Study On HPLC Efficient Detection Separation Of Phenolics Compounds And Antioxidant Capacity From Crataegus Pinnatifida Leaves

Crataegus pinnatifida Bge.Var.Major N.E.Br.?C.pinnatifida??Rosaceae?,and there is a variety of natural bioactive in it.Many of the bioactive components in the leaves have antioxidant activity.Currently,there are few reports on the simultaneous determination of various phenolic compounds and antioxidant activity in leaves of red leaves.This thesis focused on the high efficient extract of active component phenolic from C.pinnatifida leaves and evaluation of antioxidant activities of extracts.In this study,an accurate method using high pressure liquid chromatography?HPLC?has been established for the simultaneous determination of chlorogenic acid?CA?,vitexin-4?-o-glucoside?VG?,vitexin-2?-o-rhamnoside?VR?,orientoside?ORT?,rutin?RT?,vitexin?VIT?and hyperoside?HYP?from C.pinnatifida leaves.Box-Behnken design?BBD?of response surface methodology?RSM?was used to optimize ultrasound-assisted extraction?UAE?method on a basis of single factor.The effects of maceration extraction?ME?,heat-refluxing extraction?HRE?methods and UAE method method on the content of the phenolic compounds were investigated.The antioxidant activity was evaluated by DPPH radical scavenging?DPPH?,ABTS radical scavenging?ABTS?and reducing power tests.1.The HPLC compounds were used to test the phenolic compounds in the samples.At the detection wavelength of 350 nm,within 32 min,HPLC method for the simultaneous determination of seven phenolic compounds was established.HPLC method was successfully developed to detect phenolic in sample.Under this system,the retain time of CA,VG,ORT,RT,VR,VIT and HYP were 8.82 min,10.69 min,14.88 min,18.59 min,21.16 min,23.89 min and31.55 min,respectively.This method has good separation,linear range,limite of detection?LOD?and limit of quantity?LOQ?quantitativeand detection limit,repeatability,stability and recovery.The results of the method are reliable and accurate,and can be used for CA,VG,ORT,RT,VR,VIT and HYP analysis.2.The factors influencing in UAE were analyzed by single factors analysis.The optimum condition was ratio of solid/liquid 1:15,extraction temperature 40?,ethanol concentration40%and extraction time 25 min.Under the optimal condition,the extraction yields of C.Pinnatifida were 5.73 mg/g,6.20 mg/g,6.28 mg/g and 6.34 mg/g,respectively.Under optimized conditions?extraction temperature 41?,extraction time 31 min,ratio of liquid/solid 15 and ethanol concentration 39%?,the yields of CA,VG,VR,ORT,RT,VIT and HYP were 0.46 mg/g,0.38 mg/g,4.37 mg/g,0.033 mg/g,0.036 mg/g,0.30 mg/g and 1.19,mg/g,respectively.3.The contents of phenolic compounds in C.pinnatifida leaves were investigated by ME,HRE and UAE method.The results showed that the extraction efficiency of phenolic compounds using UAE method was higher than that of traditional ME?4.862 mg/g?and HRE?3.938 mg/g?,UAE?6.017 mg/g?is more suitable for the extraction of phenolic compounds from C.pinnatifida leaves.4.DPPH,ABTS and reducing power tests were used to evaluate and compare the antioxidant contents of components in the leaves of the C.Pinnatifida.Additionally,antioxidant activities of UAE extracts in DPPH,ABTS and reducing power tests were determined as 0.69 mg/mL(IC₅₀ value),0.86 mmoL Trolox and 0.24 mg/mL(IC₅₀ value),respectively.The results showed that the extracts of the leaves of the C.Pinnatifida obtained from the three methods had certain antioxidant activity,but the extract obtained by ultrasonic extraction had better antioxidant activity,which could be the importance of the research on the development of low cost natural antioxidants.Overall,the determination method of phenolic compounds in leaves of red leaves is fast,efficient and simple.The present results showed that UAE was an efficient and alternative method for the extraction of phenolic compounds form C.pinnatifida leaves and its extracts were potential antioxidant ingredients or additives in food industry using potential preservation properties.