Targeted Metabolomic Analyses Of The Flowers,Fruits,Stems And Leave Of Camptotheca Acuminata Using UPLC-Q-TOF-MS

Camptotheca acuminata Decne is a characteristic plant resource in China.The secondary metabolite,camptothecin,can specifically inhibit the activity of topoisomerase I,thus it displays anticancer activity.Because of the complex structure and low abundance of camptothecin biosynthetic intermediates,it is challenging to delineate the downstream pathway.Thus leaves,flowers,fruits and stems in annual period were collected from mature C.acuminata during 2017.3-2018.3 and their extracts were evaluated by UPLC-QTOF-HRMS.Detected metabolites were firstly screened and characterized through in-house database alignment and the results for quinoline and indole alkaloids were further verified by the MS/MS fragmentation pattern.CPT biosynthetic intermediates were thoroughly mined and identified to build their metabolic pathways and accumulation patterns.The results are listed as follows:The thorough literature survey and non-target metabolomics was periodically conducted and four hundred and eighty-seven metabolites,including alkaloids(138),terpenes(141),polyphenols(152)and other classes of metabolites(56),were summarized in an In-house SCAU-Phytometa Resource.Leaves,flowers,fruits and stems(44 sample groups)were collected from mature C.acuminata and classified by UPLC-QTOF-HRMS based metabolomics.One hundred metabolites including forty-seven alkaloids,fifteen terpenes,thirty-two polyphenols and six other metabolites were rapidly identified through the in-house database alignment at first glance.Thirty-three alkaloids classified into five groups including camptothecin group(CG1-13),pumiloside group(PG1-5),strictosidinic acid group(SG1-3),vincosamide group(VG1-7),and a new hybrid group,vincosamide-camptothecin group(VC1-5)were mined and further characterized by MS/MS analyses.Two untapped biosynthetic precursors,2-hydroxypumiloside(PG2)and 16-hydroxy-15,16-dihydrocamptothecoside(CG3),along with sixteen new alkaloids were identified in this study.According to PCA and PLS-DA analyses,thirty discriminating metabolites were obtained which can be used to identify different tissue parts.Using the fragmental biosynthetic evidences as guidance,thirty one of these alkaloids were used to delineate a more detailed version of metabolic pathway for CPT and its derivatives according to their structure characteristics in a chemical point of view.Possible enzymes involved in CPTs biosynthesis were prompted according to this metabolic map,and their enrichment patterns were also obtained through hierarchical clustering analysis.Purposefully mining of enzymes involved in the downstream biosynthetic pathway of CPT could be initiated with the help of this guiding metabolic map...