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Objective: To identify the transcriptional activity and expression profile of PcMYB1, encoding a new R2R3-MYB transcription factor from Polygonum cuspidatum, and evaluate the biological functions of PcMYB1 in transgenic Arabidopsis thaliana. Methods: The yeast one-hybrid system assay was conducted to test the transcriptional activity of PcMYB1. The tissue-specific expression profiles of PcMYB1 and the gene expression of P. cuspidatum leaves in response to UV-C irradiation were analyzed by RT-PCR analysis. To assess the biological functions of PcMYB1, the gene was expressed in A. thaliana under the control of CaMV 35S promoter. To obtain information about the lignin composition, cross-sections of the basal part of the inflorescence stem of wild-type and transgenic A. thaliana plants were treated with Wiesner staining, and the lignin content was measured by acetyl bromide method. RT-PCR analysis was used to determine expression levels of the genes encoding the enzymes of lignin biosynthesis. Results: After expression of reporter and effector constructed in yeast, β-galactosidase assays showed that the transcriptional activation activity of VP16 domain was reduced markedly when fused to PcMYB1 protein, indicating that PcMYB1 has transcriptional repression activities. Expression pattern analysis showed that PcMYB1 was widely expressed in all tissues examined, but predominantly in leaves. PcMYB1 showed a peak of transcription at 6 h post UV-C treatment. The transgenic lines with reduction in height was 24.07% shorter than the wild-type plants. Wiesner staining of lignin in stem cross-sections revealed the typical intense red stain of secondary cell walls in wild-type plants, but less intense staining was detected in transgenic plants, and lignin accumulation was significantly decreased (about 14.81%) in transgenic plants stems. The expression of genes involved in the lignin biosynthetic pathway, including AtC4H, AtC3H, AtF5H, AtCOMT, and AtCAD, were down-regulated in transgenic lines compared to wild-type plants. Conclusion: Taken together, this study provided the evidence for the biological functions of PcMYB1 as a negative regulator of lignin pathway.
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Objective: To investigate the inhibitory effect and targets of enriched components from Polygonum cuspidatum on HIV-1 in vitro. Methods: Four extracts of P. cuspidatum were screened by HIV-1 multi-target screening system based on the surface plasmon resonance. The highly active components were obtained by NHS-activated HiTrap conjugated with integrase. The anti-HIV activity of the sample was determined with TZM-bl infection assay and PBMCs infection assay. The inhibition of HZ60-IN on integrase 3’ processing was detected by fluorescence resonance energy transfer analysis. High-throughput ELISA was used to determine the effect of enriched active ingredients of P. cuspidatum (HZ60-IN) on integrase chain transfer; Effects of HZ60-IN on reverse transcriptase and protease were detected by kit. Results: HZ60-IN displayed higher affinity with integrase. HZ60-IN demonstrated potent antiviral activity against NL4.3 and 1084i strains in TZM-bl cells with the IC50 of (31.94 ± 8.96) and (38.07 ± 11.25) μg/mL, respectively. HZ60-IN showed significant inhibition on HIV-1 NL4.3 strains in PBMCs from two donors. HZ60-IN acted on the integrase with the IC50 of (6.54 ± 1.69) μg/mL for 3’ processing and (2.56 ± 0.97) μg/mL for strand transfer activity. It showed weak effects on the entry stage of HIV infection, with weak inhibitory activity on reverse transcriptase and no effect on protease activity. Conclusion: HZ60-IN showed significant inhibitive effect on HIV-1 replication and might specifically interfere with integrase activities.
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Objective: To construct the RNAi expression vector of Polygonum cuspidatum chalcone synthase (PcCHS1) gene, and to obtain the transgenic plants in which PcCHS1 expression was down-regulated. Methods: According to known sequence (EF090604) of PcCHS1 gene in GenBank, right primers were designed and the conserved sequence was cloned. The conserved fragment (574 bp) targeting at PcCHS1 gene was inserted into the expression vector pYLRNAi in both forward and reverse directions, and RNA interference (RNAi) expression vector pYLRNAi-PcCHS1 was constructed. Using the method of Agrobacterium-mediated transformation, the expression vector was used to transform the shoot tips of P. cuspidatum, and transgenic plants were obtained. The expression of PcCHS1 was confirmed by Northern blotting and the accumulation of polydatin was detected by HPLC. Results: RNAi expression vector of PcCHS1gene was constructed successfully, and five transgenic plants were obtained. Northern blotting analyses indicated that the expression levels of PcCHS1 were significantly down-regulated in the transgenic plants. Polydatin concentration in the transgenic plants was up to 3.8 times higher than that in non-transformed control plants. Conclusion: Transgenic P. cuspidatum plants with down-regulated expression of PcCHS1 gene were obtained successfully. The content of polydatin in the transgenic P. cuspidatum was significantly increased by RNAi against PcCHS1. This work might establish an experimental basis for the effective application of PcCHS1 in improving polydatin accumulation in P. cuspidatum.
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Objective: To establish the chromatography fingerprint of Polygoni Cuspidati Rhizoma et Radix with hyphenated technique of HPLC-DAD-ELSD and to evaluate Polygoni Cuspidati Rhizoma et Radix from 10 different origins. Methods: Luna C18 (2) column (250 mm × 4.6 mm, 5 μm) was used. Mobile phase was acetonitrile-H2O; Flow speed was 1.0 mL/min; Temperature of column was set at 35℃; Detective wavelength was at 254 nm; Injection volume was 10 μL. The temperature of drift tube was 109℃ and the flow speed was 3.0 L/min. Results: The chromatography fingerprint of Polygoni Cuspidati Rhizoma et Radix from 10 different origins was established. In the chromatography fingerprint with HPLC-DAD of Polygoni Cuspidati Rhizoma et Radix, 19 common peaks were demarcated and the similarities of Polygoni Cuspidati Rhizoma et Radix were between 0.938-0.993. In the chromatography fingerprint with HPLC-ELSD of Polygoni Cuspidati Rhizoma et Radix, 14 common peaks were demarcated and the similarities of Polygoni Cuspidati Rhizoma et Radix were between 0.905-0.999. Polydatin, resveratrol, emodin-8-O-β-D-glucoside, physcion-8-O-β-D-glucoside, emodin, and physcion were identified. Conclusion: The method is accurate and stable, which can be used as the evidence for the quality evaluation of Polygoni Cuspidati Rhizoma et Radix.
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Objective: To investigate the chemical constituents in the flowers of Polygonum cuspidatum. Methods: The components were separated by means of solvent extraction, repeated chromatography with silica and Sephadex LH-20 column. The structures were determined by spectral analysis and physicochemical properties. Results: Sixteen compounds were isolated from the ethyl ether extract and methanol extract from the flowers of P. cuspidatum and identified as β-sitosterol (1), aloe-emodin (2), physcion (3), emodin (4), daucosterol (5), chrysophanol (6), luteolin (7), kaempferol (8), anthraglycoside B (9), rhein (10), apigenin (11), hesperetin (12), 4-hydroxyacetophenone (13), rutin (14), sucrose (15), and genistein (16). Conclusion: Compounds 2, 8, 12, 13, 15, and 16 are obtained from the flowers of P. cuspidatum for the first time.
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Objective: To study the anti-complementary anthraquinones from Polygonum cuspidatum and their action targets. Methods: The anti-complementary activity-directed isolation was carried out with the hemolysis test as guide. All isolates were evaluated for their in vitro anti-complementary activities. The action targets of the main bioactive constituents were also examined using complement-depleted sera. Results: Ten anthraquinones and three other compounds were isolated from the EtOAc fraction of P. cuspidatum extract, including physcion (1), chrysophanol (2), questin (3), emodin-8-O-β-D-glucoside (4), emodin (5), rhein (6), fallacinol (7), citreorosein (8), xanthorin (9), isorhodoptilometrin (10), 2, 5-dimethyl-7-hydroxychromone (11), 7-hydroxy-4-methoxy-5-methylcoumarin (12), and 5, 7-dihydroxy-1-isobenzofuranone (13). Compounds 9 and 10 were isolated from the the plants of Polygonaceae for the first time, and compound 9 was the alizarin-type anthraquinone first obtained from P. cuspidatum. Compounds 3-9 showed the anti-complementary activity in different degrees, and compound 7 exhibited the most significant activity against the classical and alternative pathway [CH50 = (6 ± 2) μg/mL, AP50 = (50 ± 5) μg/mL]. The study on the preliminary mechanism revealed that compound 4 interacted with C1q, C2, and C9 in complement activation cascade, while compound 7 acted on C1q, C2, C4, and C9. Conclusion: The anthraquinones are main anti-complementary constituents in P. cuspidatum; and fallacinol (7) is a potential complement inhibitor with strong activity and definite targets, which should be further studied in future.
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Objective:To investigate the intervention effect of the compound polygonum cuspidatum sieb.et Zucc.grain on endothelium dependent dysfunction in patients with coronary artery disease.Methods:Twenty-four patients with both confirmed diagnosis of coronary artery disease(diagnosed by coronary arteriography) and endothelium dependent dilation(EDD) [expressed by flow mediated dilation(FMD) examined through high-resolution ultrasound] were enrolled into this investigation.They were divided into Group CAD,and Group HZ at random.Each group consisted of 12 patients.Group HZ,was administered the compound polygonum cuspidatum sieb.et Zucc.grain 10 grams per time,three times a day for 14 consecutive days.EDD0 and EDD14 were examined before and after the administration of the compound polygonum cuspidatum sieb.et Zucc.grain for 14 days.Group of normal control(Group NC) consisted of 12 age-and sex-matched healthy people.Results:EDD0 and EDD14 were(11.76?0.95)% and(11.83?0.70)% in Group NC,(5.37?1.15)% and(5.50?1.01)% in Group CAD which had significant difference compared with Group NC,(5.62?1.22)% and(9.84?1.83)% in Group HZ which were significantly different from those in Group NC.In GroupHZ,EDD14 was higher than EDD0 and had no significant difference compared with Group NC.Conclusion:The compound polygonum cuspidatum sieb.et Zucc.grain can improve endothelium-dependent dysfunction in patients with coronary artery disease.
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Object To determine the content of resveratrol glucoside and resveratrol in radix and rhizome of Polygonum cuspidatum Sieb. et Zucc. yielded in Hanzhong Region. Methods The HPLC method was used to assay resveratrol glucoside and resveratrol directly, using Eclipse XDB C 8 column and acetonitrile water as the mobile phase, the UV detection wavelength was 303 nm, with a flow rate of 1 mL/min. Results The content of resveratrol glucoside was 2 5% and resveratrol was 0 43% in the radix and rhizome. Conclusion The content of resveratrol glucoside is higher than that in the former refernece. The content of resveratrol in the radix and rhizome of P. cuspidatum in Hanzhong Region is first reported.
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Objective To study the chemical constitients of Polygonum cuspidatum.Methods The compounds were isolated through column chromatography and their structures were elucidated through physicochemical and spectral analyses.Results Seven compounds were obtained from the diethyl ether fraction of ethanol extract and identified as physcion(Ⅰ),emodin(Ⅱ),ambrettolide(Ⅲ),?-sitosterol(Ⅳ),oleanolic acid(Ⅴ),coumarin(Ⅵ),and 2-ethoxy-8-acetyl-1,4-naphthoquinone(Ⅶ).Conclusion Compound Ⅶ is a new compound named as cuspidatumin A,and compound Ⅲ is obtained from P.cuspidatum for the first time.
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Object To study the chemical structures and bioactivity of the water-soluble constituents from Polygonum cuspidatum Sieb. et Zucc. Methods To isolate the constituents by reverse phase methods, and characterize their structures by the analysis of chemical property and spectral data. Results Six compounds were isolated from the 60% aqueous acetone extract from the rhizome of P. cuspidatum. Their structures were elucidated as reveratrol (Ⅰ); piceid (Ⅱ); 2, 3-dihydro-2-(4′-O-?-D-glucopyranosyl-3′-methoxy-phenyl)-3-hydroxymethyl-5-(3-hydroxypropyl)-7-methoxybenzofuran (Ⅲ); 2, 6-dimethoxy-p-hydroquinone-1-O-?-D-glucopyranoside (Ⅳ); 5, 7-dihydroxy-isobenzofuran (Ⅴ) and 5, 7-dihydroxy-isobenzofuran-7-O-?-D-glucopyranoside(Ⅵ), respectively. Conclusion Compounds Ⅲ-Ⅵ are isolated from the plant for the first time. Compounds Ⅰ-Ⅵ show no DNA cleavage activity. Compound Ⅱ exhibits weak cytotoxicity against two human cancer cell lines (KB and MCF-7) in vitro.
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Object To investigate the effects on the active components in Polygonum cuspidatum Sieb. et Zucc. and its different processed products. Methods The qualitative analysis of piceid and emodin are taken by TLC, and content of emodin was determined by TLCS and HPLC. Results The contents of emodin significantly increased in samples fried with rice wine, vinegar and salt compared with the crude. Conclusion Different methods and ajuvants made a certain influence on the contents of emotion in P. cuspidatum.
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Objective:To study the interventional effect of polygonum cuspidatum sieb.et Zucc.on NOS system in vivo.Methods:Thirty-six Japanese rabbits were randomized into 6 groups:NC Group (fed normal control diet),HC group (fed atherogenic diet),SHZ group,MHZ group,LHZ group(fed atherogenic diet and three different doses of polygonum cuspidatum sieb.et Zucc.separately)and L-Arg group (fed atherogenic diet and L-arginine). At the different feeding time,the following contents were surveyed:1.serum NO,NOS,plasma ET;2.EDD and NEDD of external iliac artery in vivo;3.the area of AS plaque in thoracic aorta;4.NOS activity and the expression of iNOS mRNA and eNOS mRNA on the wall of the abdominal artery.Results:In group HC,EDD and NEDD of external iliac arteries were damaged at 1 week and 6 weeks,separately;the levels of serum NO,plasma ET and activity of NOS were elevated significantly;AS lesion was serious;the activity of NOS and expression of iNOS mRNA on the medial layer of the artery wall were much higher,the activity of NOS and expression of eNOS mRNA on endothelial cells were much lower.Except NEDD,polygonum cuspidatum sieb.et Zucc.could not only adjust the activities and expressions of disordered NOS systems on AS vessels,but also improve AS lesion in a dose-effect manner.Large dose of polygonum cuspidatum sieb.et Zucc.had the strongest effect.Conclusion:Polygonum cuspidatum sieb.et Zucc.can improve the function of the disordered NOS and lessen AS lesion on AS vessels in a dose-dependent manner.