Isolation And Preparation Of Polyphenols From Camellia Oleifera Meal Based On Antioxidant Activity

Camellia oleifera Abel meal is a by-product of Camellia oleifera seed after oil extraction.It has strong antioxidant activity and other activities.If the polyphenols with anti activity in Camellia oleifera meal can be extracted efficiently,the comprehensive utilization rate of Camellia oleifera meal will be greatly improved.To solve the problem of the low utilization rate of polyphenols in Camellia oleifera meal,the polyphenols in Camellia oleifera meal were extracted by ultrasonic assisted solvent extraction.The total phenol content of the extract was determined by spectrophotometry,the polyphenols were analyzed by HPLC and its free radical scavenging rate was determined by ABTS+assay.The results showed that DES8(choline chloride:malonic acid=1:2)is most suitable for extracting antioxidant polyphenols from Camellia oleifera meal,and the most kinds of DESs(Deep Eutectic Solvents)were more suitable for extracting antioxidant polyphenols from Camellia oleifera meal than ethanol.The peak areas of 15 peaks with large peak area in the liquid chromatogram were used as the variables,cluster analysis was carried out to study the source of the difference between extracts by different extraction solvents.Pearson correlation analysis and HPLC-ABTS+ analysis were used to quickly locate the substances that contribute greatly to the total phenols and the ABTS+free radicals scavenging activity.Based on the guidance of on-line HPLC-ABTS+,five chemicals with high purity and antioxidant activity were obtained by macroporous resin chromatography and semi-preparative liquid chromatography.Four compounds were identified by Waters-Q-TOF/MS and nuclear magnetic resonance(NMR)spectroscopy.The research contents and conclusions are as follows:(1)Selection of reagents for extracting polyphenols from Camellia oleifera meal based on activity.①The polyphenols of Camellia oleifera meal were extracted with 8 kinds of DES and 60%ethanol respectively.Taking the polyphenol extraction rate and ABTS+free radical scavenging rate as the evaluation indexes,DES8(choline chloride:malonic acid=1:2)was selected as the best extraction reagent.The total phenol content of the sample extract was the highest(27.352 mg GAE·g-1),and the free radical scavenging rate of 5.333 mg/mL extract was the highest(90.283%).②Nine extracts were analyzed by HPLC,and the chromatographic peaks were integrated.Taking the peak areas of 15 peaks with large peak areas in the chromatogram as variables,the cluster analysis was carried out by SPSS statistics 22 software.When the Euclidean distance was 13,60%ethanol and DES6 were clustered into one class,desl,DES3,DES4 and DES5 containing polyols were clustered into one class,DES7 and des8 containing organic acids were clustered into one class,and DES2 was clustered into one class.③The results showed that there was a positive correlation between the total chromatographic peak area,total phenol content and ABTS+ radical scavenging rate,and there was a significant positive correlation between peak 1 and peak 15 and ABTS+radical scavenging rate.④HPLC-ABTS+analysis showed that DES8 extract had the strongest ABTS+radical scavenging activity,and the corresponding compounds of peak 1 and peak 15 had strong ABTS+radical scavenging activity.(2)Optimization of polyphenol extraction process from Camellia oleifera meal.The extraction process was optimized by single factor test combined with response surface analysis.The effects of three factors including temperature,solid-liquid ratio and water content in DES on the extraction rate of total phenol and ABTS free radical scavenging rate were analyzed.The results showed that the best extraction process of polyphenols from Camellia oleifera meal was as follows:1:25 g/mL of solid-liquid ratio,59℃ of temperature and 26%of water content.Under this extraction condition,the extraction amount of polyphenols was(29.163±0.006)mg GAE·g-1,and the ABTS+radical scavenging rate of the extract was(94.055±0.263)%.(3)Enrichment and preparation of active polyphenols from Camellia oleifera meal.The polyphenols from Camellia oleifera meal in DES extract were enriched and crude separated by D101 macroporous resin and gradient eluent of different mass fractions of ethanol.The fractions of DES8-1,DES8-2,DES8-3,DES8-4,DES8-5 and DES8-6 were obtained.Semi preparative liquid chromatography combined with HPLC-ABTS+ on-line activity tracking was used to guide the separation of these active compounds,and finally five monomers with antioxidant activity were obtained.(4)The structures of monomeric compounds were analyzed by waters-Q-TOF/MS and NMR,and four monomeric compounds were identified,.They were protocatechin(chemical 1),gallic acid(chemical 2),5-O-caffeoylquinic acid(chemical 3)and kaempferol(chemical 5),respectively.