Determination of hedychenone in Hedychium yunnanense / 中国中药杂志

<p><b>OBJECTIVE</b>To establish an HPLC method for determination of hedychenone in Hedychium yunnanense.</p><p><b>METHOD</b>C18 Chromatographic column was used, acetonitrile-water (9:1) as mobile phase, at flow rate of 1.0 mL x min(-1): The wavelength for detection was 235 nm.</p><p><b>RESULT</b>The linear range of hedychenone was 5.92-29.6 microg x mL(-1)(r = 0.9999). The average recovery was 99.0%, RSD of precision was less than 2%.</p><p><b>CONCLUSION</b>The method is simple, effective and feasible, and can be used to evaluate the quality of the herb.</p>

Optimization of the expression of human DNA topoisomerase I in Pichia pastoris / 生物工程学报

Human DNA Topoisomerase I (hTopo I) has been identified to be an efficient target of many effective antitumor drugs. Natural hTopo I is not convenient to be used in screening because of its low concentration in cells. In order to fast screen new anticancer drugs targeting at hTopo I from natural compounds in vitro, hTopo I gene open reading frame (ORF) has been successfully cloned and overexpressed in Pichia pastoris. Total RNA extracted from Hela cells was reversely transcripted to synthesize cDNA with the hTopo I specific antisense primer and the hTopo I ORF was synthesized by PCR. After digestion with EcoR I and Kpn I, the synthesized fragment was inserted into pPICZaA, gave rise to pPICZalpha-hTopoI. After digestion with Sac I, the lined pPICZalpha-hTopoI was transformed into Pichia pastoris strains (KM71, X33 and SMD1168) by electroporation and integrated into their genome. After screened on YPDS plates (containing 1000 ug/mL zeocin), the high-copy recombinant strains (KM-hTopoI, X33-hTopoI and SMD-hTopol) could overexpress recombinant hTopo I, which was fused to the alpha-factor secretion signal and could be secreted into the supernatant in the culture. alpha-factor could be cleaved from the expressed protein during secretion. A higher activity amount of the enzyme was secreted by the particular strain SMD-hTopoI because of its absence of proteimase A than by other strains which possess proteinase A activity. After optimizing the fermentation conditions, a relatively higher enzyme activity in the culture supernatant could be obtained when SMD-hTopoI was induced in BMMY (pH7.25) at 20 degrees C , with addition of 0.5% (V/V) methanol and 3% (V/V) nutrient liquid every 24h. The enzyme activity reached 43 000 u/mL, the yield reached 11 mg/L, achieving approximate 10% of total protein in the culture supernatant. SDS-PAGE and Western blot analyses showed that the mass of the recombinant hTopo I was 91 kD with no glycosylation.

Effects of two metabolites of cultured marine fungus, Halorosellinia oceanicum 323, on the contraction of isolated guinea-pig ileum / 药学学报

<p><b>AIM</b>To investigate the effects of 323-A and 323-B, two isomers extracted from the metabolites of cultured marine fungus, Halorosellinia oceanicum 323, on the contraction of isolated guinea pig ileum (GPI).</p><p><b>METHODS</b>The GPI contractions were recorded with a two-channel-physiological recorder with tension transducers. Cumulative dose-response curves of contractions of isolated GPI induced by histamine (Hist), acetylcholine (ACh) and potassium chloride (KCl) were constructed, then the influences of 323-A and 323-B on each curve were observed. Furthermore, possible mechanisms underlying effects of the two compounds were explored by analyzing their influences on the biphasic contractile response to ACh, with comparison of a calcium antagonist, verapamil (Ver).</p><p><b>RESULTS</b>The data indicated that both 323-A and 323-B inhibited the contractile actions of GPI triggered by Hist, ACh and KCl in a concentration-dependent manner, with pD2' values of 5.13, 4.97, 5.36 and 5.51, 5.56, 5.62, respectively. The initial phase component of the ACh-elicited contractions, in the absence of external Ca2+, was significantly reduced by 323-A, 323-B, as well as Ver, whereas the subsequent sustained tonic contractions induced by adding Ca2+ to the bath solution were almost unaffected.</p><p><b>CONCLUSION</b>These results suggest that 323-A and 323-B have calcium antagonistic effects similar to that of Ver in mechanisms, and they might have potential to be developed as calcium antagonists.</p>