OBJECTIVETo investigate the selective removal of tannins from Polygonum cuspidatum extracts by using collagen fiber adsorbent, and to evaluate the adsorption and desorption performances of collagen fiber adsorbent to tannins.

METHODThe adsorbent was prepared from bovine skin collagen fiber through crosslinking reaction of glutaraldehyde, and then used for the selective removal of tannins from P. cuspidatum extracts. Gelatin-turbidity method, gelatin-ultraviolet spectrometry method and HPLC were used for detection of tannins in the solutions. Ethanol-water solutions with varying concentration were used to test their desorption ability of tannins in order to choose proper desorption solution. On the basis of batch experimental results, the column adsorption and desorption tests were carried out, by using gelatin-turbidity method for detection of tannins.

RESULTThe collagen fiber adsorbent exhibited excellent adsorption selectivity to tannins. It was found that tannins of P. cuspidatum were completely removed, while nearly no adsorption of active components (resveratrol as representative) was found. Moreover, the collagen fiber adsorbent could be regenerated by using 30% ethanol-water solution and then reused.

CONCLUSIONThe collagen fiber adsorbent can be considered as a promising material for selective removal of tannins from P. cuspidatum extracts.

Adsorption ; Collagen ; chemistry ; Fallopia japonica ; chemistry ; Plant Extracts ; analysis ; Tannins ; isolation & purification

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

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

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

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

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*

OBJECTIVETo establish a microwave extraction and UPLC method for simultaneous determination of polydatin, resveratrol, anthraglycoside B, emodin and physicion contained in Polygoni Cuspidati Rhizoma et Radix, in order to provide scientific basis for improving quality standards of Polygoni Cuspidati Rhizoma et Radix.

METHODThe test solutions were prepared in a MDS-8 closed microwave system at 160 degrees C with methanol as the solvent. The UPLC analysis was performed in a Waters Acquity UPLC system. A BEH C18 column (2. 1 mm x 100 mm, 1.7 microm) was adopted for gradient elution with acetonitrile and water as the mobile phase. The temperature of column was 30 degrees C, and the detection wavelength was 226 nm.

RESULTThe five active components can be completely extracted in 10 minutes and separated completely in 12 minutes according to UPLC analysis, with a good linearity (r > or = 0. 999 6) within the linear ranges. The average recovery rate was 97.00%-103.7% with RSD < or = 2. 2%. Despite a large difference in content among tested components from Polygoni Cuspidati Rhizoma et Radix, the total content of the five major constituents was relatiely stable (3.683 3%-7.1031%).

CONCLUSIONThe microwave extraction-ultra performance liquid chromatography method in simultaneous determination for contents of five major bioactive components contained in polygoni cuspidati rhizoma et radix is so rapid and highly reproducible that it can be used for quality control and assessment of Polygoni Cuspidati Rhizoma et Radix.

Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Fallopia japonica ; chemistry ; Medicine, Chinese Traditional ; Microwaves ; Plant Roots ; chemistry ; Plant Stems ; chemistry ; Quality Control

OBJECTIVETo optimize the ultrahigh pressure extraction (UPE) process of polydatin and resveratrol from Polygonum cuspidatum by using uniform design.

METHODOn the basis of single factor screening, the uniform design was adopted for getting the optimal technique parameters. The optimum result of UPE was compared with conventional extractions.

RESULTThe optimal conditions of UPE for polydatin and resveratrol were that the solvent was 55% ethanol, the ratio of solvent to material( mL: g) was 30, the extraction pressure was 170 MPa, and the extraction time was 120 second. With this extracting process, the extraction yield of polydatin and resveratrol were 14.29 and 2.53 mg x g(-1), respectively. The extraction yield of polydatin was 46.1% higher than the heat reflux extraction, 6.4% higher than the ultrasonic extraction and 28.5% higher than the microwave extraction, while the yield of resveratrol was 67.5% higher than the heat reflux extraction, 29.7% higher than the ultrasonic extraction and 24.6% higher than the microwave extraction, respectively.

CONCLUSIONAs a novel extraction technology for Chinese herbal medicine, the UPE procedure has higher extraction yield, lower extracting temperature, shorter extacting time and less power consumption. The UPE has provided a brand-new method for extraction of polydatin and resveratrol from P. cuspidatum.

Calibration ; Chemical Fractionation ; methods ; Fallopia japonica ; chemistry ; Glucosides ; analysis ; isolation & purification ; Pressure ; Reproducibility of Results ; Solvents ; chemistry ; Stilbenes ; analysis ; isolation & purification ; Time Factors

AIMTo establish an optimal HPLC method for the determination of the (E)- and (Z)-diastereomers of resveratrol and resveratrol glucoside from the roots of Polygonum cuspidatum.

METHODSThe determination was conducted by using reversed-phase high liquid chromatography. Nucleodur 100-5 C18 column (250 mm x 4.6 mm ID, 5 microm), and a mobile phase program of gradient elution with isopropyl alcohol and water at a flow rate of 0.6 mL x min(-1) was employed. The fluorescence detection wavelengths were: lambda(ex) 334 nm, lambda(em) 404 nm.

RESULTSA good linear relationship was obtained under the optimum condition. The average recoveries were 96.7%, 99.1% for the (E)-diastereomers of resveratrol and resveratrol glucoside, 91.1%, 93.7% for the (Z)-diastereomers of resveratrol and resveratrol glucoside, respectively. The RSD of E-resveratrol and its glucoside were 1.34% and 0.72%, respectively. The RSD of Z-resveratrol and its glucoside were 1.27% and 2.08%, respectively.

CONCLUSIONThis method is accurate and reliable for the quantity analysis of the (E)- and (Z)-diastereomers of resveratrol and resveratrol glucoside in the roots of Polygonum cuspidatum.

Chromatography, High Pressure Liquid ; methods ; Fallopia japonica ; chemistry ; Fluorescence ; Glucosides ; analysis ; chemistry ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Sensitivity and Specificity ; Stereoisomerism ; Stilbenes ; analysis ; chemistry

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