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

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

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*

To increase the content of active constituent--RE and PD of Polygonum cuspidatum hairy root, through Ri-mediated gene transformation technology, modified high salt low pH method was used to distill genome DNA of grapevine (Vitis raparia). Primer was designed according to sequence of Genebank (AF128861). Through PCR amplification obtain RS gene sequence was obtained. Binary vector pCAMBIA1300-35S-RS was constructed. Frost thawing method was used to transform Agrobacterium rhizogenes ATCC11325. Scratched aseptic seedling leaf of Polygonum cuspidatum was contaminated subsequently. DNA conformity and mRNA expression of RS gene were investigated by PCR and RT-PCR respectively. RE and PD in transgenic hairy root were determined by HPLC. For the first time successfully inducement acquires transformed RS gene hairy root of Polygonum cuspidatum. Content of active constituents--RE and PD were 17 - 187 microg x g(-1) DW and 836 - 1 970 microg x g(-1) DW, respectively, the non-transgenic hairy root was 0 - 130 microg x g(-1) DW and 190 - 320 microg x g(-1) DW. In the different root selected, the content of PD was much higher than that in non-transformed hairy roots of Polygonum cuspidatum, the highest content is 5 times, but the content of RE has not increased apparently.
Acyltransferases ; genetics ; metabolism ; DNA Primers ; DNA, Plant ; genetics ; Drugs, Chinese Herbal ; analysis ; Fallopia japonica ; genetics ; metabolism ; Genetic Vectors ; Glucosides ; analysis ; Molecular Sequence Data ; Plant Roots ; genetics ; metabolism ; Plants, Genetically Modified ; Plants, Medicinal ; genetics ; metabolism ; Rhizobium ; genetics ; Stilbenes ; analysis ; Transformation, Genetic

OBJECTIVETo study the release feature of Res-nanoliposomes in vitro and clarify the difference in absorption of Res-nanoliposomes from varied intestinal segments and the absorptive mechanism in vivo.

METHODDialytic method was used to determine resveratrol release rate of Res-nanoliposomes in vitro. An in situ rat perfusion method was used to investigate the intestinal absorption of Res-nanoliposomes.

RESULTResveratrol release from nanoliposomes in vitro fitted the log-normal distribution equation and had a property of sustained release. Compared with other intestinal segments, significantly high percentage of Res-nanoliposomes was absorbed in ileum (P < 0.001). The absorption rate constants (ka) of Res-nanoliposomes in intestine were not significantly different.

CONCLUSIONRes-nanoliposomes could sustain to release drug in vitro. The absorption was a first-order process with the passive diffusion mechanism. The Res-nanoliposomes could promote the absorption of Res in rat small intestine.

Administration, Oral ; Animals ; Delayed-Action Preparations ; Drug Compounding ; Fallopia japonica ; chemistry ; Ileum ; metabolism ; Intestinal Absorption ; Liposomes ; Male ; Nanoparticles ; Particle Size ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Stilbenes ; administration & dosage ; isolation & purification ; pharmacokinetics

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

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

OBJECTIVETo investigate the effects of Tongfu Huoxue decoction on experimental intracelebral hemorrhage and the associated machenisms.

METHODThe cerebral hemorrhage model in rats was induced by local injection of type VII collagenase and they were randomly divided into four groups. The treated groups were treated with Naoxuekang and Tongfu Huoxue decoction. The control groups were only treated with water. The changes of neurological defect were observed. The content of brain water, MDA, NO and the activity of SOD were measured.

RESULTThe cerebral hemorrhage rats showed hemiplegia, and the hemorrhage brains showed celebral edema, higher quotient of brain and content of brain water, suggesting the hemorrhage model was established successfully. After the treatment of Tongfu Huoxue decoction, the hemorrhage rats showed smaller hemorrhage volume, the brain tissue from the hemorrhage rats had lower MDA content and the quotient of brain, and also had higher activity of SOD and content of NO.

CONCLUSIONTongfu Huoxue decoction has treatment effects on cerebral hemorrhage.

Animals ; Arctium ; chemistry ; Brain ; metabolism ; pathology ; Cerebral Hemorrhage ; chemically induced ; drug therapy ; metabolism ; pathology ; Collagenases ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; therapeutic use ; Fallopia japonica ; chemistry ; Male ; Malondialdehyde ; metabolism ; Nitric Oxide ; metabolism ; Phytotherapy ; Plants, Medicinal ; chemistry ; Prunus ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the effect of Qianlie Huichun capsular on Fas expression and cell apoptosis of prostate gland tissue in the model rat.

METHODHypertrophy of prostate model rat was established by injecting testosterone to gelding male rats. After being treated with Qianlie Huichun ig 30 days, the rats were killed and prostate glands were resected for examination. The Fas expression was examined by immunobistochemical SABC. The cell apoptosis and the peak of cell apoptosis in the prostate gland of the rats were examined by flow cytometry. Compared with model group, the weight of prostage gland tissue in groups treated with Qianlie Huichun capsular was light (P < 0.01).

RESULTCompared with model group, the Fas expression in all treatment groups increase(P < 0.05, P < 0.01). Compared with normal and model groups, cell apoptosis in all treatment groups increase at different level(P < 0.01).

CONCLUSIONThe Qianlie Huchun capsular increases the Fas expression and cell apoptosis of model rats, and shows a definite treatment effect on the hypertrophy of prostate by promoting the apoptosis of prostate cell.

Animals ; Apoptosis ; drug effects ; Arthropods ; chemistry ; Capsules ; Cuscuta ; chemistry ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Fallopia japonica ; chemistry ; Male ; Materia Medica ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Prostate ; metabolism ; pathology ; Prostatic Hyperplasia ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; fas Receptor ; metabolism