Untargeted Metabolomics Profiling of Growth-Year-Associated Changes in Relative Metabolite Abundance in Tetrastigma hemsleyanum Tuberous Roots

Background/Objectives:Tetrastigma hemsleyanum is a traditional medicinal plant whose tuberous roots are rich in flavonoids and phenolic acids, but how the relative abundance of these constituents changes across growth years remains poorly resolved. Methods: Tuberous roots from five growth years (S1–S5; three independently grown plants per year) were analyzed by UHPLC–Orbitrap-MS-based untargeted metabolomics. Annotations were graded using Metabolomics Standards Initiative (MSI) criteria: of 349 curated entries, 252 were Level 2 and 56 Level 3, and none reached Level 1 because authentic standards were not analyzed. Results: Principal component analysis showed non-linear differentiation, with first-year roots the most distinct. Combined VIP, fold-change, and t-test criteria identified 264 differential entries between adjacent growth years, all remaining significant after Benjamini–Hochberg correction (q < 0.01). Flavonol-related features (e.g., quercetin-related signals, myricetin, and rutin) were relatively abundant at S1; flavone C-glycoside, flavan-3-ol, and phenolic-acid features (e.g., vitexin, catechin, and ferulic acid) at S3; and amino-acid, organic-acid, and nucleotide features at S4. KEGG mapping of 46 differential annotations indicated flavonoid- and amino-acid-related pathways. Conclusions: Given the small sample size, relative peak-area data, and putative annotations, these results are hypothesis-generating; they identify candidate stages and constituents for targeted quantification rather than an optimal harvest year.