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Article in Chinese | WPRIM | ID: wpr-776412


A quantitative nuclear magnetic resonance method(qNMR) was established for determination of the absolute content of febrifugine. Proton nuclear magnetic resonance spectroscopy of febrifugine was obtained in DMSO-d₆ with hydroquinone as the internal standard substance on a Bruker Ascend 600 MHz superconducting nuclear resonance spectrometer at 298 K. The specific parameters were as follows: the observing frequency was 600 MHz,spectra width was 7 211 Hz, pulse width was 9.70 μs, pulse sequence was zg30,scan times was 32 and relaxation time was 2 s. The proton signal peaked at δ 7.71 for febrifugine and δ 6.55 for hydroquinone were selected as the quantification peaks. Linear regression of quantitative peak area ratio of febrifugine-hydroquinone versus their mass ratio yielded a correlation coefficient of 0.999 6 and a regression equation of +0.008 6.The linear range of febrifugine was 2.17-17.07 g·L⁻¹,the precision RSD was 0.78%(=6),the repeatability RSD was 1.2%(=6),and the contents of three batches of febrifugine sample were 94.91%,95.09% and 95.52%,respectively. The content of febrifugine was 96.44% determined by high performance liquid chromatography(HPLC). The relative error of the content of febrigugine determinted by qNMR and HPLC methods was 1.27%. The results showed that the internal standard method of proton nuclear magnetic resonance spectroscopy could be used to determine the absolute content of febrifugine.

Magnetic Resonance Spectroscopy , Piperidines , Protons , Quinazolines
Article in Chinese | WPRIM | ID: wpr-350123


To develop the HPLC method for simultaneous determination of febrifugine and isofebrifugine in Dichroa febrifuga root, and on the basis of this, the feasibility of quantitative analysis of multi-component by a single-marker (QAMS) model for the determination of the two alkaloids was investigated. The chromatographic separation was performed on an octadecyl bonded silica gel column with mixed solvent consisting of acetonitrile-water-glacial acetic acid-triethylamine (9∶91∶0.36∶0.745) as mobile phase at a flow rate of 1.0 mL•min⁻¹. The detection wavelength was set at 225 nm, and the column temperature was set at 30 ℃. The linear range of febrifugine and isofebrifugine were 10.7-426 ng and 10.6-424 ng, respectively. Their average recovery were 98.33% (RSD 2.7%) and 100.4% (RSD 1.8%), respectively. On the basis of this established method, febrifugine was used as the internal reference substance to calculate the relative correction factors (RCF) and the relative retention values (RRV) of isofebrifugine to febrifugine. Through a series of methodology evaluations, the two alkaloids were simultaneously assayed only by quantitative determination of febrifugine. This result played the part of demonstration role for the application of QAMS model in the determination of isomers.

Article in Chinese | WPRIM | ID: wpr-335877


To investigate the stability and degradation kinetics of febrifugine. The results showed that within 24 hours, febrifugine content was decreased by only 1% in mobile phase solvent, but its content was decreased to be 90% of the initial content in the water, methanol, 50% methanol and 10% acetonitrile solution. When the pH value of the solution was between 3 and 7, the retention rate of febrifugine in 24 hours was over 98%, but its content was decreased by about 12% in alkaline solution (pH 9.0). The higher the temperature, the worse the stability of febrifugine. At 40-80 ℃, the content of febrifugine was decreased to be 60% of its initial content in 10 hours, but the content was decreased by only 5% in 10 h at 20 ℃.However, no matter 40 ℃or 60 ℃, febrifugine was mainly transformed into isofebrifugine, and the total content of febrifugine and isofebrifugine was equal to their initial total content in 10 hours, while incase of 80 ℃, the total content was decreased to be 83.33% in 10 h, which suggested that the structure of febrifugine was absolutely changed, not just isomerized to be isofebrigugine at high temperature. Light had a significant impact on the stability of febrifugine. Under bright light, the content of febrifugine was reduced by about 23% in 108 h, but it only decreased by about 10% in the natural light or darkness. In artificial gastric fluid (pH 1.4) and artificial intestinal fluid (pH 6.8), the content of febrifugine was decreased by less than 5% in 10 h. After storage at high temperature(60 ℃), high humidity [(75±1)%] and strong light (3 000 lx) conditions for 10 d, the content of solid febrifugine was decreased by 0.27%, 7.6% and 5.39%, respectively. The degradation of febrifugine basically complied with the first-order reaction kinetic process in the following conditions: in water, methanol, 50%methanol and 10% acetonitrile solvents, alkaline solution (pH>7), different light intensity and different temperatures (20,40 ℃). Therefore, no matter the isolation and purification of febrifugine or the production of the related preparations, it should be done fast in the acidic solution, low temperature and dark conditions, while the febrifugine solid should be kept in dry and dark conditions.

Article in Chinese | WPRIM | ID: wpr-272745


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.

Chinese Journal of Hepatology ; (12): 649-653, 2008.
Article in Chinese | WPRIM | ID: wpr-279714


<p><b>OBJECTIVES</b>Programmed death-1 (PD-1) up-regulation impairs virus-specific CD8+ T-cell responses during chronic viral infection. Whether PD-1 expression influences the virus-specific CD8+ T cells in humans with acute viral infection remains largely undefined. This study aims to characterize the PD-1 expression during acute hepatitis B (AHB), and further addresses the association between the PD-1 dynamics and memory T-cell formation during acute HBV infection.</p><p><b>METHODS</b>Peripheral HBV-specific CD8+ T cells from 11 HLA-A2-positive AHB patients were longitudinally quantitatively analyzed, and PD-1, memory markers CCR7, CD45RA and CD127 and activation marker CD38 on HBV-specific CD8+ T cells were measured using flow cytometric assay. Serum ALT, HBsAg, HBsAb and HBV-DNA levels were evaluated for each subject.</p><p><b>RESULTS</b>All 11 AHB patients examined had multiple pentamer-positive CD8+ T-cell responses in their early phase of HBV infection. Specifically, their PD-1 on pentamer-positive CD8+ T-cells was significantly up-regulated at the onset of their disease. Following their disease resolution, the dynamic decrease in PD-1 expression was found to correlate with the phenotypic development of memory CD8+ T cells, indicated by the increases in CCR7, CD45RA and CD127 and decrease in CD38.</p><p><b>CONCLUSION</b>PD-1-mediated negative signaling may be closely associated with memory T-cell formation during acute self-limited hepatitis B.</p>

Acute Disease , Adult , Antigens, CD , Metabolism , Apoptosis Regulatory Proteins , Metabolism , Female , Hepatitis B , Allergy and Immunology , Metabolism , Humans , Immunologic Memory , Male , Middle Aged , Programmed Cell Death 1 Receptor , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Metabolism , Young Adult