| Camellia oleifera cake is the residue after degreasing Camellia oleifera seeds,with high yield and low utilization rate.However,Camellia oleifera cake exist many active components,such as Camellia saponin,polyphenols,polysaccharide,peptides,those play role of antioxidant,anti-inflammatory and anti-cancer activity.In particular,the antioxidant active components rich in Camellia oleifera cake can be used as hydrogen donor and singlet oxygen quencher to protect cells from oxidative stress.Therefore,improving its utilization can increase Camellia oleifera seed value of by-product.In this paper,taking Camellia oleifera cake degreased from Camellia oleifera seed as raw material,the extraction process of antioxidant active components was optimized.Through the fractional extraction of methanol extract,the best antioxidant polar parts were determined.The antioxidant active components in Camellia oleifera cake were screened by on-line antioxidant screening technology and liquid chromatography-mass spectrometry.The main research results are as follows:1.The extraction process of antioxidant active components of methanol extract of Camellia oleifera cake was optimized.Taking DPPH radical scavenging ability as the index,the single factor investigation was carried out on methanol concentration,extraction temperature,ultrasonic time,ultrasonic power and solid-liquid ratio.The factors that have the greatest impact on DPPH scavenging activity were determined by Plackett-Burman experiment,and the above factors were selected for response surface optimization experiment.Finally,we concluded that the best extraction process was that we extracted antioxidant active components using 60%methanol at ultrasonic power of 250 W and with temperature of 42℃ for 15 min,what’s more,the solid-liquid ratio was 1:17 g/mL.The extracted antioxidant components were detected and found that it showed good DPPH,ABTS free radical scavenging ability,reduction ability and oxidation free radical absorption ability(ORAC).When the concentration was 2.0 mg/mL,the DPPH free radical scavenging rate could reach 82.85%,The measured ORAC value was 692.8±41.63 μM TE/g DW.2.The methanol extract of Camellia oleifera cake was fractionated with petroleum ether,ethyl acetate and n-butanol.The contents of total phenols,total flavonoids and Camellia saponins in different polar extraction phases were compared,and the antioxidant activity of each extraction phase was determined.It was found that the ethyl acetate extraction phase had obvious enrichment effect on flavonoids and polyphenols compared with other extraction phases,and the n-butanol part had better enrichment effect on Camellia saponins.In terms of DPPH、ABTS free radical scavenging ability and reduction ability,the overall antioxidant activity of ethyl acetate extraction phase was the strongest.Therefore,ethyl acetate extraction phase was selected for subsequent antioxidant component research.Through correlation analysis,it was found that the antioxidant activity had a strong correlation with the content of total flavonoids and total phenols in each extraction phase of Camellia oleifera cake,and a weak correlation with the content of Camellia saponin.3.We used the rapid mixing element mixer instead of the reaction coil to build a system with good selectivity for the detection of antioxidant active components,and compared the established HPLC-DPPH with HPLC-ABTS.It was found that HPLCABTS had higher sensitivity for the detection of antioxidant active components.The conditions of the established online detection system were optimized,and the detection conditions of HPLC-ABTS were finally established as follows:InertSustain?C18 column(4.6 mm × 250 mm,5μm),mobile phase:A was methanol,B was 0.2%formic acid aqueous solution;The samples were eluted by gradient.Column temperature was 25℃;detection wavelength of ultraviolet detector was 280 nm;injection volume was 20 μL;flow rate was 1 mL/min;post column analysis system:the flow rate of ABTS free radical reaction solution(the concentration of 0.9 ± 0.02 absorbance at 734 nm)was 0.8 mL/min,the detection wavelength was 734 nm,and the flow cell volume of the mixer was 0.5 mL.Using this system,twenty-one components with potential antioxidant activity in the ethyl acetate extraction phase of methanol extract of Camellia oleifera cake were screened.4.The ethyl acetate extraction phase of Camellia oleifera cake was preliminarily separated and purified by silica gel column chromatography.Finally,10%methanol and 20%methanol(methanol:dichloromethane)eluent were determined to enrich the main antioxidant components in the ethyl acetate extraction phase of Camellia oleifera cake.Then the antioxidant active components in the ethyl acetate extraction phase of Camellia oleifera cake methanol extract were identified and analyzed by HPLC-ABTS combined with UPLC-MS/MS-PDA.In this experiment,eighteen compounds contained in the ethyl acetate extraction phase of Camellia oleifera cake were analyzed,of which thirteen compounds were components with antioxidant activity,including four phenolic acids and nine flavonoids,and their chemical structures were confirmed.They are Gallic acid,Catechins,Epicatechins,Caffeic aldehyde,Protocatechuic acid,Vanillic acid,Apigenin8-C-glucoside,Naringin-7-O-glucoside,Kaempferin B,Kaempferin B isomers,Kaempferol-3-O-rutoside,Kaempferol-3-O-glucoside and Kaempferol.After analysis,Gallic acid and Kaempferol were the main antioxidant components in the ethyl acetate extraction phase of methanol extract of Camellia oleifera cake.In this paper,the mixer was used instead of peek reaction coil as the mixing element to establish an online antioxidant screening system with high sensitivity and good selectivity.It could reduce the phenomena of insufficient peak capacity and overlapping detection components caused by excessive dead volume outside the column,which could accurately determine the antioxidant active components.Using this method combined with HPLC-MS/MS analysis,it was found that the main antioxidant components in the ethyl acetate extraction phase of Camellia oleifera cake were Gallic acid and Kaempferol,but there were still eight antioxidant substances whose specific structures had not been determined,which need to be further studied in the future. |
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