Antiviral activities of Polygonum perfoliatum L. extract and related phenolic acid constituents against hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is an important public health problem. Polygonum perfoliatum L. is a traditional medicinal herb and has been reported to have pharmacological activities such as anti-inflammatory, antibacterial, and antiviral. In this study, the antiviral activities and mechanisms of Polygonum perfoliatum L. extract against HBV and the effective components were investigated. The results showed that the total extract of Polygonum perfoliatum L. reduced the levels of HBV e antigen (HBeAg) secretion and the viral covalently closed circular DNA (CCC DNA) formation, but had little or no negative effects on viral capsid assembly and pregenomic RNA packaging. Further fractionation showed that the water extract (WE) fraction exerted comparable anti-HBV activities with the total extract, especially in inhibiting the CCC DNA formation and HBeAg production, indicating that the effective antiviral components are mainly distributed in this fraction. Further study showed that the phenolic acids constituents, protocatechuic acid, and gallic acid, but not ethyl caffeate, which is reported enriched in the WE fraction, showed strong anti-HBV activities in inhibiting viral core DNA synthesis, CCC DNA formation, and HBeAg production. These results suggested that the Polygonum perfoliatum L. total extract and the related phenolic acids like protocatechuic acid and gallic acid could inhibit HBV replication and also indicated the potential utility of Polygonum perfoliatum L. and related constituents as sources of novel antivirals against HBV.

Functional Characterisation of New Sesquiterpene Synthase from the Malaysian Herbal Plant, Polygonum Minus.

Polygonum minus (syn. Persicaria minor) is a herbal plant that is well known for producing sesquiterpenes, which contribute to its flavour and fragrance. This study describes the cloning and functional characterisation of PmSTPS1 and PmSTPS2, two sesquiterpene synthase genes that were identified from P. minus transcriptome data mining. The full-length sequences of the PmSTPS1 and PmSTPS2 genes were expressed in the E. coli pQE-2 expression vector. The sizes of PmSTPS1 and PmSTPS2 were 1098 bp and 1967 bp, respectively, with open reading frames (ORF) of 1047 and 1695 bp and encoding polypeptides of 348 and 564 amino acids, respectively. The proteins consist of three conserved motifs, namely, Asp-rich substrate binding (DDxxD), metal binding residues (NSE/DTE), and cytoplasmic ER retention (RxR), as well as the terpene synthase family N-terminal domain and C-terminal metal-binding domain. From the in vitro enzyme assays, using the farnesyl pyrophosphate (FPP) substrate, the PmSTPS1 enzyme produced multiple acyclic sesquiterpenes of ß-farnesene, α-farnesene, and farnesol, while the PmSTPS2 enzyme produced an additional nerolidol as a final product. The results confirmed the roles of PmSTPS1 and PmSTPS2 in the biosynthesis pathway of P. minus, to produce aromatic sesquiterpenes.

Production of anthraquinones, phenolic compounds and biological activities from hairy root cultures of Polygonum multiflorum Thunb.

Polygonum multiflorum Thunb. is a highly important medicinal plant producing anthraquinones (emodin and physcion) and phenolic compounds which has pharmaceutical use. In vitro seedling explants such as roots, internodals, nodals and leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed roots were induced from internodals and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 30 g/l sucrose showed highest accumulation of biomass (99.05 g/l FW [fresh weight] and 10.95 g/l DW [dry weight]) and highest production of anthraquinones content (emodin 211.32 µg/g DW and physcion 353.23 µg/g DW) were observed at 20 days. Nearly 9.5-fold increment of biomass was evident in suspension cultures at 20 days of culture and hairy root biomass produced in suspension cultures possessed 3.7- and 3.5-fold higher content of emodin and physcion, respectively, when compared with the untransformed control roots. MS basal liquid medium was superior for the growth of hairy roots and production of anthraquinones compared with other culture media evaluated (SH, B5 and N6), with MS-basal liquid medium supplemented with 30 g/l sucrose was optimal for secondary metabolite production. A total of 23 polyphenolic compounds were identified and quantified from P. multiflorum untransformed and hairy roots, which includes hydroxybenzoic acids, hydroxycinnamic acids, flavonols and other groups of phenolic compounds. The ultra-performance liquid chromatography (UPLC) analysis of the phenolic compounds profile revealed that pyrogallol, hesperidin, naringenin and formononetin were higher in hairy roots compared to untransformed roots. The total phenolics, flavonoids content, antioxidant and antimicrobial activity was high in hairy roots compared to untransformed roots. This is the first report for the production of anthraquinones (emodin and physcion), phenolic compounds and biological activities from hairy root cultures of P. multiflorum.

[Study on sequence characterized amplified region (SCAR) markers of Polygonum capitatum].

OBJECTIVE: To establish sequence characterized amplified region markers of Polygonum capitatum. METHOD: The random primer was screened through RAPD to obtain the specific RAPD marker band, and the band was separated, extracted, cloned and sequenced. The specific primers were designed for conventional PCR reaction on the basis of the specific band, and the SCAR marker was acquired. RESULT: Screening from 50 RAPD primer, only C29 primer had 2 specific bands could distinguish P. capitatum from P. nepalense, then 4 pairs of specific primers were designed based on the 2 sequences of RAPD marker bands, and only 1 pair primer (Z1-2) was successfully converted into SCAR marker after repeated tests. CONCLUSION: The Z1-2 primer, could be used as an effective SCAR mark to identify Z300 DNA for P. capitatum. The SCAR mark was established and can be used as a molecular marker to distinguish P. capitatum from P. nepalense

[Optinization of rapid propagation technique and induction and identification of autotetraploid of Polygonum multiflorum].

OBJECTIVE: To establish and optimize the rapid propagation system of Polygonum multiflorum, as well as explore method for induction and identification of autotetraploid. METHOD: Propagation medium was optimized by orthogonal test. The buds were immersed in colchicine solution with different concentrations for different time to select induction conditions for autotetraploid of P. multiflorum. RESULT: The most appropriate propagation medium was MS medium supplemented with 1.0 mg x L(-1) 6-BA, 0.3 mg x L(-1) NAA, and 0.4 mg x L(-1) PP333. That the buds were soaked in 0.2% colchicine solution for 30 h, or soaked in 0.3% colchicine solution for 18 h, was optimal condition to induce autopolyploid of P. multiflorum with induction rate as high as 16.7%. CONCLUSION: Rapid propagation of P. multiflorum could be achieved by tissue culture. Furthermore, colchicine was an effective inducer of polyploidy, and 25 tetraploid lines were obtained through chromosome identification. The experiment laid a foundation for the wild resource conservation, superior varieties breeding of P. multiflorum.

[Analysis on chloroplast DNA sequences of Polygonum capitatum of different geographical population].

OBJECTIVE: To investigate the relationship between the variation of chloroplast DNA gene sequences and the geographical origins of Polygonum capitatum in order to provide the molecular evidence for its excellent germplasm resources. METHOD: PCR direct sequencing was applied to detect the chloroplast psbA-trnH, trnL-trnF gene sequence of 11 samples collected from 11 populations of P. capitatum. RESULT: The psbA-trnH gene sequence of P. capitatum from different populations was 402 bp in length, there were 6 variable sites. TrnL-F gene sequence was 875 bp, there were 5 variable sites. The clusters diagram by UPGMA method showed that P. capitatum groups in Yunnan and Guizhou existed a considerable variation. CONCLUSION: P. capitaturni which is located in the east of Yunnan and the west of Guizhou is helpful of screening the germplasm resources.

[Biotransformation of daphnetin by suspension transgenic hairy roots of Polygonum multiflorum].

OBJECTIVE: To investigate the biotransformation of daphnetin by suspension transgenic hairy root of Polygonum multiflorum and provide a biotechnological method for large-scale production of the daphnetin-8-O-beta-D-glucoside using this new culture system. METHOD: Daphnetin was added into the media of suspension to culture 36 h. The biotransformation product was detected with TLC and HPLC, and isolated by various chromatographic methods. The influence of co-cultured time on conversion ratio, content of degradation product and the reason for the degradation of product II were investigated using HPLC. RESULT: One biotransformation product, daphnetin-8-O-beta-D-glucoside, was obtained, the optimal co-cultured time in suspension hairy root of P. multiflorum was 36 h with the highest biotransformation molar ratio of 32.11%, the sucrose medium (sucrose-only) can increase the biotransformation molar ratio to 72.44%. The result demonstrated that the degradation products of the product II was induced by the MS medium. CONCLUSION: The potential application of suspension transgenic hairy root of P. multflorum in the sucrose-only medium on generating daphnetin-8-Obeta-D- glucoside could be prospective.

[Genetic diversity of Polygonum capitatum from Guizhou populations by ISSR markers].

OBJECTIVE: To detect genetic diversity of 48 population of Polygonum capitatum in Guizhou province. METHOD: The genetic diversity of 48 representational populations of P. capitatum including 240 individuals had been investigated by ISSR marker technique. RESULT: The genetic diversity had been revealed as follow: A total of 8 293 bands were produced in 240 individuals, of which 7 962 bands were common in the 48 population. The value of the average percentage of polymorphic bands (PPB) was 79.09%, Nei's genetic diversity index (H(e)) was 0.245 8, Shannon's information index (I) was 0.396 2, and genetic differentiation index (G(st)) was 0.238 0 at population level, respectively. The genetic differentiation index (G(st)) was 0.072 2, genetic differentiation coefficient by Shannon's diversity (I(st)) was 0.044 2 within the population levels. Groups cluster analysis based on the UPGMA method indicated that although the 48 populations could be divided into 3 groups and the P. capitatum seed sources. The groups cluster showed that a cross clustering of P. capitatum between the southwest and southeast populations in Guizhou province, and no significant correlation was found between geographical and genetic distance among them. CONCLUSION: The genetic diversity of P. capitatum is relatively high at the population levels, while low within the population levels, a significant degree of genetic differentiation occurs among the populations. The groups cluster analysis indicated they has not apparent genetic variation in regional pattern between the place of origin populations and the migrate populations.

Biotransformation of thymol by hairy roots of transgenic Polygonum multiflorum.

OBJECTIVE: The exogenous substrate, thymol, was firstly biotransformed by using suspension hairy roots of transgenic Polygonum multflorum, and its biotransformed situation was also investigated. METHODS: After five days co-cultivated period, the transformed product was isolated by Thin Layer Chromatograph and Column Chromatograph, with the structure elucidated by physic-chemical methods and spectra data. Meanwhile, the time course of biotransformation (T-C) for thymol was also measured by HPLC to illuminate its bio-transformed situation. RESULTS: The glycosylated product, namely DMP, was isolated and purified, which structure was determined as 5-methyl-2-(1-methylethyl) phenyl- beta-D-glucopyranoside. And the distribution of DMP in the medium or culture was varied in different co-cultivated periods, and for five days co-cultivated period, it mainly existed in the medium. CONCLUSION: The hairy roots of Polygonum multiflorum were able to convert the aromatic exogenous substrate, thymol, into its glycoside. Furthermore, the time course indicated the relationship between DMP and co-cultivated period.

[SRAP study on genetic diversity of radix plygoni multiflori in chongqing].

OBJECTIVE: To detect the polymorphisms of Radix Plygoni Multiflori in chongqing by means of a new marker system SRAP. METHOD: Different shaples of Radix Plygoni Multiflori from major production areas were collected. The SRAP was used to asses divergence among 16 populations. The data were analyzed using unweighted pairgroup method, based on arithmetic averages (UPGMA) bootstrap analysis. Cluster analyses was performed by using DPSv3.01 software, the alkaloid was extracted from P. ternate with chlorolform. RESULT: 104 combinations generated 250 polymorphie bands, the cluster analysis indicated that 16 materials could be distinguished into two main groups and one special type, Nei&Li similarity coefficient ranged from 0.23-0.99, and the average distance is 0. 44. CONCLUSION: The results of the study showed a potential application of SRAP fingerprinting for identification of Radix Plygoni Multiflori.

Barriers to sexual reproduction in Polygonum viviparum: a comparative developmental analysis of P. viviparum and P. bistortoides.

Polygonum viviparum is widely distributed in arctic and alpine regions of the northern hemisphere. Fruit set has never been observed in North American populations and has been reported only very rarely in Europe. Although this species is extremely well studied, the impediments to successful fruit production are unknown. We investigated the sexual reproductive process in P. viviparum growing in the southern Colorado Rocky Mountains. For comparison, we also examined this process in the sympatric congener P. bistortoides, in which reproduction is exclusively sexual. Lack of viable fruit production in P. viviparum has no single developmental explanation; defects occur in each of the processes and structures associated with sexual reproduction studied, yet, these processes and structures also appear to function normally in at least some flowers or individuals. Development is abnormal in many ovules of P. viviparum, however, comparison with P. bistortoides shows that these abnormalities do not contribute to differences in seed production between the two species. The virtual absence of sexual reproduction in P. viviparum appears to be due largely to a low rate of fertilization and to embryo/fruit abortion.