Differential Constituents in Roots, Stems and Leaves of Polygonum multiflorum Thunb. Screened by UPLC/ESI-Q-TOF-MS and Multivariate Statistical Analysis.

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.

Stable isotope labeling and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucopyranoside biosynthetic pathway characterization in Fallopia multiflora.

MAIN CONCLUSION: 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-ß-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, 13C-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 [13C9]L-PHE and [13C1]L-PHE confirmed that the p-coumaric moiety of PD and THSG was derived from PHE. The results of the feeding experiments with [13C] SB and [2, 3-13C2] 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.

[Polyketone Reaction in Biosynthetic Pathways of 2, 3, 5, 4'-Tetrahydroxy Stilhene-2-O-ß-D-glucoside in Polygonum multiflorum by Biocatalysis].

OBJECTIVE: 2, 3, 5, 4'-Tetrahydroxy stilbene-2-O-ß-D-glucoside (THSG), the active ingredient of Polygonum multiflorum, its polyketone reaction in the biosynthesis pathways was studied by biocatalysis method. METHODS: The substrates 4-coumaroyl-CoA and malonyl-CoA were catalyzed in vitro by the crude enzyme extracted from Polygonum multiflorum callus, then the products were verified by HPLC and LC-MS methods. And the crude enzyme was analyzed by ammonium sulfate precipitation method and SDS-PAGE. RESULTS: HPLC chromatogram showed the same retention time of both the product and resveratrol standards; LC-MS spectra showed that the m/z of product was 227, which was consistent with resveratrol standards under the mode of negative ion; Ammonium sulfate (AS) precipitation method showed AS of 40% - 70% had catalytic activity,and 50% - 60% was the optimum; SDS-PAGE showed protein bands were obviously different among different AS concentration between 20% - 80%, the protein band of 42 kDa was found in AS of 50% - 60%, which had the same molecular weight with stilbene synthase. CONCLUSION: The product of polyketone reaction in the biosynthesis of THSG is resveratrol rather than THSG, so it is speculated that THSG is the conversion product of resveratrol instead of the direct product of the polyketone reaction.

Transcriptome profiling and digital gene expression analysis of Fallopia multiflora to discover putative genes involved in the biosynthesis of 2,3,5,4'-tetrahydroxy stilbene-2-O-ß-D-glucoside.

The compound 2,3,5,4'-tetrahydroxy stilbene-2-O-ß-d-glucoside (THSG) synthesized by Fallopia multiflora (F. multiflora) exhibits pharmacological potency. However, the mechanistic details of its biosynthesis pathway are still vague. To clear this ambiguity, we performed de novo transcriptome assembly and digital gene expression (DGE) profiling analyses of F. multiflora using the Illumina RNA-seq system. RNA-seq generated approximately 70 million high-quality reads that were assembled into 65,653 unigenes (mean length=750 bp), including 26,670 clusters and 38,983 singletons. A total of 48,173 (73.4%) unigenes were annotated using public protein databases with a cut-off e-value above 10(-5). Furthermore, we investigated the transcriptome difference of four different F. multiflora tissues using DGE profiling. Variations in gene expression were identified based on comparisons of transcriptomes from various parts of a high-level THSG- and a low-level THSG-producing F. multiflora plant. Clusters with similar differential expression patterns and enriched metabolic pathways with regard to the differentially expressed genes putatively involved in THSG biosynthesis were revealed for the first time. Our data provides the most comprehensive sequence resource regarding F. multiflora so far. Taken together, the results of this study considerably extend the knowledge on THSG production.