Ultrasound-assisted Aqueous Extraction Of Clanis Bilineata Oil And Oxidation Protective Effect On Nerve Cells

Insects are rich in nutrients,such as amino acids,fatty acids and protein,which are all kinds of human essential nutrients.Therefore,people are choosing a variety of edible insects for the food industry,the pharmaceutical industry.Clanis bilineata(CB)is belonging to the family Sphingidae(Lepidoptera).Interestingly,it is also regarded as an important economic insect because its larvae contain a large percentage of protein and oil.C.bilineata can be raised by artificial way on a large-scale in different regions of China such as Jiangsu,Anhui,Sandong provinces.It opens up the development of CB in the food and pharmaceutical industries.1.Detection of Clanis bilineata amino acids,containing 7 kinds of essential amino acids,namely,methionine,phenylalanine,valine,threonine,lysine,isoleucine and leucine.The protein and fat composition of CB were detected.The protein was 68.31%,and the crude fat fraction of Soxhlet extraction was 24.43%.It was found that CB was a high protein insect.Ultrasound-assisted aqueous extraction was used to extract oil from CB,compared with Soxhlet extraction,physicochemical and antioxidant properties of oil extracted by UAAE were studied for the first time.Compared with SEO,UAAEO showed lower acid value(AV),peroxide value(PV),anisamine value(PAV),and higher content of polyunsaturated fatty acids as well as excellent thermal stability.By GC-MS,a total of 19 kinds of fatty acids,including eight kinds of saturated fatty acids(SAFA),three kinds of unsaturated fatty acids(MUFA),five polyunsaturated fatty acids(PUFA)and three kinds of odd carbon fatty acids(OCFA)were found.According to DPPH free radical scavenging and ?-carotene bleaching experiments,UAAEO showed stronger antioxidant capacity than SEO.Compared with SEO(ICso?80.47?g/mL),UAAEO exhibited stronger efficacy in scavenging the DPPH radicals(P<0.05,IC50=76.23?g/mL).?-carotene bleaching test is often used as a accurate and simple method for the determination of antioxidant capacity of oil.According to P-carotene bleaching test,UAAEO(IC50=1.56mg/mL)showed stronger resistance to peroxidation than SEO(IC50=2.67mg/mL).In addition,by the determination of the lipid class,the lower layer of ultrasound-assisted aqueous extraction contains more phospholipid,which is the most favorable confirmation for the subsequent experimental data.2.Chooosing ultrasonic power,extraction time,ultrasonic temperature,and ultrasonic time interval four variables,response surface methodology(RSM)was used to optimize Ultrasound-assisted aqueous extraction,got the highest oil extraction rate is 19.47%.The optimal conditions are ultrasonic interval time,ultrasonic temperature,ultrasonic power and ultrasonic time of 2 s,40?,400 W,and 50 min,respectively.Finally,we use the best condition to reproduce the prediction ability of the model.The experimental value of refined oil is(18.91+0.20g/100g),is very close to the predicted value(18.62g/100g).The results showed that there was a highly a high degree of consistency between the predicted values of the regression models and the experimental values(R2=0.9826,R2adj=0.9652).Therefore,this model can effectively predict the oil of Clanis bilineata(CB).3.Referring to the qualitative analysis of lipids in Chapter 1,the lower layer obtained by ultrasound assisted aqueous extraction has a high content of phospholipids,and there have been many reports about the protective effect of concentrated phospholipids on PC 12 cells damaged by H2O2.Therefore,we established a PC 12 cell model damaged by 750?M H2O2 and MTT method was used to measured the cell survival rate.Now,the protection group of 500-1250 ?g/mL can obviously improve the cell survival rate.The activities of antioxidant enzymes(SOD,CAT)and antioxidants(GSH)in the samples were measured.The results showed that the enzyme activity in the sample protection group was significantly higher than that in the injury group,and the GSH content was also higher.The morphology of cells can be observed by inverted phase contrast microscope.The morphology of cells in the sample group is more similar to that in the control group.The survival rate of cells was observed by fluorescence microscope.Compared with the model group,the percentage of injured cells in the sample group decreased significantly.Caspase-3 enzyme activity which plays an important role in the process of cell apoptosis,was measured.It was found that the enzyme activity in the sample group was significantly lower than that in the injury group.The apoptosis rate of the cells was detected.It can be seen that the sample protection group can reduce the cell apoptosis rate(16.%)compared with the injury group(31.9%).4.The lower emulsion were further identified.The content of phospholipid in the lower layer was determined by high-performance liquid chromatography.The phosphatidylcholine(PC)200mg/g and phosphatidyl ethanolamine(PE)100mg/g were identified.The results confirmed that the lower substance played a protective role on the PC 12 cells damaged by H2O2.The content of lower fatty acids was determined by gas phase.It contained 6 kinds of saturated fatty acids(including 2 odd carbon fatty acids),4 monounsaturated fatty acids and 5 polyunsaturated fatty acids,especially the content of alpha linolenic acid.And the use of LC-MSMS,MaLdi-TOF-TOF to determine the protein of the underlying emulsion.