Metabolomic analysis of differential metabolites among different cultivars of Citrus grandis‘Tomentosa’flowers

Abstract: 【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 trichomes, 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 trichome coverage between the ZM, FM, and GQ have significant implications for their medicinal properties 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. Therefore, 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 value 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 extracted 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 Reaction Monitoring (MRM) mode of triple quadrupole mass spectrometry. Metabolite annotation was conducted using an in-house database developed by Biotree company. Using adonitol as an internal standard, 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 chromatography- 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 Partial Least Squares Discriminant Analysis (OPLS-DA) was utilized to compare the strains pairwise. Differential 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 detected a total of 978 metabolites. The PCA score plot showed that the contributions of principal components 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, terpenoids, and alkaloids. From the ZM vs FM comparison, 34 metabolites were identified as differentially expressed, with 25 upregulated and 9 downregulated. From the ZM vs GQ comparison, 48 metabolites 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 metabolites, 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, vicenin-2, and narirutin. GC-TOF/MS detected a total of 236 metabolites, primarily including organic oxides, 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 differences 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-Oglucoside and citric acid, and downregulation of caffeic acid, trans-4-hydroxycinnamic acid, ferulic acid, 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 Dcellobiose, caffeic acid, α-sophorose, and D-(-)-fructose. From the GQ vs FM comparison, 7 differential metabolites were identified, with upregulation of naringenin-7-O-glucoside, ferulic acid, pyruvic acid, 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 components 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, rutin, 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 metabolites include phenolic acids such as vanillic acid, caffeic acid, ferulic acid, and trans-4-hydroxycinnamic 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.