Metabolomic analysis of differential metabolites among different culti-vars of Citrus grandis'Tomentosa'flowers
[Objective]Citrus grandis'Tomentosa'(CGT)is an ancient variant of pomelo[C.grandis(L.)Osbeck]native to Huazhou,Guangdong Province.It is the raw material source for the traditional Chinese medicine Huajuhong(Exocarpium Citri Grandis).Based on the morphology of the fruit tri-chomes,CGT can be divided into different local strains:Zhengmao(ZM),Fumao(FM),and Guangqing(GQ).ZM fruits have dense and long trichomes on the surface,while FM fruits have relatively short and sparse trichomes,and GQ fruits have a smooth surface without trichomes.The differences in tri-chome coverage between the ZM,FM,and GQ have significant implications for their medicinal proper-ties and potential applications in traditional Chinese medicine.The effective components in the flowers of CGT are similar to those in its fruits,possessing expectorant and anti-inflammatory effects.There-fore,locals often collect excess flowers and use them to make flower tea.The floral morphology of dif-ferent strains of CGT is nearly indistinguishable,yet their differences in composition and medicinal val-ue remain largely unexplored.The objective of this study is to explore the variances in composition among the flowers of ZM,FM,and GQ,aiming to establish a basis for the utilization of these CGT flowers in medicine and healthcare.[Methods]Flowers from the three varieties of CGT,namely ZM,FM,and GQ,were collected during the same growth period.Metabolites from the flowers were extract-ed using 25％methanol-water with 2-chlorophenylalanine as the internal standard for widely targeted metabolomics based on UPLC-MS/MS.Mass spectrometry data was collected using the Multiple Reac-tion Monitoring(MRM)mode of triple quadrupole mass spectrometry.Metabolite annotation was con-ducted using an in-house database developed by Biotree company.Using adonitol as an internal stan-dard,metabolites from the flowers were extracted with 25％methanol-water,followed by oximation and silylation derivatization,and then subjected to un-targeted metabolomics analysis using gas chro-matography-time of flight mass spectrometry(GC-TOF/MS).Principal Component Analysis(PCA)was employed to reveal the overall differences among the three strains.Subsequently,Orthogonal Par-tial Least Squares Discriminant Analysis(OPLS-DA)was utilized to compare the strains pairwise.Dif-ferential metabolites were screened based on Variable Importance in Projection(VIP)values in the OPLS-DA model,fold change(FC),and p-values from student's t-test.[Results]UPLC-MS/MS de-tected a total of 978 metabolites.The PCA score plot showed that the contributions of principal compo-nents 1 and 2 were 44.4％and 8.5％,respectively.The sample points for the ZM,FM,and GQ groups were clearly separated,indicating significant compositional differences among flowers of the three CGT strains.OPLS-DA analysis revealed 63 differential metabolites,mainly flavonoids,coumarins,ter-penoids,and alkaloids.From the ZM vs FM comparison,34 metabolites were identified as differential-ly expressed,with 25 upregulated and 9 downregulated.From the ZM vs GQ comparison,48 metabo-lites were identified,with 37 upregulated and 11 downregulated.From the FM vs GQ comparison,58 metabolites were identified,with 34 upregulated and 24 downregulated.Among these differential me-tabolites,ten effective components of CGT flowers were found in higher concentrations in the FM and ZM strains compared to the GQ strain.These components included bergapten,diosmin,meranzin,kaempferol-3-O-rutinoside,rutin,Vitexin-2"-O-glucoside,leteolin-7-O-rutinoside,vanillic acid,vicen-in-2,and narirutin.GC-TOF/MS detected a total of 236 metabolites,primarily including organic ox-ides,organic acids,lipids,and benzene derivatives.The PCA score plot showed that the contribution rates of principal components 1 and 2 were 39.8％and 17.1％,respectively,indicating significant differ-ences among the ZM,FM,and GQ strains.OPLS-DA analysis revealed 11 differential metabolites.From the FM vs ZM comparison,6 metabolites were identified,with upregulation of naringenin-7-O-glucoside and citric acid,and downregulation of caffeic acid,trans-4-hydroxycinnamic acid,ferulic ac-id,and pyruvic acid.From the GQ vs ZM comparison,8 metabolites were identified,with upregulation of naringenin-7-O-glucoside,shikimic acid,citric acid,and sedoheptulose,and downregulation of D-cellobiose,caffeic acid,α-sophorose,and D-(-)-fructose.From the GQ vs FM comparison,7 differen-tial metabolites were identified,with upregulation of naringenin-7-O-glucoside,ferulic acid,pyruvic ac-id,sedoheptulose,and trans-4-hydroxycinnamic acid,and downregulation of D-cellobiose and D-(-)-fructose.[Conclusion]The flowers of the three strains of CGT,ZM,FM,and GQ,exhibit significant differences in their composition.Flavonoids and coumarin compounds are the primary effective compo-nents in both the fruits and flowers of CGT.This study revealed that the flowers of the ZM and FM strains contain notably higher concentrations of 7 flavonoids(diosmin,kaempferol-3-O-rutinoside,ru-tin,vitexin-2"-O-glucoside,luteolin-7-O-rutinoside,vicenin-2,and narirutin)and 2 coumarins(bergat-en and meranzin)compared to those of the GQ strain.Additionally,the identified differential metabo-lites include phenolic acids such as vanillic acid,caffeic acid,ferulic acid,and trans-4-hydroxycinnam-ic acid,as well as primary metabolites like citric acid,shikimic acid,fructose,7-methylguanine,uridine,and eicosapentaenoic acid.These differential metabolites are involved in various metabolic pathways,including the tricarboxylic acid cycle,polyphenol biosynthesis,purine metabolism,and fructose and mannose metabolism.
万方数据
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