ABSTRACT
An ultra performance liquid chromatography method has been developed for determination of flavones in different origin and different parts from Bupleurum smithii var. parvifoliaum. The separation was performed at 30 degrees C on an Acquity UPLC BEH C18 column eluted with methanol and 0.1% phosphoric acid solution as mobile phases in gradient elution. The detection wavelength was set at 256 nm and the flow rate was 0.4 mL x min(-1). There flavones of rutin, quercetin and isorhamnetin were separated well, the linear calibration curves were obtained over the ranges of 0.106-1.06 microg for rutin (r = 0.999 8, n = 6), 0.004 87-0.048 7 microg for quercetin (r = 0. 999 7, n = 6), 0.022 0-0.220 microg for isorhamnetin (r = 0.999 8, n = 6). The mean recoveries of the three compounds were 99.3%, 97.8%, 98.9% with RSD of 2.4%, 3.0%, 3.2%. The result showed that the method is convenient and feasible for determination of the flavone content in B. smithii var. parvifoliaum.
Subject(s)
Bupleurum , Chemistry , China , Chromatography, High Pressure Liquid , Methods , Drugs, Chinese Herbal , Reference Standards , Flavones , Quality ControlABSTRACT
<p><b>BACKGROUND</b>To evaluate the inhibitive effect of combination of 3TC with Ara-AMP against HBV in vitro.</p><p><b>METHODS</b>Used 2.2.15 cell as target cell. With radioimmunological technique and blot slot, the inhibitive effect of 3TC, Ara-AMP and the combination of both against the HBsAg, HBeAg and intracellular HBV DNA were investigated.</p><p><b>RESULTS</b>The inhibitive ratio of Ara-AMP against HBsAg, HBeAg was 45.48% and 41.46% respectively when its concentration was 400.0 microgram/ml. Although 3TC also has inhibitive effect in its experimental concentration, its effect is weaker. When Ara-AMP 50.0 microgram/ml was combined with 3TC 1.25 and 5.00 microgram/ml respectively, the inhibitive ratio against HBsAg were 19.92% and 17.32% respectively. Compared with using same concentration 3TC alone, the difference of results was significant (P<0.05). But when compared with using the same concentration Ara-AMP alone, the difference of results had no statistical significance (P <0.05). Remarkable inhibitive effects of combination of 3TC with Ara-AMP against HBeAg were n ot found. When 3TC 5.00 microgram/ml was combined with Ara-AMP 12.5 and 50.0 microgram/ml respectively, the inhibitive ratio against HBV DNA was 45.90% and 50.36% respectively. Comparing the content of HBV DNA in these groups with that of control group and the groups using the same concentration 3TC and Ara-AMP alone, the differences were significant (P <0.05).</p><p><b>CONCLUSIONS</b>Combination of 3TC with Ara-AMP could enhance the inhibitive effects against HBV DNA.</p>
Subject(s)
Humans , Antiviral Agents , Pharmacology , DNA, Viral , Metabolism , Dose-Response Relationship, Drug , Drug Synergism , Hepatitis B virus , In Vitro Techniques , Lamivudine , Pharmacology , Vidarabine Phosphate , PharmacologyABSTRACT
OBJECTIVE:To establish an HPLC method for determining the contents of Quercetin and Isorhamnetin in Bupleurum.smithii var.parvifolium Shan et Y.Li.METHODS:The HPLC was performed on Kromasil column C18(250mm? 4.6mm,5? m),using methanol-0.4% phosphoric acid(55∶ 45)as mobile phase,with flow rate at 1.0mL? min-1 and detection wavelength at 256nm.RESULTS:The linear range of Quercetin was 0.08～ 0.40? g(r=0.999 6),with an average recovery rate of 101.02%(RSD=1.53%);that of Isorhamnetin was 0.06～ 0.30? g(r=0.999 2),with an average recovery rate of 101.26%(RSD=2.95%).CONCLUSION:This method is simple and accurate with good reproducibility.It is suitable for the quality control of Bupleurum.smithii var.parvifolium Shan et Y.Li.
ABSTRACT
Aim To screen antibodies of novel purinoceptor as a marker for further study of the purinoceptor. Method BALB/c mice were immunized for 4 times with rat aortic endothelial cell. Then the phage display system was used to construct a single-chain Fv fragment (ScFv) cDNA library from the total RNA of immunized mice. The characteristics of novel purinoceptor not existing on vascular smooth muscle cell but on aortic endothelium were used to enrich the aortic endothelium specific antibodies. Induced with IPTG, these antibodies were secreted into the periplasm of E. coli. The functional experiment of novel purinoceptor named organ bath experiment was used to screen out the positive ScFv from the soluble expressed antibodies. Immunohistochemistry experiment was used for positive ScFv identification. Results The total mouse anti-rat endothelium lgG is 1 ∶16 000. 8?106 mouse anti-rat endothelium ScFv cDNA library was successfully constructed. After 4 times of rat endothelium and rat smooth muscle cells screening, 2 500 ScFv cDNA binding membrane of aortic endothelium was enriched. After 4 times of functional screening, a phage-ScFv named B inhibiting the adenosine induced NO dependent construction by 83.4%?21.6% was selected from the expressed antibodies. Immunohistochemistry experiment showed that ScFv-B combined with aortic endothelium specifically and functional experiment showed that ScFv-B did not have any effect on adenosine induced ileum contraction, indicating that ScFv-B specifically binding to the novel purinoceptor. Conclusions ScFv-B binding specifically to the novel purinoceptor was selected by phage display technique and functional screening experiment which provide a good marker for further study of the novel purinoceptor.