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Objective To establish a method for the simultaneous determination of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A by HPLC, and test 16 batches of samples from 14 manufacturers. Methods The test was performed on Kinetex EVO C18 column (150 mm × 4.6 mm, 5 μm) with the column temperature at 35 ℃ . The gradient elution was adopted with the mobile phase of acetonitrile and 0.3% phosphoric acid aqucous at a flow rate of 1.0 ml/min. The detection wavelength of (R,S)-Epigoitrin and Phillyrin were set as 236 nm, the detection wavelength of Forsythoside A, Cholorogenic Acid and Isochlorogenic Acid A were set as 327 nm. Results The good linear relationships were displayed within the linear range of 0.050 45-2.018 00 μg for Forsythoside A (r=0.999 9), 0.018 21-0.728 40 μg for Phillyrin (r=0.999 9), 0.010 16-0.406 40 μg for (R,S)-Epigoitrin (r=0.999 9), 0.006 60-0.263 90 μg for Cholorogenic Acid (r=0.999 9) and 0.0040 44~0.161 76 μg for Isochlorogenic Acid A ( r=0.999 5). The RSDs of reproducibility and stability tests were lower than 2%; recoveries were 97.01%, 98.28%, 99.35% and 96.21%, RSD were 3.19%, 1.19%, 0.81%, 2.88% and 2.96%. The content ranges of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A from 16 batches of samples from 14 manufacturers were 0.057 43-1.508 71 mg/g, 0.017 72-0.350 15 mg/g, 0.005 68-0.177 13 mg/g, 0.007 53-0.226 33 mg/g and 0.00308-0.11908 mg/g. Conclusions The established method is simple and accurate, and has a good repeatability. It can be used for the quality analysis of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A. The content of the tested chemical components from 16 batches of samples from 14 manufacturers have significant differences which indicate that a reinforcement of the quality control is needed.
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Using split plot design, a pot experiment with sand culture was conducted to investigate the effects ofnitrogen and sulfur combined application on nutritional components and active component of Isatis indigotica at seedling stage under different N (5，15，25 mmol·L⁻¹)and S(0.00，1.25，2.50，5.00，7.50 mmol·L⁻¹) levels. The results showed thatthe two elements had obvious effects and the leaf and root dry weights of I. indigotica seedlings increased greatly at N₂ level. Under the same nitrogen concentration, the leaf and root dry weights increased firstly and decreased with the rising of sulfur concentrations in which S₂ was conducive to the growth and biomass accumulation. Soluble sugar, soluble protein, soluble amino acids contents were the highest in N₁, N₂ and N₃ treatments, respectively. The influence of sulfur concentrations on nutritional components was same as biomass, but the peak of different nutritional components was diversity in different nitrogen levels. The effects on secondary metabolites (total flavones, indigo, indriubin, epigotrin contents) were not obvious significantly, in which these indexes by N₁S₃，N₁S₂，N₃S₀，N₃S₁were the highest, respectively. In conclusion, the combination of nitrogen and sulfur of N₂S₂(N 15 mmol·L⁻¹ and S 2.5 mmol·L⁻¹) was beneficial to the growth and secondary metabolites accumulation of I. indigotica. These results could provide a theoretical basis for rational fertilization and cultivation of I. indigotica seedling.
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The method of quantitative nuclear magnetic resonance spectroscopy (qNMR) for determination of epigoitrin in Radix Isatidis was established based on solid phase extraction (SPE).The twice ultrasonic extraction method using pure water was used for fully extracting epigoitrin in sample, and then the extraction was enriched and concentrated by poly-Sery MCX SPE cartridge.The effect of sample pretreatment and qNMR experimental conditions was investigated.The qNMR experiment conditions were selected using DMSO as solvent, calibrated 2,3,5-triiodobenzoate as internal standard, and P1(pulse width)=14.1 μs, d1(pulse delay time)=5 s, NS(number of scan)=256.The .1H-NMR peaks of δ 5.365-5.399 (H-7b, d, 1H) of epigoitrin were chosen as the quantitative peaks.Method validation was performed including precision (intra-day precision RSD was 0.5%, and the inter-day precision was 0.8%), linearity (correlation coefficient r>0.9991), LOD (0.05 mg/g, standard curve method) and LOQ (0.19 mg/g, S/N≥150).The recoveries of the SPE-qNMR were 97.4%-101.7%.The result showed that the method was stable, accurate and reliable.With this method the epigoitrin in a real Radix Isatidis was determined to be <0.19-1.26 mg/g.SPE combining with qNMR could extend the application field of qNMR, especially in the detection of low-content component in complex samples.
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Objective To research the anti-influenza effect of active ingredients in Compound Yizhihao Granule (CYG). Methods The cytotoxicity and cytopathic effects (CPE) were observed under the phase-contrast microscope, besides 50% toxicity concentration (TC50) and 50% inhibitory concentration (IC50) were also calculated using Reed-Muench method, then the antiviral activity in vitro according to Selection Index (SI = TC50/IC50) was evaluated. In PR8 virus-infected mice, survival time, death rate, and lung index were observed in order to evaluate the protective effect. Besides, the effective ingredients were determined using HPLC method, and their contents were calculated by external standard method. Results CYG could inhibit the influenza virus-induced CPE, with IC50 of 4.6 mg/mL (equal to herbal extracts 262.2 μg/mL), and no direct cytotoxic effect at this concentration. PR8-infected mice were ig given CYG, the lung index and mortality were significantly reduced, and survival time was obviously prolonged. HPLC analysis indicated CYG contained many kinds of antivirus active components, including rupestonic acid, epigoitrin, and adenosine. Conclusion CYG is an effective natural anti-influenza medicine. Its antiviral effect should be the synergic effect of a variety of antiviral active ingredients.
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Objective:To optimize the flash extraction process of Isatidis Radix by Box-Behnken experimental design. Methods:A flash extraction method was used with the extraction voltage, material-liquid ratio and extraction time as the independent variables. A Hassan’s method was used to calculate the normalized value(OD) of comprehensive evaluation of the dry extract yield and the epi-goitrin extraction rate in order to establish the mathematical relationship between the comprehensive evaluation OD and the independent variables, and the response surface method was used to predict the best process conditions. Results:The optimum process conditions were as follows:voltage of 88 V, material-liquid ratio of 19. 62, and extraction time of 2. 03 min. The rate of dry extract and epigoitrin extraction was 37. 902% and 0. 1887%, respectively. Conclusion:The measured value is close to the predicted one,which indicates the comprehensive flash extraction parameters optimized by Box-Behnken experimental design can be used for the extraction for Isatidis Radix.
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Objective: To investigate the key factors in the process of IsatidisRadix (IR)preparation. The content changes of its main chemical ingredients (epigoitrin, uridine, guanosine, adenosine, and indirubin) and the correlation with its pharmacological activity had been studied, which would providetheoretical foundation for further optimization of process. Methods: The process of IR preparation (extracting-concentrating-alcohol sinking-drying-granulation) was simulated. HPLC was employed to determine the contents of the chemical ingredients in each step of IR preparation, and the clearance rate of OH•was used as the model to investigate the antioxidant activity. Cluster analysis, partial least sqμares (PLS), and multiple linear regression (MLR) were used to analyze the correlation between its chromatogram and pharmacological activity. Results: The content changes of chemical ingredients from IR preparation were generally decreased on the whole, and the highest loss rate of chemical ingredients occurred in the concentrating-drying process; Two metrological methods had been used toanalyze the correlationand the chromatogram ofindirubin, guanosine, and adenosine, which was positively related with the clearance rate of OH•.Conclusion: Each step in the process of IR preparation had the significant effect on the content of its chemical ingredients and pharmacological activity. The concentrating and drying process may be the key process which would finally influence the quality of preparation. Indirubin, guanosine, and adenosine may be the effective substances of IR with antioxidant activity.
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Objective To establish HPLC method for determination of (R,S)-epigoitrin in Banlangen granules and discuss the content limitation. Methods The samples were separated on SHIMADZU VP-ODS (150 mm×4. 6 mm, 5 μm) and the mobile phase consisted of methol-0. 02% phosphoric acid solution (7:93) at a flow rate of 1. 0 mL. min-1 . The detection wavelength was set at 245 nm and the injection volume (in automatic sampler) was 10 μL. The content limitation was assessed according to the transfer rate and statistical data of the results. Results (R,S)-epigoitrin showed a good linear relationship at concentration of 0. 058 7 – 150. 349 5 μg·mL-1(r = 0. 999 7, n = 7). The average recovery rate was 98. 72% , 98. 40% and 98. 60% , respectively; RSD was 1. 84% , 0. 50% and 1. 82% , respectively. The content limitation of (R,S)-epigoitrin was unreasonable according to the transfer rate and the statistical data of the results. Conclusion The method is easy and simple to operate, accurate and stable in results, and highly specific, thus it is applicable for the quality control of Banlangen granules. The content limitation should be determined on the basis of pharmacokinetic and pharmacodynamic data.
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Objective To investigate the relationship between pharmacokinetics(PK) and pharmacodynamics PD of epigoitrin,a main component in total alkaloids of Radix Isatidis,in yeast-induced febrile rats with the combined PK-PD model.Methods The plasma concentration of epigoitrin after ig administration with total alkaloids of Radix Isatidis was determined by HPLC method and the body temperature was recorded by electronic thermometer.The individual PK parameters were fitted using one compartmental model.The PD parameters were fitted by three kinds of PK-PD binding models,such as indirect inhibition-Kin model,indirect stimulation KoutPD model,and Sigmoid-Emax model.Results The main PK parameters t1/2,Cmax,AUC were(4.94?0.84) h,(4.01?0.21) ?g/mL,(28.37?2.42) ?g?h/mL and(5.71?0.91) h,(4.15?0.25) ?g/mL,(30.35?2.58) ?g?h/mL in both normal and febrile rats,respectively.The relationship between pharmacological effects and effect compartment concentration was better fitted with the indirect inhibition-Kin PD model.The corresponding PD parameters were Kin=(0.70?0.10) h-1,Kout=(0.54?0.12) h-1,R0=1.33?0.16,IC50=(0.94?0.66) mg/L.ConclusionThe PK parameters of epigoitrin in total alkaloids of Radix Isatidis show that there is no significant difference in PD behavior in vivo in both normal and febrile rats.Relationship between in vivo PK and PD of epigoitrin in febrile rats is established using indirect inhibition Kin model.
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AIM: To determine epigoitrin and 2,4(1H,3H)-quinazolinedione in Radix Isatidis and its preparation by HPLC-DAD. METHODS: HPLC was carried out with an Agilent 1200,equipped with ZORBAX SB-C_(18) column(4.6 mm?250 mm,5 ?m).The mobile phase of acetonitrile:(0.1% phosphoric acid+0.03% triethylamine)aqueous solution=12∶88.The flow rate was 0.7 mL/min.The detection wavelength was set at 224 nm.The column temperature was at 30 ℃. RESULTS: The linear ranges of epigoitrin was 0.040 9 ?g-1.103 8 ?g;2,4(1H,3H)-quinazolinedione was 0.010 6 ?g-0.169 6 ?g,The recoveries of epigoitrin and 2,4(1H,3H)-(quinazolinedione) were 98.44%(n=9),with RSD of 1.43% and 98.67%,with RSD of 1.65%,respectively. CONCLUSION: The method is simple,sensitive and reliable.It can be used for quantitative determination of Radix Isatidis and its preparation.