Non-Invasive Detection of Anti-Inflammatory Bioactivity and Key Chemical Indicators of the Commercial Lanqin Oral Solution by Near Infrared Spectroscopy.

Quality control methods of current traditional Chinese medicine (TCM) preparation is time-consuming and difficult to assess in terms of overall efficiency of the drug. A non-destructive rapid near-infrared spectroscopy detection system for key chemical components and biological activity of Lanqin oral solution (LOS), one of the best-selling TCM formulations, was established for comprehensive quality evaluation. Near infrared spectral scanning was carried out on 101 batches of commercial LOS under the penetrated vial state and traditional state. RAW 264.7 cells were cultured to detect the anti-inflammatory ability of LOS, and the reference concentrations of epigoitrin, geniposide, and baicalin were obtained by HPLC. The quantitative models were optimized by three kinds of variable selection methods. The correlation coefficients of prediction value of the models were greater than 0.94. The system also passed the external validation. The performance of the non-invasive models was similar to the traditional models. The established non-destructive system can be applied to the rapid quality inspection of LOS to avoid unqualified drugs from entering the market and ensure drug effectiveness. The biological activity index of LOS was introduced and predicted by NIRs for the first time, which provides a new idea about the quality control of TCM formulations.

An efficient approach for simultaneously obtaining oil and epigoitrin from Orychophragmus violaceus seeds by microwave-mediated immiscible binary solvent extraction.

Microwave-mediated immiscible binary solvent extraction (MIBSE) was applied to simultaneously extract oil and epigoitrin from Orychophragmus violaceus seeds. The upper phase of n-hexane was used to obtain oil, and the lower phase of ethanol solution was used to obtain epigoitrin. Factors potentially affecting the yields of oil and epigoitrin were systematically investigated. The optimum conditions were an ethanol volume fraction of 65%, liquid-solid ratio of lower-phase of 20 mL/g, liquid-solid ratio of upper-phase of 12 mL/g, microwave irradiation power of 393 W, and microwave irradiation time of 29 min. The actual yields of oil and epigoitrin were 34.08% ± 1.38% and 11.86 ± 0.47 mg/g, respectively. GC-MS analysis illustrated that the seed oils obtained by MIBSE and Soxhlet extraction exhibited similar fatty acid compositions. The separated epigoitrin was determined by HPLC analyses, which obtained a purity of 91.25% ± 3.83%, follwed by NMR determinations.

Evaluation of potential herb-drug interactions between oseltamivir and commonly used anti-influenza Chinese medicinal herbs.

ETHNOPHARMACOLOGICAL RELEVANCE: According to Traditional Chinese Medicine theory, influenza is categorized as a warm disease or Wen Bing. The Wen Bing formulas, such as Yin-Qiao-San and Sang-Ju-Yin, are still first-line herbal therapies in combating variant influenza virus. To continue our study on the pharmacokinetic and pharmacodynamic interactions between Wen Bing formulas and oseltamivir (OS), the first-line western drug for the treatment of influenza, further interactions between OS and the eight single herbs and their relevant marker components from Wen Bing formulas were investigated in the current study. AIM OF STUDY: To establish an in-vitro screening platform for investigation of the potential anti-influenza herbs/herbal components that may have pharmacokinetic and pharmacodynamic interactions with OS. MATERIALS AND METHODS: To screen potential inhibition on OS hydrolysis, 1 µg/mL of OS is incubated with herbs/herbal components in diluted rat plasma, microsomes and human recombinant carboxylesterase 1(hCE1) under optimized conditions. MDCK-WT and MDCK-MDR1 cell lines are utilized to identify potential modification on P-gp mediated transport of OS by herbs/herbal components. Caco-2 cells with and without Gly-Sar inhibition are performed to study the uptake of OS via PEPT1 transporters. Modification on OAT3 mediated transport is verified by the uptake of OS on HEK293-MOCK/HEK293-OAT3 cells. Anti-virus effects were evaluated using plaque reduction assay on H1N1 and H3N2 viruses. Potential pharmacokinetic and pharmacodynamic interaction between OS (30 mg/kg) and the selected herb, Radix Scutellariae (RS), at 300-600 mg/kg were carried out on rats. All samples are analyzed by an LC/MS/MS method for the contents of OS and OSA. A mechanistic PK model was developed to interpret the HDI between OS and RS in rats. RESULTS: Our developed platform was successfully applied to screen the eight herbal extracts and their ten marker components on metabolic inhibition of OS and modification of OS transport mediated by P-gp, OAT3 and PEPT1. Results from six in-vitro experiments were analyzed after converting raw data from each experiment to corresponding fold-change (FC) values, based on which Radix Scutellariae (RS) were selected to have the most HDI potential with OS. By analyzing the plasma and urine pharmacokinetic data after co-administration of OS with a standardized RS extract in rats using an integrated population pharmacokinetics model, it is suggested that RS could inhibit OS hydrolysis during absorption and increase the absorbed fraction of OS, which leads to the increased ratio of OS concentration versus that of OSA in both rat plasma and urine. Never the less, the anti-virus effects of 2.5 h post-dose rat plasma were not influenced by co-administration of OS with RS. CONCLUSION: A six-dimension in-vitro screening platform has been developed and successfully applied to find RS as a potential herb that would influence the co-administrated OS in rats. Although co-administered RS could inhibit OS hydrolysis during absorption and increase the absorbed fraction of OS, which lead to the increased ratio of OS concentration versus that of OSA in both rat plasma and urine, the anti-virus effect of OS was not influenced by co-administered RS.

Epigoitrin, an Alkaloid From Isatis indigotica, Reduces H1N1 Infection in Stress-Induced Susceptible Model in vivo and in vitro.

Stress has been proven to modulate an individual's immune system through the release of pituitary and adrenal hormones such as the catecholamines, growth hormone, and glucocorticoids. These signal molecules can significantly alter the host immune system and make it susceptible to viral infection. In this study, we investigate whether epigoitrin, a natural alkaloid from Isatis indigotica, provides protection against influenza infection by reducing the host's susceptibility to influenza virus under stress and its underlying mechanism. To support it, the mouse restraint stress model and the corticosterone-induced stress model were employed. Our results demonstrated that epigoitrin significantly decreased the susceptibility of restraint mice to influenza virus, evidenced by lowered mortality, attenuated inflammation, and decreased viral replications in lungs. Further results revealed that epigoitrin reduced the protein expression of mitofusin-2 (MFN2), which elevated mitochondria antiviral signaling (MAVS) protein expression and subsequently increased the production of IFN-ß and interferon inducible transmembrane 3 (IFITM3), thereby helping to fight viral infections. In conclusion, our study indicated that epigoitrin could reduce the susceptibility to influenza virus via mitochondrial antiviral signaling.

Simultaneous determination of Forsythoside A and other four components in Xiao'er-Ganmao-Keli from multivendor by HPLC / 国际中医中药杂志

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.

[Effects of nitrogen and sulfur combined application on nutritional components and active components of Isatis indigotica at seedling stage].

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.

Effects of nitrogen and sulfur combined application on nutritional components and active components of Isatis indigotica at seedling stage / 中国中药杂志

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.

Hepatoprotective Effect of Aqueous Extract from the Seeds of Orychophragmus violaceus against Liver Injury in Mice and HepG2 Cells.

Orychophragmus violaceus (O. violaceus) is a kind of edible wild herb in north China and its seeds have medical potential, however, the effect of O. violaceus seeds on liver injury and the mechanism of action remains poorly understood. Thus, the purpose of the present study is to investigate the effect of O. violaceus seeds on liver injury and further explore the molecular mechanism of the beneficial effects using aqueous extract from the seeds of O. violaceus (AEOV). Mice were orally administrated with saline, AEOV, and biphenyldicarboxylate for 4 days, and were then injected subcutaneously with 0.1% carbon tetrachloride (CCl4) dissolved in corn oil. Sixteen hours later, mice were sacrificed and blood samples were collected. Then, the serum was separated and used for biochemical assay. Livers were excised and were routinely processed for histological examinations. Enzyme activities and protein levels in liver homogenates were detected using commercial kits or by western blot analysis. Additionally, the hepatoprotective effect of AEOV in vitro was evaluated using epigoitrin, the major alkaloid compound isolated from AEOV. We found that AEOV attenuated liver injury induced by CCl4 as evidenced by decreased levels of alanine aminotransferase (ALT) and aminotransferase (AST) in serum, improvement of liver histopathological changes, and substantial attenuation of oxidative stress and inflammation via regulation of nuclear factor-erythroid 2-related factor-2 (Nrf2) and nuclear factor κB (NFκB) pathways. These effects of AEOV were comparable to that of biphenyldicarboxylate which was commonly used as a hepatoprotective reference. Moreover, pretreatment of HepG2 cells with epigoitrin improved cell viability, decreased lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels, increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, attenuated the NFκB pathway, and elevated the Nrf2 pathway after exposure to H2O2. These results suggest that AEOV could effectively prevent CCl4-induced liver injury in mice via regulating the Nrf2 and NFκB pathways, and reveal the cytoprotective effects of epigoitrin against H2O2-induced oxidative stress in HepG2 cells.

Optimization of Flash Extraction for Isatidis Radix by Box-Behnken Experimental Design / 中国药师

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.

Determination of Epigoitrin in Radix Isatidis by Solid PhaseExtraction-Quantitative Nuclear Magnetic Resonance Spectroscopy / 分析化学

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.

Anti-flu Effect of Compound Yizhihao Granule and Its Effective Components / 中草药·英文版

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.

Correlation between chemical composition in processof Isatidis Radix preparation and its pharmacological activity / 中草药

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.

Determination of (R,S)- Epigoitrin in Banlangen Granules by HPLC / 医药导报

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.

In vitro antioxidant and anti-inflammatory activities of Radix Isatidis extract and bioaccessibility of six bioactive compounds after simulated gastro-intestinal digestion.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Isatidis called "Ban-Lan-Gen" is one of the most commonly-used traditional Chinese medicines for antiviral, anti-inflammatory, antioxidant and antipyretic purposes. Investigate the bioaccessibility of uridine, epigoitrin, adenosine, clemastanin B, indigoticoside A and isolariciresinol as well as the antioxidant and anti-inflammatory activities during an in vitro gastro-intestinal digestion of the Radix Isatidis extract (RIE). MATERIALS AND METHODS: High performance liquid chromatography (HPLC) technique was adopted to determine the bioaccessibility of six bioactive compounds in RIE. Antioxidant activities of RIE in different digestive stages were determined by 1,1-Diphenyl-2-picrylhydrazyl (DPPH), superoxide anion and hydroxyl radical scavenging abilities. Anti-inflammatory activity was assayed by the inhibitions of inflammatory cytokines such as nitrous oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor α(TNF-α) producted by lipopolysaccharide (LPS) stimulated RAW264.7 cells. RESULTS: The bioaccessibility of uridine, epigoitrin, adenosine, clemastanin B, indigoticoside A and isolariciresinol were 15.38%, 18.28%, 24.01%, 6.50%, 8.65% and 17.78%, respectively. Also, the digestion products still possessed certain antioxidant activities. The antioxidant activity was highly correlated with lignans (clemastanin B, indigoticoside A and isolariciresino). The anti-inflammation activity of the three samples decreased in the order: IN sample (the solution that had diffused into the dialysis tubing)>Nondigested sample (RIE solution)>Gastric sample (post-gastric digestion)>OUT sample (material that remained in the gastro-intestinal tract). CONCLUSIONS: Results obtained in this research reveal the amount of bioactive compounds from RIE that could be available for absorption in vivo. The antioxidant activity decreased significantly but the anti-inflammatory activity was enhanced in serum-available fraction after gastro-intestinal digestion in vitro. This study could provide a scientific basis for a deeper pharmacological activity study of Radix Isatidis and a simple method for pharmacodynamic material basis research.

Pharmacokinetic-pharmacodynamic binding model of febrile rats in vivo for study on epigoitrin from total alkaloids of Radix Isatidis / 中草药

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.

Determination of main antiviral alkaloidal constituent in Radix Isatidis and its preparation by HPLC-DAD / 中成药

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.