Extraction,Separation And Purification Of Polyphenols From Olive Leaves And Their Application In Walnut Oil

Olive leaves contain a variety of antioxidant substances,among which the content of oleuropein and its antioxidant effect are particularly prominent,so it has gradually attracted people’s attention.In this paper,olive leaves were used as raw materials,and polyphenols from olive leaves were extracted by solvent.The main components of olive leaf polyphenols were qualitatively and quantitatively analyzed,and oleuropein was isolated,purified and structurally identified.The free radical scavenging ability of crude phenolic extracts and oleuropein and their antioxidant properties in walnut oil were discussed in order to provide reference for the development and utilization of olive leaves.The main findings are as follows:（1）Single factor and response surface methodology were used to investigate the effects of ethanol volume fraction,extraction time,solid-liquid ratio(M _{olive leaf}:V _{ethanol solution})and water bath temperature on the yield of olive leaf polyphenols.The results showed that the ethanol volume fraction 50%,extraction time 1.0 h,solid-liquid ratio 1:40（g/m L）,water bath temperature 72℃,the yield of olive leaf polyphenols was 5.86%.The olive leaf polyphenols were qualitatively and quantitatively analyzed by high performance liquid chromatography.The results showed that the content of oleuropein in olive leaves was 32.57 mg/g,accounting for 55.58%of olive leaf polyphenols;1.55 mg/g,accounting for 2.65%of olive leaf polyphenols;the content of luteolin-7-O-glucoside is 3.54 mg/g,accounting for 6.04%of olive leaf polyphenols;the content of rutin is 3.62 mg/g,accounting for 6.18%of olive leaf polyphenols.Oleuropein,hydroxytyrosol,luteolin-7-O-glucoside and rutin accounted for70.45%of olive leaf polyphenols.（2）The macroporous resin purification method was used to investigate the effects of loading concentration and adsorption time on the adsorption effect of polyphenols,as well as the effects of elution concentration,elution volume and elution speed on the purification effect of polyphenols.The results showed that:using D-101 macroporous resin,the loading concentration is 0.25 g/ml,the adsorption time is 2.5 h,the eluent is 44%ethanol solution,the elution volume is 5 BV,and the elution speed is 2 BV/h.The polyphenols were detected by high performance liquid chromatography,and the results showed that the purity of oleuropein after purification was 73.12%;after removing the ethanol from the aforementioned product purified by the macroporous resin,it was extracted 3 times with petroleum ether and 5 times with ethyl acetate.,the ethyl acetate was removed by rotary evaporation at 40°C,and purified by silica gel column chromatography using ethyl acetate:methanol（4:0.45 v/v）as the developing solvent.Infrared spectroscopy and mass spectrometry were used to characterize the structure of the obtained material,and the results showed that the structure characteristics of the material finally obtained by silica gel column chromatography were consistent with those of oleuropein,and the material was oleuropein.Detected by high performance liquid chromatography,the purity of oleuropein was 97.08%.（3）The DPPH method and ABTS method were used to evaluate the free radical scavenging ability of olive leaf polyphenols and oleuropein.BHA and VC were used as control.The IC₅₀ values of free radical scavenging ability were 0.3209 mg/m L,30.13μg/m L,23.12μg/m L,23.27μg/m L,respectively.The order of DPPH free radical scavenging ability was:VC≈BHA>oleuropein>olive leaf polyphenols;IC₅₀ values of ABTS free radical scavenging ability are 0.239 mg/m L,23.75μg/m L,25.28μg/m L,23.57μg/m L,respectively.The order of ABTS free radical scavenging ability is:VC>oleuropein>BHA>olive leaf polyphenols.（4）Different concentrations of crude polyphenol extracts and oleuropein were added to walnut oil to study their effects on the oxidation stability of walnut oil.The results showed that when the concentration of olive leaf polyphenols was 0.02%,the oxidation stability of walnut oil was the best.The order of antioxidant capacity of olive leaf polyphenol crude extract,oleuropein,TBHQ,BHA and VC is:TBHQ>olive leaf polyphenol>oleuropein>BHA>VC,indicating that olive leaf polyphenol has strong antioxidant capacity in walnut oil.