Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.
Amino Acid Sequence ; Fallopia japonica/metabolism* ; Polyketide Synthases/chemistry* ; Acetone ; Mutagenesis, Site-Directed ; Flavonoids/metabolism* ; Acyltransferases/metabolism*

BACKGROUND: To observe the effects of Chinese medicine (CM) Polygonum cuspidatum (PC) on adenosine 5'-monophosphate-activated protein kinase (AMPK), forkhead box O3α (FOXO3α), Toll-like receptor-4 (TLR4), NACHT, LRR and PYD domains-containing protein 3 (NLRP3), and monocyte chemoattractant protein-1 (MCP-1) expression in a rat model of uric acid-induced renal damage and to determine the molecular mechanism. METHODS: A rat model of uric acid-induced renal damage was established, and rats were randomly divided into a model group, a positive drug group, and high-, medium-, and low-dose PC groups (n=12 per group). A normal group (n=6) was used as the control. Rats in the normal and model groups were administered distilled water (10 mL•kg) by intragastric infusion. Rats in the positive drug group and the high-, medium-, and low-dose PC groups were administered allopurinol (23.33 mg•kg), and 7.46, 3.73, or 1.87 g•kg•d PC by intragastric infusion, respectively for 6 to 8 weeks. After the intervention, reverse transcription polymerase chain reaction, Western blot, enzyme linked immunosorbent assay, and immunohistochemistry were used to detect AMPK, FOXO3α, TLR4, NLRP3, and MCP-1 mRNA and protein levels in renal tissue or serum. RESULTS: Compared with the normal group, the mRNA transcription levels of AMPK and FOXO3α in the model group were significantly down-regulated, and protein levels of AMPKα1, pAMPKα1 and FOXO3α were significantly down-regulated at the 6th and 8th weeks (P<0.01 or P<0.05). The mRNA transcription and protein levels of TLR4, NLRP3 and MCP-1 were significantly up-regulated (P<0.01 or P<0.05). Compared with the model group, at the 6th week, the mRNA transcription levels of AMPK in the high- and medium-dose groups, and protein expression levels of AMPKα1, pAMPKα1 and FOXO3α in the high-dose PC group, AMPKα1 and pAMPKα1 in the mediumdose PC group, and pAMPKα1 in the low-dose PC group were significantly up-regulated (P<0.01 or P<0.05); the mRNA transcription and protein levels of TLR4 and NLRP3 in the 3 CM groups, and protein expression levels of MCP-1 in the medium- and low-dose PC groups were down-regulated (P<0.01 or P<0.05). At the 8th week, the mRNA transcription levels of AMPK in the high-dose PC group and FOXO3α in the medium-dose PC group, and protein levels of AMPKα1, pAMPKα1 and FOXO3α in the 3 CM groups were significantly up-regulated (P<0.01 or P<0.05); the mRNA transcription levels of TLR4 in the medium- and low-dose PC groups, NLRP3 in the high- and low-dose PC groups and MCP-1 in the medium- and low-dose PC groups, and protein expression levels of TLR4, NLRP3 and MCP-1 in the 3 CM groups were down-regulated (P<0.01 or P<0.05). CONCLUSION PC up-regulated the expression of AMPK and its downstream molecule FOXO3α and inhibited the biological activity of TLR4, NLRP3, and MCP-1, key signal molecules in the immunoinflammatory network pathway, which may be the molecular mechanism of PC to improve hyperuricemia-mediated immunoinflflammatory metabolic renal damage.
AMP-Activated Protein Kinases ; physiology ; Animals ; Chemokine CCL2 ; blood ; Disease Models, Animal ; Fallopia japonica ; Forkhead Box Protein O3 ; physiology ; Hyperuricemia ; complications ; Kidney Diseases ; drug therapy ; etiology ; Male ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Uric Acid

The aim of this paper was to study the effect and mechanism of alcohol extract from Polygonum cuspidatum(PCE) on acute gouty arthritis in C57 BL/6 mice through NLRP3/ASC/caspase-1 axis. The model mice which injected with ankle joint injection of sodium urate crystals(MSU) were orally administrated with three different concentration of PCE, with colchicine as positive control. HE staining was used for observing the morphological changes of synovial tissue; concentration of IL-1β, IL-6 and TNF-α secreted by synovial tissue of the ankle joint were detected by ELISA; mRNA and protein expression of NLRP3, ASC and caspase-1 in synovial tissue were detected by RT-PCR and Western blot respectively. The results showed that the swelling degree of ankle joint in model mice were significantly elevated; expression of IL-1β, IL-6 and TNF-α were significantly increased; mRNA and protein expression of NLRP3, ASC and caspase-1 also significant increase, compared with normal control group. The swelling degree of ankle joint significantly relief; expression of IL-1β, IL-6 and TNF-α in joint synovium significantly decrease; mRNA and protein expression of NLRP3, ASC and caspase-1 were significantly decrease in PCE treatment group compared with model group. Our research implied that alcohol extract from P. cuspidatum had positive effect on acute gouty arthritis in mice, and the regulation of NLRP3/ASC/caspase-1 axis may be its mechanism.
Animals ; Ankle Joint ; physiopathology ; Arthritis, Gouty ; drug therapy ; CARD Signaling Adaptor Proteins ; metabolism ; Caspase 1 ; metabolism ; Fallopia japonica ; chemistry ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein ; metabolism ; Plant Extracts ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism ; Uric Acid

Anti-Helicobacter pylori activity guided fractionation led to the isolation of five anthraquinones, two stilbenes and one naphthoquinone from the EtOAc fraction of Polygonum cuspidatum, using silica gel column chromatography, Sephadex-LH20, MPLC and recrystallization. The chemical structures were identified to be physcion (1), emodin (2), anthraglycoside B (3), trans-resveratrol (4), anthraglycoside A (5), polydatin (6), 2-methoxy-6-acetyl-7-methyljuglone (7) and citreorosein (8) by UV, ¹H-NMR, ¹³C-NMR and mass spectrometry. Anti-Helicobacter pylori activity including MIC values of each compound was evaluated. All of the isolates exhibited anti-H. pylori activity of which MIC values were lower than that of a positive control, quercetin. Compounds 2 and 7 showed potent growth inhibitory activity. Especially, a naphthoquinone, compound 7 displayed most potent antibacterial activity with MIC₅₀ value of 0.30 µM and MIC₉₀ value of 0.39 µM. Although anti-H. pylori activity of this plant was previously reported, this is the first report on that of compounds isolated from this species. From these findings, P. cuspidatum roots or its isolates may be useful for H. pylori infection and further study is needed to elucidate mechanism of action.
Anthraquinones ; Chromatography ; Emodin ; Fallopia japonica* ; Mass Spectrometry ; Plants ; Polygonum* ; Quercetin ; Silica Gel ; Stilbenes

The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation.
Butanones ; Catalytic Domain ; Crystallization ; Fallopia japonica ; enzymology ; Polyketide Synthases ; genetics ; metabolism

The present study investigated the chemical composition of ethylacetate extracts from an endophytic actinomycete Streptomyces sp. A0916 and its host Polygonum cuspidatum. A comparative analysis of the antimicrobial and antioxidant properties of the extracts was also conducted. 32 compounds of P. cuspidatum and 23 compounds of Streptomyces sp. A0916 were isolated and identified by GC/MS. Antimicrobial activities of the extracts were evaluated using eight microbial strains (3 Gram-positive bacteria, 3 Gram-negative bacteria, and 2 fungi). The Streptomyces sp. A0916 extracts showed a wide range of antimicrobial activities and presented greater antimicrobial effectiveness than the P. cuspidatum extracts. The minimum inhibitory concentration (MIC) of Streptomyces sp. A0916 extracts against the ampicillin-resistant strain Enterococcus faecium SIIA843 was 32 μg·mL(-1). Furthermore, the extracts had greater antimicrobial effect against Gram-positive bacteria than Gram-negative bacteria. Finally, the antioxidant activity of the Streptomyces sp. A0916 extracts was equal to that of the P. cuspidatum extracts. In conclusion, our results suggest that the endophytic actinomycetes of the medicinal plants are an important source of bioactive substances.
Anti-Infective Agents ; chemistry ; isolation & purification ; pharmacology ; Antioxidants ; chemistry ; isolation & purification ; pharmacology ; Bacteria ; drug effects ; Fallopia japonica ; chemistry ; microbiology ; Fungi ; drug effects ; Plant Extracts ; chemistry ; isolation & purification ; pharmacology ; Streptomyces ; chemistry ; classification ; genetics ; isolation & purification

OBJECTIVETo observe the effect of detoxifying and blood circulation activating Chinese herb extraction of polygonum cuspidatum and hawthorn on carotid intima-media thickness (IMT), plaque integral and plaque stability related serum indexes of patients with carotid atherosclerosis.

METHODSixty and four cases of carotid artery atherosclerosis patients were assigned randomly to 2 groups: detoxifying and blood circulation activating treatment group (treatment group, 32 cases) and control group (32 cases). Patients in treatment group were treated with capsules of extraction of polygonum cuspidatum and hawthorn, 1 pill po, bid (dosage of administration: polygonum cuspidatum extraction 5.33 mg x kg(-1) x d(-1), hawthorn extraction 5.0 mg x kg(-1) x d(-1)); patients in control group were treated with lovastatin 20 mg po, qd (dosage of administration: 0.33 mg x kg(-1) x d(-1)). The course of treatment was six months. To observe changes of IMT, plaque integral, and detect the level of plaque stability related serum indexes such as Hs-CRP, MMP-1 and TIMP-1.

RESULTAfter 6 months of treatment, in control group one patient quit the clinical trial because of liver dysfunction and one patient was rejected because of having not followed the therapeutic regimen. 32 cases in treatment group and 30 cases in control group were analyzed. The results showed that IMT and plaque integral of treatment group decreased significantly after the treatment (P < 0.05, P < 0.01), and there was no significant difference compared with control grope. Serum Hs-CRP, MMP-1 and MMP-1/TIMP-1 decreased after the treatment (P < 0.05, P < 0.01), and the treatment group was superior to control group in decreasing serum Hs-CRP (P < 0.05).

CONCLUSIONDetoxifying and blood circulation activating Chinese herb extraction of polygonum cuspidatum and hawthorn has good effect of anti-atherosclerosis and promoting plaque stability. Its mechanism might be related with anti-inflammation and inhibiting degradation of extracellular matrix, and deserves further studies.

C-Reactive Protein ; metabolism ; Carotid Artery Diseases ; blood ; drug therapy ; Crataegus ; chemistry ; Drugs, Chinese Herbal ; adverse effects ; pharmacology ; therapeutic use ; Fallopia japonica ; chemistry ; Female ; Humans ; Male ; Matrix Metalloproteinase 1 ; blood ; Middle Aged ; Safety ; Tissue Inhibitor of Metalloproteinase-1 ; blood

Resveratrol is a natural phytoalexin with special pharmacological and health functions. Stilbene synthase (STS) is a key and rate-limiting enzyme in the biosynthesis of resveratrol that is present only in a limited number of plants. The content of resveratrol from Polygonum cuspidatum is more than 1000 times higher than grapes and peanuts. We speculate that the catalytic ability of different STS may be one of the reasons causing differences in the content of resveratrol. To verify the above speculation, Vitis vinifera stilbene synthase gene (VvSTS) was amplified according to overlap PCR protocol with genomic DNA as template. VvSTS and PcSTS (PcPKS5) were analyzed through heterologous expression in Escherichia coli. The expression products were purified with Ni-NTA sepharose affinity chromatography and desalted through PD-10 column. The molecular weight of the two fusion proteins was about 43 kDa. Enzyme reaction and product analysis showed that the two products were resveratrol. The enzyme kinetic analysis showed that the catalyze efficiency (Kcat/Km) of PcPKS5 was 2.4 times of the VvSTS. Our findings confirms that STS from certain plants has much higher catalytic capability.
Acyltransferases ; metabolism ; Fallopia japonica ; enzymology ; Recombinant Fusion Proteins ; biosynthesis ; Stilbenes ; metabolism ; Vitis ; enzymology

An HPLC method has been developed to determine polydatin in giant knotweed rhizome. In order to systematically validate the method, specificity, precision, linearity of reference solution and test solution, repeatability, reproducibility, accuracy, stability and robustness were measured. In the robustness test, a one-variable-at-a-time procedure was applied to evaluate the influence of slight variations in method factors, including the flow rate, the column temperature, the extraction time, and etc., on the assay result of polydatin. No significant differences were found when the process parameters changed during the experimental domain. And system suitability test limits were defined based on the robustness test. Results showed that the developed method was accurate, reproducible and robust.
Chromatography, High Pressure Liquid ; methods ; Drug Stability ; Fallopia japonica ; chemistry ; Glucosides ; analysis ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Rhizome ; chemistry ; Sensitivity and Specificity ; Stilbenes ; analysis

Through searching some domestic or abroad literatures of rhizoma polygoni cuspidati in recent years, the paper summarized its pharmacological effects, including antibacterial, antiviral, anti-inflammatory, analgesic, cardiovascular system protection, liver protection, anti tumor, improving immunity pharmacology and so on. These studies indicated Polygoni Cuspidati Rhizoma was a kind of drugs with exploiting and using value. [Key words]
Animals ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Fallopia japonica ; chemistry ; Rhizome ; chemistry