrochemical titration analysis method of scavenging DPPH free radicals.Based on electrochemical oxidation mechanism of plant polyphenols, a novelmicro-electrochemical titration method to detect micro-electrochemical titration curve.According to micro-electrochemical titration curve, the stoichiometricrelationship ofgallic and DPPH was determinated with a stoichiometric proportion of1:3（gallic acidto DPPH）,1:2（caffeic acid to DPPH）, which was coincidences with the results ofspectrophotometry.