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.

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.

Biphasic modulation of α-ENaC expression by lipopolysaccharide in vitro and in vivo.

Acute lung injury (ALI) is characterized by pulmonary edema, in which the epithelial sodium channel (ENaC) has a critical role in the clearance of edema fluid from the alveolar space. Lipopolysaccharide (LPS), frequently employed to induce ALI in experimental animal models, has been reported to regulate ENaC expression and alveolar fluid clearance. The role of LPS in regulating ENaC expression is currently controversial, with increases and decreases reported in ENaC expression in response to LPS treatment, as well as reports that ENaC expression is not affected by LPS induction. The present study aimed to systematically analyze the regulation of α­ENaC expression in LPS models of ALI at different pathological stages in vitro and in vivo. ENaC expression was observed to increase ≤8 h after LPS treatment, and to decrease thereafter. This finding may explain the contradictory data regarding α­ENaC expression in response to LPS in the lung. The results of the present study, in combination with those of previous studies, indicate that the modulation of α-ENaC expression may not be a direct genetic response to LPS exposure, but a general response of the lung to the pathological changes associated with inflammation, hypoxia and endothelial and epithelial damage involved in the development of ALI. The findings of this study may have potential clinical significance for understanding the pathogenesis of ALI and improving patient outcome.

Network pharmacology analyses of the antithrombotic pharmacological mechanism of Fufang Xueshuantong Capsule with experimental support using disseminated intravascular coagulation rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Xueshuantong (FXST) Capsule is developed on a traditional Chinese medicine remedy, with a four-herb formula of Panax notoginseng, Radix astragali, Salvia miltiorrhizae and Radix scrophulariaceae. It has been used for treatment of the clinic cardiovascular disease for many years. MATERIALS AND METHODS: Due to its complexity of compositions and polypharmacological effects, it often complicates understanding of the mechanisms of action. In the present work, we have constructed an integrated model of system pharmacology to investigate the polypharmacological mechanisms of FXST formulation for treatment of thrombosis disease. RESULTS: The predicted results showed that 22 ingredients in FXST were closely associated with 41 protein targets related to blood coagulation, fibrinolysis and platelet aggregation. Through analysis of the compound-protein target association, significant cross-targets between each herb indicated the multiple active chemical ingredients might interact with the same target simultaneously and thus explained the synergistic mechanisms of the principle of Traditional Chinese medicines (TCMs) as ''Jun (emperor) - Chen (minister) - Zuo (adjuvant) - Shi (courier)''. To validate the polypharmacological effects predicted by our network pharmacology (NetPharm) analysis, we have carried out experimental investigation the effects of FXST on the disorders of the blood coagulation system in a lipopolysaccharide-induced disseminated intravascular coagulation (DIC) rat model. The results showed that FXST could significantly ameliorate the activation of coagulation system, which is congruent with the cross-target prediction by NetPharm approach. CONCLUSIONS: The combined investigations provide more insight into better understanding of the pharmacological mechanisms of FXST, and may also offer an alternative avenue to further explore the chemical and pharmacological basis of TCMs.

Chinese medicinal formula Fufang Xueshuantong capsule could inhibit the activity of angiotensin converting enzyme.

Fufang Xueshuantong (FXST) capsule, a Chinese medicinal formula composed of four herbals - Panax notoginseng, Radix Astragali, Radix Salvia Miltiorrhizae and Radix Scrophulariaceae, has been used to treat cardiovascular diseases for many years, but the pharmacological mechanisms underlying its effects has not been clarified. This study investigates if a connection between FXST and angiotensin converting enzyme (ACE) might be an explanation for its pharmacological effects. ACE inhibition assay was performed on FXST capsule, 50% ethanol extracts from the four herbals and three selected saponins most abundant in P. notoginseng (Ginsenoside Rg1, Ginsenoside Rb1 and Notoginsenoside R1) using a biochemical test. Reversed-phase high-performance liquid chromatography of liberated hippuric acid from the ACE assay was conducted to determine the inhibitory effect. As a result, FXST and extracts from P. notoginseng showed a significant and dose-dependent inhibition on ACE activity with the IC50 values of 115 µg/ml and 179 µg/ml, respectively. But extracts from the other three herbals and the three selected saponins had no significant effect on ACE inhibition. Compared to other reported plant extracts, FXST could be considered as an effective ACE inhibitor. The inhibition of ACE activity supports the traditional use of FXST on blood circulation and the inhibitory property of FXST is mainly caused by P. notoginseng.

[Molecular authentication of Fallopia multiflora by PCR-RFLP based on the trnL-trnF analysis].

OBJECTIVE: To establish a method for the molecular authentication of Fallopia multiflora. METHODS: The trnL-trnF regions of Fallopia multiflora and its closely related species and/or adulterants were sequenced and analyzed. RESULTS: It was found that the trnL-trnF sequence divergences between Fallopia multiflora and its closely related species and/or adulterants were 2.1%-22%. While the intra-species trnL-trnF divergences of Fallopia multiflora were 0%-1.5%. Based on the trnL-trnF regional variations, an endonuclease Xba I (T CTAGA) restriction site specific to Fallopia multiflora was detected. The Fallopia multiflora trnL-F polymerase chain reaction product could be cleaved by Xba I into two pieces, 804-819 bp and 256 bp each, whereas the restriction endonuclease could not digest the trnL-trnF polymerase chain reaction product of its closely related species or adulterants. The restriction patterns analyzed for restriction enzyme Xba I were found to be identical in all Fallopia multiflora individuals from different geographical regions in China. CONCLUSION: The assay based on polymerase chain reaction amplification of the trnL-trnF fragment of chloroplast DNA and subsequent restriction fragment length polymorphism can be used as a general test to identify Fallopia multiflora.

An effective method of RNA isolation from Fallopia multiflora tuberous roots.

To isolate high-quality total RNA from Fallopia multiflora tuberous roots is difficult because of the presence of high levels of carbohydrates, phenolics, and other secondary metabolites. Since several procedures specialized for RNA isolation from polysaccharides and phenols rich tissues have resulted in poor yields, in this study, we developed a modified protocol that was derived from the traditional CTAB method. The protocol was able to produce high-quality and intact RNA from the tuberous roots of F. multiflora. The yield of total RNA was more than 0.15 mg/g fresh weight, with an A260/A280 ratio of 1.9-2.0. The obtained RNA was of sufficient quality and suitable for downstream application such as reverse-transcription polymerase chain reaction (RT-PCR), Northern hybridization, and cDNA library construction. The protocol may also have wider applicability for total RNA isolation from other plant species with tuberous roots.

Overexpression of a resveratrol synthase gene (PcRS) from Polygonum cuspidatum in transgenic Arabidopsis causes the accumulation of trans-piceid with antifungal activity.

Although resveratrol-forming stilbene synthase (STS) genes have been well characterized in many plant species, there are only a few descriptions about STS genes from Polygonum cuspidatum Sieb. et Zucc, an important medicinal crop in Asian countries. To evaluate the biological functions of a Polygonum cuspidatum resveratrol synthase gene (PcRS), the PcRS gene was expressed in Arabidopsis under the control of Cauliflower mosaic virus (CaMV) 35S promoter. Integration and expression of transgene in the plant genome of Arabidopsis was confirmed by Southern blot and Northern blot analyses. Transgenic plants accumulated a new compound in both the leaves and seeds, which was identified as trans-piceid by high-pressure liquid chromatography (HPLC) and electrospray mass spectrometry (HPLC-ESI-MS). Overexpression of PcRS in transgenic Arabidopsis caused restriction of Colletotrichum higginsianum colonization by inhibition of spore production, resulting in enhanced resistance against C. higginsianum. So, the PcRS gene could be deployed in other crop plants to significantly enhance resistance to fungal pathogens and improve the nutritional quality. In addition, altered seed coat pigmentation and significant reduction in anthocyanin levels were observed in transgenic Arabidopsis, while the expression of endogenous chalcone synthase (CHS) gene was not down-regulated. These results suggest that additional STS activities cause a lack of precursors for CHS which leads to the disturbance of the subsequent flavonoid biosynthesis steps in Arabidopsis.

[Analysis and authentication of Fallopia multiflora and its adulterants by matK sequence].

OBJECTIVE: To identify Fallopia multiflora from its adulterants by comparing their matK sequences. METHODS: Genomic DNA of different materials was extracted using modified cetytrimethyl ammonium bromide (CTAB) method. The double-strand matK genes were amplified using PCR method and then sequenced. The data were analyzed in Clustral W and MEGA 4.0 software package. RESULTS: Besides F. multiflora var. ciliinerve, the matK sequences of other adulterants show distinct differences with F. multiflora, whether for nucleotides substitutions or genetic distances; and the specific identifying sites for distinguishing F. multiflora and other Fallopia adulterants were found through further comparative analysis. Moreover, the 3 inspected materials were successfully authenticated by comparing the matK sequences. CONCLUSION: matK sequences can be used for the molecular identification between F. multiflora and its adulterant species.

[A comprehensive overview of type III polyketide synthases from plants: molecular mechanism and application perspective--a review].

Type III polyketide synthases (PKSs) from plants produce a variety of plant secondary metabolites with notable structural diversity and biological activity. These metabolites not only afford plants the ability to defend against pathogen attack and other external stresses, but also exhibit a wide range of biological effects on human health. Several plant PKSs have been identified and studied in recent years. This paper summarized what was known about plant PKSs and some of their aspects such as molecular structure, reaction mechanisms, gene expression and regulation, and transgenic engineering. The review provides information for manipulating polyketide formation and further increasing the scope of polyketide biosynthetic diversity, as well as new avenues for developing transgenic engineering of type III PKSs.