Stable isotope labeling and 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucopyranoside biosynthetic pathway characterization in Fallopia multiflora

Abstract

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.

Overexpression of a stilbene synthase gene from Fallopia multiflora causes trans-piceid accumulation in transgenic Arabidopsis

“…1), THSG may be synthesized by the hydroxylation of trans-resveratrol to form the corresponding tetrahydroxystilbene. This hypothesis was recently confirmed by our intro studies using stable isotope labeling and biocatalytic methods (Xia et al 2017). Considering the results of previous and present study, we presumed that if FmSTS1 was cotransformed with a hydroxylase from F. multiflora, THSG may probably be produced in transgenic Arabidopsis.…”

“…THSG is the main component distributed in F. multiflora. Our team has performed considerable research aimed at characterizing the THSG biosynthesis mechanism (Xia et al 2017). We previously showed that the expression pattern of FmSTS1 analyzed by Northern blot corresponded well with the THSG distribution in different tissues of F. multiflora, implying that FmSTS1 may play a crucial role in THSG biosynthesis (Sheng et al 2010).…”

“…Pharmacokinetic studies confirmed that stilbenes are the main characteristic components of F. multiflora. Twenty-one stilbene compounds have been separated from F. multiflora, including transresveratrol and a variety of 2, 3, 5, 4′-tetrahydroxystilbene glucosides (Lin et al 2015, Xia et al 2017. Therein, 2, 3, 5, 4′-tetrahydro xystilbene-2-O-β-D-glucopyranoside (THSG, the structure is shown in Fig.…”

“…Therein, 2, 3, 5, 4′-tetrahydro xystilbene-2-O-β-D-glucopyranoside (THSG, the structure is shown in Fig. 1) has a similar structure with trans-resveratrol and is considered as the most important active component in F. multiflora (Xia et al 2017). In Chinese medicine, THSG is often used as a chemical marker for the quality control of F. multiflora (Zhao et al 2014).…”

2,3,5,4′-Tetrahydroxy-stilbene-2-O-beta-d-glucoside induces autophagy-mediated apoptosis in hepatocytes by upregulating miR-122 and inhibiting the PI3K/Akt/mTOR pathway: implications for its hepatotoxicity

“…Stilbene includes 2,3,5,4 0 -tetrahydroxy-stilbene-2-O-b-D-glucoside (TSG) (Ruan et al 2019). TSG is the main active chemical constituent of PMR, which has therapeutic effects on the nervous, cardiovascular, digestive, endocrine, and respiratory systems (Xia et al 2018). Recently, a great number of studies have shown that TSG may induce hepatocyte damage and cause liver damage (Xing et al 2019).…”