Isolation and Identification of Bioactive Constituents from Stem Barks of Illicium difengpi / 中草药·英文版

Objective: To isolate and identify bioactive constituents from the stem barks of Illicium difengpi. Methods: The chemical constituents were isolated and purified by repeated silica gel, Sephadex LH-20, recrystallization, and preparative HPLC techniques. The structures of the compounds were identified on the basis of spectral data including NMR, MS, and IR. Results: Two sesquiterpene lactones, majucin (1) and anisatin (2), two steroids, β-sitosterol (3) and daucosterol (4), three carboxylic acids, 2-ethyldecanoic acid (5), shikimic acid (6), and 3,4-dihydrobenzoic acid (7), and a flavonoid, quercetin (8), were successively isolated from the stem barks of I. difengpi. Conclusion: Apart from compound 3, other seven compounds are reported in this plant for the first time. The results suggested that the current studies on I. difengpi is still far from being well known and therefore more studies need to be done for better understanding of this plant. © 2014 Tianjin Press of Chinese Herbal Medicines.

Ribosome display screening of a novel human anti-IgE scFv fragment / 药学学报

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

Studies on chemical constituents of Caragana spinifera / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of the whole plant Caragana spinifera.</p><p><b>METHOD</b>The chemical constituents were isolated and repeatedly purified on silica gel column. The structures were elucidated by the NMR spectra and physico-chemical properties.</p><p><b>RESULT</b>Six compounds were isolated and identified as (6aR, 11aR) 4,9-dimethoxy-3-hydroxypterocarpan, (6aR,11aR)-4, 9-dihydroxy-3- methoxypterocarpan (melilotocarpane B), 5, 4'-dihydroxy-7-methoxyisoflavone, 7-hydroxy4'-methoxyisoflavone, 6, 7-dihydroxy4'-methoxyisoflavone, beta-sitosterol respectively.</p><p><b>CONCLUSION</b>All compounds were isolated from the plant for the first time.</p>

Study on relationship of laxative potency and anthraquinones content traditional Chinese drugs / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the laxative potency and anthraquinones content of six kinds of traditional Chinese drugs (TCDs) like Rheum tanguticum, Polygonum cuspidatum, R. palmatum, R. officeinale, Semen Cassiae and Radix Polygoni Multiflori.</p><p><b>METHOD</b>The half effective dose (ED50) was applied to determine the laxative potency and the content of anthraquinones was evaluated by RP-HPLC.</p><p><b>RESULT</b>The ED50 for the six kinds of TCD was 0.458, 0.686, 0.925, 1.004, 1.047, 1.986 g x kg(-1), respectively, and the sequence of laxative potency was R. tanguticum > P. cuspidatum > R. palmatum > R. officeinale > Semen Cassiae > Radix Polygoni Multiflori. In terms of the HPLC quantitative determination, the content of combined anthraquinones was 2.82% ,1.64%, 1.44%, 0.82%, 0.15%, 0.019%, respectively,and the sequence was R. tanguticum > Polygoni cuspidatum > R. palmatum > P. cuspidatum > Semen Cassiae > Radix Polygoni Multiflori.</p><p><b>CONCLUSION</b>There is a great difference in laxative potency between TCDs, and the relationship between laxative potency and the content of combined anthraquinones was found. The bioassay may be utilized to evaluate and control the quality of TCD with the chemical methods.</p>

Expression of endo-beta-mannanase gene from Trichoderma reesei in Pichia pastoris / 生物工程学报

Complete mannanase gene with two introns was cloned from Trichoderrna reesei by PCR. The two introns were then removed by overlap extension PCR. The gene encoding the mature mannanase protein was inserted into the expression vector pPIC9K, downstream of a alpha-factor signal peptide sequence. The resultant recombinant vector was named pM242. After linearized with Sac I , pM242 was transformed to Pichia pastoris GS115 by electroporation. After screening, the recombinant strain Gpmf25 that expresses the secretory protein at high level was obtained. The activity of the recombinant mannanase reached 12.5 IU/mL. Optimum pH and temperature for the recombinant enzyme were 5.0 and 80 degrees C, respectively. The enzyme was stable at pH 5.0-6.0 and maintained over 50% of original activity after incubation at 70 degrees C for 30 min.