Study On Chemical Compositions And Metabonomics Of The Peels Of Mandarins And Oranges By UPLC-QTof-MS

Citrus is the world's most widely cultivated fruit crops,with abundant germplasm resources,mainly including mandarin(Citrus reticulata),orange(Citrus sinensis),lemon(Citrus limon),pummelo(Citrus grandis),grapefruit(Citrus paradisi)and citron(Citrus medica).Citrus fruit are widely used for food and medicine,containing a variety of biologically active ingredients,including flavonoids,carotenoids,phenolic acids,and limonoids and so on.They show good preventive and therapeutic effects on cancer,inflammation,atherosclerosis,obesity and diabetes.Researches show that the types and contents of chemical constituents in Citrus peels are generally higher than those in pulps.Therefore,it is of great significance to analyze and study the chemical constituents of citrus peels for the development of their medicinal values.In this study,the investigations of polymethoxylated flavonoids,non-targeted metabolic profile analysis,evaluation of in vitro antioxidant activity,and different metabolites among Citrus varieties were carried out in mandarins and oranges using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-QTof-MS)and Progenesis QI.Simple and rapid determination of polymethoxylated flavonoids in the peels of mandarins and oranges.Polymethoxylated flavonoids(PMFs)refer to a group of natural products of flavones attached with four or more methoxy groups on their basic 2-phenyl chromone skeleton.They are a special class of flavonoids contained in Citrus fruits and have a stronger anticancer effect than common flavonoids.In this study,a simple,rapid and sensitive method for qualitative and quantitative analysis of PMFs in C.reticulata and C.sinensis was established by UPLC-QTof-MS.Here,a MS quantitative method was established and systematically validated in linearity,precision,and recovery.The results show that the method is stable and reliable,and is suitable for the analysis of PMFs in Citrus.The main results are as follows:(1)We identified 42 kinds of PMFs 14 Citrus through analyzing the MS/MS spectra of PMFs,compared with the standard references and the spectra in the literature.Among them,33 were polymethoxylflavones and 9 were polymethoxy flavanones.(2)The contents of 13 kinds of PMFs was determined at the same time.Among them,the contents of nobiletin,sinensetin,tangerin and 3,5,6,7,8,3,4'-heptamethoxyflavone were higher than those of the others.And the kinds and contents of PMFs in Citrus reticulata were generally higher than those in Citrus sinensis.(3)All 13 standards showed good linearity between concentrations and the corresponding MS responses with correlation coefficients>0.99.The linear ranges are from 1.25 ng/mL to 1.0?g/mL with the LOD of 75 pg/mL and the LOQ of 0.25 ng/mL.The developed method showed good repeatability with interday relative standard deviations(RSDs)?2.79%,and intraday RSDs?5.43%.The accuracy of the method was measured by recovery experiments,and the result ranged from 94.52%to 106.20%with RSDs?9.50%.Studies on chemical constituents and in vitro antioxidant activity of Citrus reticulata(C.reticulata)and Citrus sinensis(C.sinensis)Citrus contains a variety of active compounds,including flavonoids,phenolic acids,terpenoids,carotenoids and so on.In this study,the untargeted metabolic profiles of 10 kinds of C.reticulata and 6 C.sinensis were performed,and the fragmentation behavior of flavonoids was summarized.ABTS,DPPH and FRAP methods were used to evaluate the antioxidant activity of these16 Citrus in vitro,and the correlation was analysed between the antioxidant activity and the chemical components.The main results are as follows:(1)92 chemical constituents were found in 16 Citrus species,including 4flavonoids,22 polymethoxylated flavones,10 polymethoxylated flavanones/chalcone,33flavonoid glycosides,9 acylated flavonoid glycosides,8 kinds of limonoids,3 kinds of coumarins and 3 kinds of other compounds.The types and contents of compounds in C.reticulata are more than those in C.sinensis.And the kinds and contents of compounds in wild C.reticulata are more than those in cultivated C.reticulata.MSJ and XP contained the most types of constituents,while S26 and CY contained the least types of constituents and the lowest contents among them.(2)The APC index was used to evaluate the comprehensive antioxidant capacity of Citrus.The antioxidant capacity of CX was the strongest,and MSJ ranked second,and both of them belong to wild Citrus.The weakest was XF,followed by S26,and both of them belong to oranges.Overall,the antioxidant capacity of C.reticulata was stronger than that of C.sinensis,and wild C.reticulata was stronger than cultivated C.reticulata.(3)In the positive ion mode,the results of correlation analysis showed that 8compounds were significantly correlated with the antioxidant capacity.Among them,the compound that mainly affects the antioxidant capacity of the ABTS method was natsudaidain-3-O-HMG-glucoside,and the compound with a significant positive correlation with the antioxidant capacity of the DPPH method was Caffeicacyl-hydroxyl-methoxylflavanone-O-diglycoside.There were 7 compounds with extremely significant positive correlations with the antioxidant capacity of the FRAP method,followed by Caffeicacyl-hydroxyl-methoxylflavanone-O-diglycoside;Dihydroxyflavone-dimethoxyflavone-O-glucoside;5,7,4'-Trimethoxyflavone;(2R)-Hesperidin,Swertisin-5-O-?-D-glucoside;5,7,3',4'-Tetramethoxyflavone-6-O-?-D-glucoside;1-Feruloyl-?-D-glucopyranoside.In the negative ion mode,there were 9 compounds with significant positive correlation with antioxidant capacity.Among them,Natsudaidain-3-O-(HMG)-glucoside and Isopyrenin were found to be highly significant positively related to ABTS method,and seven compounds that affected the antioxidant capacity of FRAP were Caffeicacyl-hydroxyl-methoxylflavanone-O-diglycoside;5,7,3',4'-Tetramethoxyflavone-6-O-?-D-glucoside;Dihydroxyflavone-dimethoxyflavone-O-glucoside;Tricin-5-glucoside/Tricin-7-glucoside;(2R)-Hesperidin;Swertisin-5-O-?-D-glucoside;Hesperetin-7-O-glucoside.Analysis of metabolic differences of Citrus using metabonomics Metabonomics is a method to study the changes of all metabolites produced by external stimuli to biological systems(cell,tissue or organism).In this section,UPLC-QTof-MS and Progenesis QI were used to perform untargeted metabonomics analysis on C.reticulata and C.sinensis varieties to find metabolic differences among different Citrus species.The main results are as follows:(1)10 kinds of C.reticulata and 6 C.sinensis fingerprints were collected using the UPLC-QTof-MS platform.QC samples are used for evaluating the stability of system.In the positive ion mode,the number of peaks RSD?30%accounted for 91.34%of the whole data;in the negative ion mode,the number of peaks RSD?30%accounted for88.79%of the whole data.Principal component analysis(PCA)of QC samples gathered well,proved that the data sampling method is reliable and stable.(2)In multivariate statistical analysis,unsupervised pattern recognition,principal component analysis(PCA),was used to observe the dispersion and cluster of all samples.In the positive ion mode,R~2X[1]=0.5051,R~2X[1]=0.1519,the first principal component(PC1)and the second principal component(PC2)explained 65%of the original variables,and the C.reticulata and C.sinensis can be separated on PC1 without outlier.Partial least squares discriminant analysis(PLS-DA)and orthogonal partial least squares discriminant analysis(OPLS-DA)are supervised pattern recognition techniques.They are mainly used to find differences between groups.In OPLS-DA,two groups were completely separated from PC1.R~2Y=99%and Q~2=90%proved that the models had good interpretability and predictability,and the data obtained were reliable.Potential biomarkers can be screened by important variable projections(VIP)and S-Plot.Variables with VIP?1,|p|?0.1,FC>2,Anova(p)<0.05 and|p(corr)|>0.5 were selected as markers.As a result,5 potential markers were screened for discrimination in the positive mode and 6 in the negative mode.