| In this paper,the response surface method was used to optimize the extraction process of flavonoids from seeds of Passiflora Edulis Sims,and the flavonoids were purified by macroporous resin column chromatography.By building an experimental model of D-galactose oxidative damage in mice,in vivo antioxidant capacity was investigated,by measuring DPPH,ABTS,OH radical scavenging rate and total reduction capacity to evaluate the antioxidant activity of the purified flavonoids in vitro.The components of flavonoids in seeds were speculated by means of color reaction,ultraviolet spectrum,liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-tandem mass spectrometry.The specific research results are as follows:（1）The extraction process of flavonoids from seeds of Passiflora Edulis Sims was studied by using organic solvent reflux methodology and ultrasonic-microwave-assisted methodology.Optimized by single factor and orthogonal test,the optimum technological conditions for organic solvent refluxing extraction as follows:70%ethanol volume fraction,1:60 of ratio between solid and liquid,extraction temperature 90℃,extracting time 2.5 h,extracted flavonoids content in the seeds of 151.34 mg/g.Through the single factor and response surface optimization test,the quadratic multiple linear regression equation was used for fitting,and the optimal process conditions for ultrasonic-microwave-assisted methodology as follows:80℃of extraction temperature,73.78%of ethanol concentration,1:56.02 of ratio between solid and liquid,300 w of ultrasonic power,50 min of ultrasonic time,160 w of microwave power,90 s of microwave time,by which the yield of flavonoids reached 117.53 mg/g.Compared with the reflux extraction with organic solvents,the extraction time of ultrasonic-microwave-assisted methodology was shortened by 2/3,but the flavonoid extraction content was reduced by 22.34%.（2）The flavonoids from seeds of Passiflora Edulis Sims extracted by ultrasonic-microwave-assisted was used as raw material,the adsorption and analytical properties of six types of macroporous resins for flavonoids in seeds were compared,and AB-8 resins were selected as fast equilibrium resins.The column chromatography conditions,such as sample concentration,maximum sample loading,amount of water detergent and amount of eluent,were investigated.Finally,the sample concentration was determined to be 1.0mg/m L,the maximum sample loading was 270m L,the amount of water detergent was 150m L,and the amount of eluent was 90m L.By single factor and orthogonal optimization test,the optimal purification process was obtained as follows:loading flow rate of 9r/min,loading p H3,elution flow rate of 12r/min,ethanol volume fraction of 70%,flavonoids recovery rate is reach 89.67%.By comparing the purity and the liquid phase analysis diagram of samples before and after purification,it can be seen that the purity of crude flavonoids purified by AB-8 macroporous resin increased from58.58%to 72.84%.（3）DPPH,ABTS+·,·OH radical scavenging rate and total reduction capacity of samples before and after purification were determined to compare the antioxidant activity of them in vitro.The results showed that DPPH,ABTS+·,·OH radical scavenging rate and total reduction capacity increased with the increase of sample concentration.The IC50values of DPPH,ABTS+·and·OH radical scavenging rates of the purified products were lower than those of the crude extracts,indicating that the purity of flavonoids was improved,and the antioxidant capacity of the samples in vitro was also improved.The animal model of oxidative damage induced by D-galactose was established,purified flavonoids were intragastrically administered.The body weight,superoxide dismutase（T-SOD）,catalase（CAT）,glutathione peroxidase（GSH-PX）,glutathione（GSH）,malondialdehyde（MDA）and protein carbonyl were measured to evaluate the antioxidant activity of purified flavonoids as exogenous antioxidants in vivo.The results showed that compared with the blank group,the body weight,T-SOD activity,CAT activity,GSH-PX activity,GSH content in liver and kidney and T-SOD activity in serum of the model group mice were significantly decreased（P<0.05）,while the MDA content,protein carbonyl content in liver and kidney and MDA content in serum were significantly increased（P<0.05）,indicating that the oxidative damage model was successfully constructed.Compared with the model group,the activities of T-SOD,CAT and GSH-PX,the content of GSH and the body weight of mice increased with the increase of flavonoids gavage dose in each dose group,while the content of MDA and protein carbonyl decreased,and the dose-effect relationship existed between the dosage of flavonoids and the antioxidant effect.Compared with the VC positive control group,purified flavonoids with medium and high doses had better effects on increasing GSH content,CAT and GSH-PX activites,and reducing MDA and protein carbonyl content.（4）The components of flavonoids in seeds were speculated by means of color reaction,ultraviolet spectrum,liquid chromatography coupled with electrospray Ioniza tion quadrupole-time of flight-tandem mass spectrometry.The results are as follows:the color reaction showed that the purified product was flavonoids with hydroxyl group at C5 of ring A.The UV spectrum showed the characteristic absorption peak of flavonoids,after the addition of diagnostic agent,the UV spectrum was used for identification and the chromogenic reaction was combined to infer that the flavonoids in the purified product has O-diphenol hydroxyl replaced in ring A and one of them at C5,ang has O-diphenol hydroxyl replaced in ring B and one of them at C4’.The absorption peak information of each functional group in the infrared spectrum indicated that the purified product has a complete basic framework of flavonoids,and it was determined that the purified product belonged to flavonoids.The purified product was analyzed by liquid and mass analysis,and the detected compound information was obtained by molecular characteristic extraction technology（MFE）.The molecular mass,molecular formula,first-order mass spectrometry information and second-order mass spectrometry information were obtained.Combined with the literature reports,the cleavage pathway was speculated,and four flavonoid glycosides were speculated,including2-（3,4-dihydroxyphenyl）-5,7-dihydroxy-6-isopentyl-4H-chromen-4-one,2-（4-hydroxyphenyl）-3,6-dimethoxy-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-4-one,5-hydroxy-2-（4-hydroxy-3-methoxyphenyl）-3-methoxy-7-（（3-methylbut-2-en-1-yl）oxy）-4H-chromen-4-one,7-（（4-methoxybenzyl）oxy）-3-（2-methoxyphe nyl）-4-oxo-4H-chromene-2-carboxylate. |
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