The THSG biosynthetic pathway in F. multiflora was characterized, and enzymatic activities responsible for the resveratrol synthesis, hydroxylation, and glycosylation reactions involved in THSG biosynthesis were confirmed in vitro. The biosynthetic origin of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside (THSG) and the enzymes involved in THSG biosynthesis in Fallopia multiflora were studied using stable isotope labeling and biocatalytic methods. UPLC-MS-based analyses were used to unravel the isotopologue composition of the biosynthetic intermediates and products, as well as to detect the products of the enzyme assay experiments. In this study, C-labeled L-phenylalanine (L-PHE), sodium pyruvate (SP), and sodium bicarbonate (SB) were used as putative precursors in the feeding experiment. Labeling of polydatin (PD) and THSG using [C]L-PHE and [C]L-PHE confirmed that the p-coumaric moiety of PD and THSG was derived from PHE. The results of the feeding experiments with [C] SB and [2, 3-C] SP suggested that PD and THSG were derivatives of resveratrol that were synthesized by glycosylation and hydroxylation. We developed methods using total crude protein extracts (soluble and microsomal) for comprehensive and simultaneous analysis of resveratrol synthase, glycosyltransferase, and hydroxylase activities in various tissue types of wild F. multiflora and callus cultures. The activity of each tested enzyme was confirmed in one or more tissue types or cell cultures in vitro. The results of the enzyme activity experiments and the distributions of PD and THSG were used to determine the main site and pathway of THSG biosynthesis in F. multiflora.
The differential constituents in leaves, stems and roots of Polygonum multiflorum Thunb. were analyzed by ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS) and by multivariate statistical analysis. The established extraction and analysis method showed relative standard deviations (RSDs) for intra-day precision of less than 3.40%, for repeatability of less than 4.06% and for stability of less than 5.10%. Principal component analysis and orthogonal projections to latent structures discriminant analysis of the UPLC/ESI-Q-TOF-MS data showed good ability to classify the leaves, stems and roots of P. multiflorum Thunb. The differential constituents, such as stilbenes, polygoacetophenoside, flavonoids and anthraquinones, accounting for variations between the leaves, stems and roots, were filtered through the variable importance in projection values and were further identified by elemental composition analysis, mass fragmentation data and retention times of available standards. Differences between the chemical compositions in the leaves, stems and roots of P. multiflorum Thunb. were closely related to their various therapeutic effects. This UPLC/ESI-Q-TOF-MS-based analytical strategy could be further utilized to evaluate the overall quality of traditional Chinese medicines and their differences of chemical constituents in different parts of the plant and/or in the plants of different geographical locations.
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