To select suitable references gene of Polygonum multiflorum for gene expression analysis in different tissues, five candidate reference genes like Actin,GAPDH,SAND,PP2A,TIP41 were selected from the transcriptome data of P. multiflorum, then the specific primers were designed. The expression stability of the five reference genes in different tissues of P. multiflorum was analyzed by Real-time quantitative PCR through avilable analysis methods such as geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, to ensure the reliability of the analysis results. The results showed that there were significant differences in the expression levels and stability of candidate genes in different tissues of P. multiflorum. Ct distribution analysis of the expression levels of candidate genes showed that the expression levels of Actin and GAPDH genes were relatively high in different tissues, while the expression levels of SAND, PP2A and TIP41 were lower. The stability of each candidate gene was analyzed by different methods. The results of geNorm analysis showed that the expression of PP2A and GAPDH was the most stable, the expression stability of SAND was the worst, the stability of PP2A was the highest in both NormFinder and Delta CT, the stability of SAND was the lowest, and the stability of Actin was the most stable in BestKeeper analysis. Through the comprehensive evaluation and analysis of the stability of candidate genes by RefFinder, it is concluded that the stability of PP2A gene is the highest, followed by GAPDH, Actin, TIP41, SAND, and SAND gene is the worst. Therefore, the PP2A gene is an ideal reference gene for the analysis of gene expression in different tissues of P. multiflorum.
Fallopia multiflora ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Genes, Plant/genetics* ; Real-Time Polymerase Chain Reaction ; Reference Standards ; Reproducibility of Results

In this study, the rhizobacteria and actinomycetes of Polygonum multiflorum were screened for the strains with indole acetic acid(IAA)-producing capacity by Salkowski method, the siderophore-producing strains by Chrome Azurol S(CAS) assay, and the strains with inorganic phosphorus-solubilizing capacity by PKO inorganic phosphorus medium. The strains were identified by morphological identification, physiological and biochemical characteristics, and 16 S rDNA sequences. Furthermore, the effect of growth-promoting strains on the seed germination and development of P. multiflorum was tested. The results showed that among 196 strains, two strains F17 and F42 were found to be capable of producing IAA and siderophore and solubilizing inorganic phosphorus simulta-neously. For F17 and F42, the results are listed below: 38.65 and 33.64 mg·L~(-1) for IAA production, 0.85 and 0.49 for siderophore-producing capacities(A_s/A_r), and 1.35 and 1.70 for inorganic phosphorus-solubilizing capacities(D/d), respectively. Comprehensive analysis revealed that strains F17 and F42 were identified as Pseudochrobactrum asacharolyticum and Bacillus aryabhattai, respectively, and both could significantly promote the seed germination of P. multiflorum.
Bacillus ; Fallopia multiflora ; Germination ; Seeds ; Soil Microbiology

To investigate the effect of Polygonum multiflorum-Andrographis paniculata intercropping system on rhizosphere soil actinomycetes of P. multiflorum, the community structure and diversity of soil actinomycetes were studied by using the original soil as the control group and the rhizosphere soil actinomycetes communities of P. multiflorum under monoculture and intercropping systems as the experimental group. In this study 655 221 effective sequences were obtained with an average length of 408 bp. OTU coverage and rarefaction curve showed that the sequencing could represent the real situation of soil actinomycetes. According to the results of alpha diversity analysis, the diversity soil actinomycetes varied as follows: original soil>intercropping soil>monoculture soil. The soil actinomycetes community structure and the relative abundance of dominant genera were significantly changed by both monoculture and intercropping, especially monoculture. OTU clustering and PCA analysis of soil samples showed that all the soil samples were divided into three distinct groups and the original soil was more similar to intercropping soil. In addition, intercropping increased the relative abundance of some beneficial actinomyces, such as Kitasatospora and Mycobacterium, which was beneficial to maintain soil health and reduce the occurrence of soil-borne diseases. The results show that, P. multiflorum-A. paniculata intercropping reduced the change of community structure and the decrease of diversity of soil actinomycetes caused by P. multiflorum monoculture, and made the actinomycete community in rhizosphere soil of P. multiflorum close to the original soil.
Actinobacteria ; Actinomyces ; Agriculture ; Andrographis ; Fallopia multiflora ; Rhizosphere ; Soil ; Soil Microbiology

A simple, specific and selective quantitative analysis of multi-components by single marker(QAMS) method for simultaneous determination of anthraquinones and anthraquinone glycosides in Polygonum multiflorum was developed. Four main anthraquinones and its glycosides, emodin, emodin-8-O-β-D-glucoside, physcion and physcion-8-O-β-D-glucoside were selected as analytes to evaluate the quality of P. multiflorum. Emodin was used as the internal standard, and the relative correction factors(RCFs) between emodin and the other three anthraquinones were calculated. Comparison of the contents of the four components in 30 batches of P. multiflorum from different regions and 12 batches decoction pieces from different manufacturers by QAMS and external standard method(ESM) showed that there was no significant difference between QAMS and ESM for quantification of the four main components by using relative error results, and the QAMS method was accurate and reliable, and had a good repeatability. In addition, compared with the results calculated by the difference method between total anthraquinone and free anthraquinone in the content determination of P. multiflorum in Chinese Pharmacopoeia, the results of direct determination combined anthraquinone by QAMS were very close to that by measured the external standard method. Therefore, simultaneous quantification of four main anthraquinones by using QAMS is suitable to evaluate the quality of P. multiflorum. Then the optimized assay method of the combined anthraquinone contents showed simple and feasible, which could be replaced and improved the quantification method of the combined anthraquinone in the current Chinese Pharmacopeia.
Anthraquinones/analysis* ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/analysis* ; Fallopia multiflora/chemistry* ; Glucosides ; Phytochemicals/analysis*

Polygonflavanol B(1), a new flavonostilbene glycoside, was isolated from the roots of Polygonum multiforum(Polygonaceae) by various column chromatography methods including macroporous resin HP-20, silica gel, Sephadex LH-20, and preparative HPLC. The structure with absolute configuration of the new compound was identified by its physicochemical properties, spectroscopic data, ECD calculation, and chemical method.
Fallopia multiflora/chemistry* ; Flavonols/isolation & purification* ; Glycosides/isolation & purification* ; Plant Roots/chemistry* ; Stilbenes/isolation & purification*

In this study, we aimed to establish a rat liver micro-tissue evaluation system to evaluate the hepatotoxicity of the main monomers in Polygonum multiflorum. Rat primary hepatocytes were isolated and purified by two-step in situ perfusion method to prepare hepatic parenchymal cells. The ultra-low adsorption plate and the inverted model were used to establish an in vitro hepatotoxicity evaluation system. After the system was established, the main monomer components(monanthone with emodin type, rhein, emodin, emodin-8-O-β-D-glucopyranoside, physcion) of P. multiflorum were selected for in vitro hepatotoxicity evaluation. This study showed that the primary cells of the liver can form liver micro-tissues in the low adsorption plate method and the mold perfusion method, with good liver structure and function, which can be used to evaluate the hepatotoxicity of the drug to be tested after long-term administration. The five monomers to be tested in P. multiflorum can significantly affect the proliferation of primary liver micro-tissues in rats in a dose-and time-dependent manner. The hepatotoxic effects were as follows: monanthone with emodin type > rhein > emodin > emodin-8-O-β-D-glucopyranoside > physcion. The results suggested that the emodin-type monoterpene and rhein might be the potential hepatotoxic components, while the metabolites of emodin-8-O-β-D-glucoside and emodin methyl ether showed more toxic risks. The rat primary hepatocyte micro-tissue model system established in this experiment could be used to achieve long-term drug administration in vitro, which was consistent with the clinical features of liver injury caused by long-term use of P. multiflorum. The experimental results provided important information and reference on the clinical application and toxic component of P. multiflorum.
Animals ; Chemical and Drug Induced Liver Injury ; Emodin ; Fallopia multiflora ; Glucosides ; Plant Extracts ; Polygonum ; Rats

Idiosyncratic hepatotoxicity of Polygonum multiflorum has attracted a great attention in the world. The most toxic part of idiosyncratic hepatotoxicity was screened by MTT assay and flow cytometry, which was the 50% ethanol elute by macroporous adsorptive resins from alcohol-extraction of P. multiflorum. The fingerprints were collected by HPLC from 50% ethanol elute of crude and processed P. multiflorum from different habitats, then 14 common peaks were determined. Spectrum-toxicity relationship was analyzed by rough set theory(RST). Two main chemical components were predicted for idiosyncratic hepatotoxicity, in which TSG was the greater contributor. Idiosyncratic hepatotoxicity of TSG was tested in vitro, and the results indicated that TSG was the most important constituent contributed to idiosyncratic hepatotoxicity of P. multiflorum. The study showed the discovery of the main chemical components for idiosyncratic hepatotoxicity, and RST was effective for analyzing the spectrum-toxicity relationship, which could be a new method used in the effective/toxic constituents field of traditional Chinese medicine.
Chemical and Drug Induced Liver Injury ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; adverse effects ; Fallopia multiflora ; chemistry ; Humans ; Medicine, Chinese Traditional ; Phytochemicals ; adverse effects

To explore the immune status of patients with drug-induced liver injury caused by Polygonum multiflorum preparations,and analyze their immune characteristics. Case-control design was used to collect the cases of drug-induced liver injury caused by P. multiflorum preparations through key specialized surveillance. Five matching factors,namely type of P. multiflorum preparations,gender,age,basic diseases and concomitant medication were controlled. According to the ratio of 1 ∶ 1,cases of patients who took P. multiflorum preparations but with no liver injury were monitored at prospective hospitals. The demographic information,disease information,medication information and laboratory examination information of the two groups were recorded,and venous blood was collected. The gene sequence was detected by high-throughput sequencing technology,and the characteristics of TCR immune repertoire of the two groups were analyzed. A total of 46 pairs of patients were enrolled in the study. The results showed significant differences in the number of CDR3 and clone species,the length of amino acid sequence in CDR3 region,the abundance of V gene and J gene,the cross-linking of V-J gene and the diversity of immune repertoire between patients with drug-induced liver injury and patients without liver injury. The immunohistochemical diversity and high-frequency V-J cross-linking characteristics of patients with liver injury caused by P. multiflorum preparations were found,which provided a reference for screening out drug users to reduce the occurrence of liver injury caused by P. multiflorum preparations.
Chemical and Drug Induced Liver Injury ; Drugs, Chinese Herbal/adverse effects* ; Fallopia multiflora ; Humans ; Polygonum ; Prospective Studies ; Receptors, Antigen, T-Cell

Polygoni Multiflori Radix (PMR) has been commonly used as a tonic in China for centuries. However, PMR-associated hepatotoxicity is becoming a safety issue. In our previous in vivo study, an interaction between stilbenes and anthraquinones has been discovered and a hypothesis is proposed that the interaction between stilbene glucoside-enriching fraction and emodin may contribute to the side effects of PMR. To further support our previous in vivo results in rats, the present in vitro study was designed to evaluate the effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside (TSG) on the cellular absorption and human liver microsome metabolism of emodin. The obtained results indicated that the absorption of emodin in Caco-2 cells was enhanced and the metabolism of emodin in human liver microsomes was inhibited after TSG treatment. The effects of the transport inhibitors on the cellular emodin accumulation were also examined. Western blot assay suggested that the depressed metabolism of emodin could be attributed to the down-regulation of UDP-glucuronosyltransferases (UGTs) 1A8, 1A10, and 2B7. These findings definitively demonstrated the existence of interaction between TSG and emodin, which provide a basis for a better understanding of the underlying mechanism for PMR-induced liver injury.
Caco-2 Cells ; Chemical and Drug Induced Liver Injury ; etiology ; Emodin ; analysis ; metabolism ; Fallopia multiflora ; adverse effects ; Glucosides ; toxicity ; Glucuronosyltransferase ; antagonists & inhibitors ; Humans ; Plant Roots ; Stilbenes ; toxicity

Toxicity of different processed was evaluated Polygoni Multiflori Radix by determining the hepatotoxic potency for selecting processing technology. Process Polygoni Multiflori Radix using high pressure steamed, Black Bean high pressure steamed, atmospheric steamed for different time. Using normal human hepatocytes (L02) as evaluation model, hepatotoxic potency as index to evaluate hepatotoxic potency of different processed Polygoni Multiflori Radix. Analysis chemical composition of some processed products by UPLC-MS. Hepatotoxic bioassay method cloud evaluate the toxicity of different Polygoni Multiflori Radix samples. Different processing methods can reduce the toxicity of Polygoni Multiflori Radix, high pressure steamed three hours attenuated was better. Different processing methods have different effects on chemical constituents of Polygoni Multiflori Radix. Comparing with crude sample, the contents of gallic acid, 2,3,5,4-tetrahydroxyl diphenylethylene-2-O-glucoside, emodin-8-O-beta glucoside and emodin were decreased in processed products with 3 kinds of different methods. The change trend of 2,3,5,4-tetrahydroxyl diphenylethylene-2-O-glucoside content was similar with hepatotoxic potency. Different processing methods can reduce the toxicity of Polygoni Multiflori Radix. Processing methods and time attenuated obvious impact on toxicity. Recommended further research on the attehuated standard control of Polygoni Multiflori Radix concocted.
Biological Assay ; Cell Line ; Chemistry, Pharmaceutical ; methods ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; toxicity ; Fallopia multiflora ; chemistry ; toxicity ; Hepatocytes ; drug effects ; Humans ; Plant Roots ; chemistry ; toxicity