Comparative study on contents of four alkaloids in homologous herbal medicines--Arecae Pericarpium and Arecae Semen / 中国中药杂志

To establish a high performance liquid chromatography (HPLC) method for simultaneous determination of four alkaloids(arecoline， guvacoline， arecaidine， and guvacine) in Arecae Pericarpium (AP) and Arecae Semen (AS), and compare the contents of these four alkaloids between different medicinal parts. The chromatographic conditions were as follows:Welch SCX（4.6 mm×250 mm， 5 μm）column, with acetonitrile-0.2% phosphoric acid solution (adjusted to pH 3.85-3.90 with ammonium hydroxide) at 50:50 as the mobile phase, at a flow rate of 0.5 mL·min⁻¹. The column temperature was set at 35 °C, and the detection wavelength was 215 nm. The results of content determination in 7 batches of AS and 10 batches of AP showed that, the contents of 4 alkaloids in AS (arecaidine 0.020%-0.045%, guvacine 0.031%-0.086%, arecoline 0.194%-0.346%, and guvacoline 0.065%-0.094%) were generally higher than those in AP (arecaidine 0.10%-0.032%, guvacine 0.006%-0.029% arecoline 0.00%-0.070%, and guvacoline 0.00%-0.020%), and most of the APs had no arecoline and arecaidine at all in fruit peel. The above results indicated that different alkaloids can be used to distinguish the different medicinal parts of Arera catechu. Arecoline， guvacoline， arecaidine， and guvacine can be used as the quality control markers of AS, while for AP, only arecaidine and guvacine were needed.

HPLC fingerprint of Zishen Yutai pills and simultaneous determination of 5 index components / 中国中药杂志

To establish the HPLC fingerprint and determine five index components (loganic acid， chlorogenic acid， loganin， sweroside and asperosaponin Ⅵ) of Zishen Yutai pills by high performance liquid chromatography, and provide a scientific basis for its quality control. The fingerprint chromatogram was analysed by the chromatographic fingerprint similarity evaluation system for tradition Chinese medicine (2012), fifteen common peaks were obtained at the wavelength of 254 nm. Different batches of Zishen Yutai pills showed a similarity of above 0.90 in HPLC fingerprint profiles. For the quantitive analysis method, The separation of five components showed good regression (>0.999 2) with linear ranges, and the mean recoveries were in the range of 97.62%-101.9%, with the RSD (=9) less than 3%. The established fingerprint and quantitative analysis methods are highly specific, simple and accurate, which can reflect the quality of Zishen Yutai pills more comprehensively, and can be used for its quality control.

Comparison of alkaloids in roots of cultivated and wild Sophora flavsecens / 中国中药杂志

To compare the contents of alkaloids in theroots of cultivated and the wild Sophora flavsecens, 22 cultivated and 17 wild samples were collected. HPLC method was employed to simultaneously determine the contents of six alkaloids (oxymatrine, oxysophocarpine, sophoridine, N-methylcytisine, matrine, and sophocarpine). Independent t-test, hierarchical clustering analysis（HCA）and principal components analysis (PCA) were applied to analyze and evaluate the cultivated and the wild S.flavsecens. With a great wide range of the inter-group, the t-test results showed that the contents difference of N-methylcytisine, matrine, and sophocarpine were statistical significance(matrineandsophocarpine P<0.05, N-methylcytisine P<0.01).However, it was not statistically significant for oxymatrine, oxysophocarpine, and sophoridine.HCA and PCA showed that there were no significant differences in the contents of alkaloids of cultivated and wild S. flavsecens. The results indicated that there were no differences in the contents of alkaloids of cultivated and wild S. flavsecens.

Humoral immune response elicited by plasmid DNA containing HGV E2 gene fragment / 生物工程学报

In order to study the feasibility of E2 gene fragment of hepatitis virus G(HGV) as a component of DNA vaccine against the hepatitis virus G infection, a 559bp DNA fragment encoding HGV E2 was cloned into plasmid pCMV-S from pThioHis-E2 in the same reading frame with HBsAg gene to form a recombinant plasmid named pCMV-S-E2. BALB/c mice of Kunming strain were immunized with purified plasmid DNA of pCMV-S-E2 by intra-muscularly inoculation. The immunizations were boosted twice at an interval of 14 days. The whole blood was collected from mice orbit on the day-8 after the last boost. Mice sera were screened by ELISA to determine the humoral immune response using E2-GST fusion protein as the immobilized antigen and the sera from mice immunized with pCMV-S as control. The result indicated that the immunization with plasmid DNA of pCMV-S-E2 could induce quite strong humoral immune response.

Expression of a DNA fragment encoding the active domain of human TNF related apoptosis inducing ligand in pichia pastoris / 生物工程学报

Human tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands which has been reported in 1995. The TRAIL protein induces apoptosis of certain types of target cells, such as transformed cells that include but are not limited to cancer cells and virus-infected cells but the normal cells. It is a type II transmembrane protein and the extracellular domain of TRAIL is the functional domain in induction of cell apoptosis. A gene fragment encoding for the active domain of TRAIL was modified with oligo-nucleotide directed mutagenesis according to the characters of Pichia pastoris expressing vector. Arginine at the position of 149 corresponding to the amino acid residue 531 which might be a potential Kex2 protease processing sites was substituted with Lysine to prevent the expressed protein from the digestion by the protease. After proved with DNA sequencing. the modified gene fragment coding soluble TRAIL domain was inserted into the Pichia pastoris expression vector pPIC9K in the same reading frame with alpha-factor secreting signal peptide. The recombinant plasmid pPIC9K - TRAIL was transferred into P. pastoris cell by spheroplast transformation. The recombinant yeasts were identified by antibiotic G418 and Southern dot blot. The transformants (His+ Mut(s)) containing multi-copy gene fragment of TRAIL were selected with increasing concentration of G418 and induced with 0.5% methanol in shaking flask to expression the active domain of TRAIL. After inducing for 3 - 4 days, the proteins in the culture supernatant was assayed with SDS-PAGE and Western blot. Two expressed protein bands whose appearant molecular weight were 19kD and 38kD, respectively, could be specifically recognized by polyclonal antibodies against human TRAIL. The 38kD protein might be a dimers of TRAIL in the culture supernatant. The amount of expressed foreign protein made up to 36% of the total proteins in the culture suprenatant. Biological activity assay, in vitro, indicated that the expressed protein could induce tumor cells apoptosis.

Expression and characterization of envelope protein 2 gene of hepatitis G virus in Pichia pastoris / 生物工程学报

A cDNA fragment locating at the putative envelop protein 2(E2) region of GBV-C/HGV fused with Schistosoma japonicum, glutathione S-transferase(GST) was amplified with PCR from plasmid pGEX-E2. The amplified DNA fragment was inserted into plasmid pGEX-5X-1, at the downstream of the coding sequences of GST, in the same reading frame with the gene of GST. The fusion gene fragment of GST-E2 was amplified with PCR, using the recombinant plasmid pGEX-5X-1-E2 as the template. The amplified 1324 bp DNA fragment of GST-E2 was inserted into Pichia pastoris expression vector pPIC9K in reading frame with alpha-factor secreting signal peptide. The plasmid pPIC9K-GST-E2 was transformed into Pichia pastoris GS115 with electroporation. The transformants (His+ Muts) were selected and induced to express the 54kD GST-E2 fusion protein, which could be specially recognized by both the antisera directed against E2 and against GST. The GST-E2 fusion protein was purified with Sepharose 4B glutathione affinity chromatography to a purity of 95%. The expression was optimized to achieve the highest expression level of GST-E2 fusion protein which was accumulated up to 50% of total proteins in the culture supernatant. The GST-E2 protein derived from the recombinant Pichia pastoris was proved possessing antigenicity and high specificity by ELISA, probed with sera from the patients infected by GBV-C/HGV.

Expression in E.Coli and Antigenicity of HGV NS5 Protein / 中国病毒学

A 880 bp cDNA localized to the putative NS5 region of HGV genome was expressed in E.coli BL21(DE3). The cDNA fragment was inserted into a plasmid pGEX-5X-1,at the downstream of the DNA sequence encoding Schistosoma japonicum glutathione S- transferase(GST),in the same reading frame with the gene of GST. A 60KD GST-NS53 fusion protein was expressed at 37℃ in a form of inclusion bodies amounting to 30 percent of total host protein whereas at 20℃ mainly in a form of soluble protein . The fusion protein was extracted and purified to homologue. The purified GST-NS53 fusion protein could be specifically recognized with either the sera from the patient infected by HGV or the antisera directed against GST.