Correlation analysis between the HPLC fingerprints and in vitro antibacterial activity of ethyl acetate extracts from Radix isatidis

Aim To study the correlation between the HPLC fingerprints and in vitro antibacterial activities of EtOAc extracts of Radix isatidis from various sources. Methods Ten batches of Radix isatidis EtOAc extracts were analyzed with HPLC and the fingerprints were established. The influence of EtOAc extracts on the thermogenic curve of growth of Escherchia coli was obtained by microcalorimetry. The chemical differences of EtOAc extracts of Radix isatidis from various sources in the HPLC fingerprints were probed with hierarchical clustering analysis and similarity analysis. The correlation between the HPLC fingerprints and in vitro antibacterial activities was analyzed with multivafiant correlation analysis. Results Close correlation existed between the HPLC fingerprints and in vitro antibacterial activities of EtOAc extracts of Radix isatidis. Conlusion The combination of HPLC fingerprints and antibacterial activities can be used to discover principle components of Radix isatidis on bioactivity.

Determination of Five Organic Acids in Radix Isatidis by Column Partition Chromatography and Capillary Zone Electrophoresis柱分配色谱-毛细管区带电泳联用分析板蓝根中五种微量有机酸(英文)

Aim To determine five organic acids in Radix Isatidis . Method The extraction method and the column partition chromatographic conditions were studied. Then a capillary zone electrophoretic method was set up for the determination. Results The linear ranges of quinazolinone acid, n anthranilic acid, benzoic acid, salicylic acid, and syringic acid were 5 52-92 0 μg·mL -1 , 5 12-102 μg·mL -1 , 2 28-84 4 μg·mL-1 , 4 78-159 μg·mL -1 , and 1 74-87 0 μg·mL -1 respectively. Conclusion The established method is accurate and simple.

Effect of Radix Isatidis on the Expression of Moesin mRNA Induced by LPS in the Tissues of Mice

To investigate the effect of the anti-endotoxic part of Radix Isatidis on the expression of moesin mRNA in murine tissues induced by lipopolysaccharide （LPS）, the sample solution of F0z2 part from Radix Isatidis was intrapefitoneally administered to experimental mice, and the lipopolysaccharide （LPS） were injected into the tail vein, and then the tissues of liver, kidney and spleen were colleted and cut into slices. The mRNA was detected by moesin mRNA hybridization in situ. The staining results were observed under microscope. It was found that moesin mRNA expression was increased in the tissues of liver, kidndy and spleen in mice treated with LPS, while in the mice pre-treated with F022 part from Radix Isatidis, the LPS-induced moesin mRNA expressions in these tissues were inhibited in a dose-dependant manner. Our study showed that F022 part from Radix Isatidis can inhibit the LPS-induced expression of moesin mRNA in the tissues of liver, kidney and spleen in mice.

Particle Sizes and Antibacterial Activity of Radix Astragalus and Radix Isatidis Ultrafine Powders

Using 9200 laser particle size analyzer and KYKY-2800 scanning electron microscope, particle sizes and cellular morphology of Radix Astragalus and Radix Isatidis ultrafine powders were observed. According to the results, the particle size of 89. 1 % of Radix Astragalus ultrafine powders ranged from 1.729 [xm to 44.938 ｜xm, Z ）50 =4.368 ｜xm; the particle size of 93.411% of Radix Isatidis ultrafine powders ranged from 1.510 [xm to 44.938 ｜xm, Z ）50 = 8 .7 2 6 [xm. Radix As-tragalus and Radix Isatidis ultrafine powders were pulverized completely without intact cellular morphology. The antibacterial activity of Radix Astragalus and Radix Isatidis ultrafine powders against chicken-derived E. coli （078） was investigated. The results indicated that Radix Astragalus and Radix Isatidis ultrafine powders exhibited higher antibacterial activity against chicken-derived E. coli （078 ） compared with the corresponding coarse powders. This study laid a solid foundation for the development and application of Chinese medicine ultrafine powder preparations.

Screening of Anti-endotoxin Components from Radix Isatidis

To find a component with the strongest anti-endotoxin effect from the four components （F021 , F022, F023, and F024 ） of chloroform extract （F02） of Radix Isatidis, the protective effects of the five components on endotoxin-challenged mice and their effects on the production of TNFα and IL-6 by macrophages from the endotoxin-challenged mice were compared. It was found that the five components all had protective effects on endotoxin-challenged mice in terms of mortality. The mortalities of the mice challenged by 2.42 mg/kg endotoxin were 30 %, 50 %, 20 %, 50 % and 60 0% after treatment with F02, F021 , F022 , F023 , and F024. The 5 components all had inhibitory effects on the production of TNFα and IL-6 by macrophages from the mice. At a concentration of LPS of 50 ng/mL, the inhibitory rates of F02, F021 1, F022, F023, and F024 for the production of TNFα and IL-6 were 76.54 %, 30. 86 %, 78.60 %, 36. 63 %, 2.06 % and 75.85 %, 18.45 %, 77. 68 %, 24. 41 %, 3. 64% and at the dose of 100 ng/mL, the inhibitory rates were 49. 65 %, 16.86%, 66.97 %, 13. 39 %, 7.16 % and 59.78 %, 1.28 %, 61.86 %, 2.08 %, 1.44 %, respectively. It is concluded that the component F022 of Radix Isatidis has the strongest anti-endotoxin effect.

Anti-Endotoxic Effects of Syringic Acid of Radix Isatidis

The anti-endotoxic effect of syringic acid (SA) isolated from Radix Isatidis (Banlangen, BLG) was studied. SA was extracted and isolated from BLG and diluted into 1 % solution. The content of SA-pretreated endotoxin (ET) was quantitatively determined using Limulus test. The ability of fever induction of ET pretreated with SA was measured using endotoxin-induced fever test in rabbits. The LPS-induced death in mice pretreated with and without SA was compared. Results showed that after pretreatment with SA, 83. 16 % of ET was destroyed, the ET-induced fever in rabbits relieved markedly and the LPS-induced death rate in mice dropped from 68 % to 20 %. It was concluded that SA isolated from BLG had anti-endotoxic effects.

Artificial neural network techniques to predict the moisture ratio content during hot air drying and vacuum drying of Radix isatidis extract

Background:To predict the moisture ratio of Radix isatidis extract during drying.Methods:Artificial neural networks were designed using the MATLAB neural network toolbox to produce a moisture ratio prediction model of Radix isatidis extract during hot air drying and vacuum drying,where regression values and mean squared error were used as evaluation indexes to optimize the number of hidden layer nodes and determine the topological structure of artificial neural networks model.In addition,the drying curves for the different drying parameters were analyzed.Results:The optimal topological structure of the moisture ratio prediction model for hot air drying and vacuum drying of Radix isatidis extract were“4-9-1”and“5-9-1”respectively,and the regression values between the predicted value and the experimental value is close to 1.This indicates that it has a high prediction accuracy.The moisture ratio gradually decreases with an increase in the drying time,reducing the loading,initial moisture content,increasing the temperature,and pressure can shorten the drying time and improve the drying efficiency.Conclusion:Artificial neural networks technology has the advantages of rapid and accurate prediction,and can provide a theoretical basis and technical support for online prediction during the drying process of the extract.

Influence of Radix Isatidis on the Endotoxin-induced Release of TNF-α and IL-8 from HL-60 Cells

Summary： The effects of active antiendotoxin chemical fraction isolated from Radix Isatidis （fraction D） on TNF-α and IL-8 secretion in HL-60 cells induced by lipopolysaccharide （LPS） were studied. The appropriate densities of cell suspension and fraction D solution were determined by MTT colorimetric method. Fraction D and LPS were added to HL-60 cell suspension with three different methods respectively. The contents of TNF-α and IL-8 in the cultured supernatant induced by LPS were detected by using ELISA method. The results showed that the absorbance （A） was directly proportional to the number of cells and the linearity was good in the range from 0.25×10^5 to 2×10^5 cell/mL cell suspension. The fraction D significantly inhibited the oversecretion of TNF-α and IL-8 in HL-60 cells induced by LPS at the concentration of 7.812 mg/mL which had no cytotoxicity. It was indicated that the antiendotoxin mechanism of the active fraction from Radix Isatidis was contributed to the inhibition of the oversecretion of cvtokines induced bv LPS.

A valid evaluation method for UPLC fingerprint analysis and moisture ratio prediction model:application to microwave vacuum drying of Radix isatidis extract

Background:Drying is a necessary component of traditional Chinese medicine extracts.The heating principle of microwave vacuum drying is different from that of the conventional heat method.However,at present,there is paucity of information on the drying process of traditional Chinese medicine extract by microwave vacuum drying,and the results of such process are unclear.Methods:To study the dynamic changes in the chemical characteristics of microwave vacuum drying under different drying conditions,ultrahigh-performance liquid chromatography fingerprint profiles were established using Radix isatidis extract as a model drug and analyzed using similarity analysis,partial least squares-discriminant analysis,and semi-quantitative analysis.In addition,a backpropagation artificial neural network model was developed to predict the moisture ratio of the drying process.Results:Qualitative results showed that the similarity between different drying conditions was greater than 0.95,and 2 amino acid components(peaks 5 and 6)affected by process fluctuations were screened out.The quantitative results showed that the mass concentration of component 1 fluctuated after drying,while that of component 2 increased.The optimal backpropagation artificial neural network model structure used to predict the moisture ratio was 5-4-1,with regression and mean squared error values of 0.996 and 0.0003,respectively,after training,which were well fitted and had a strong approximation ability.Conclusion:Upon comparison of fingerprints and the evaluation of statistical methods,common components of Radix isatidis extract had little variation under different drying conditions,and the selected components provided a reference for the establishment of process evaluation indexes.The establishment of backpropagation artificial neural network provides a theoretical basis for the application of microwave vacuum drying technology and online monitoring of moisture ratio.

Antiviral activities against influenza virus(FM1) of bioactive fractions and representative compounds extracted from Banlangen(Radix Isatidis)

OBJECTIVE: To study the antiviral activities of clemastanin B(CB), epigoitrin, phenylpropanoids portion(PEP) and the mixture of phenylpropanoids, alkaloids and organic acid fractions(PEP+ALK+OA)from Banlangen(Radix Isatidis).METHODS: The experiment consisted of four parts:therapeutic action, prophylaxsis action, inhibition of virus attachment, and direct virucidal action. Cytopathic effect(CPE) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium(MTT) were used to assess antiviral activity.RESULTS: CB, epigoitrin, PEP and PEP + ALK + OA fractions from Banlangen(Radix Isatidis) extract significantly increased the viability of MDCK cells pre-infected with the virus compared with the virus control group in all the dilutions(P < 0.01). Pretreated with either pure compounds or chemical frac-tions of Banlangen(Radix Isatidis) extract in all the dilutions significantly improved the viability of MDCK cells(P < 0.01). The inhibition of virus absorption to the host cells by CB, epigoitrin and PEP was in a dose dependent manner.CONCLUSION: CB, epigoitrin, PEP and PEP+ALK+OA exert their anti-influenza activity by inhibiting the virus multiplication, prophylaxsis and blocking the virus attachment. The primary mode of action of PEP and PEP + ALK + OA is the inhibition of virus replication. The inhibitory effects on virus attachment and multiplication are the main modes for epigoitrin.

Effect of Radix Isatidis Polysaccharides on Immunological Function and Expression of Immune Related Cytokines in Mice

Objective： To investigate the effect of Radix Isatidis polysaccharides （RIP） on the immunological function and expression of immune related cytokines in mice. Methods： Sixty mice were randomly divided into six groups, the normal group, cyclophosphamide （Cy） model group, Levamisole （positive control） group, RIP low, medium and high dose groups （0.08 g/kg, 0.16 g/kg, 0.32 g/kg, respectively）, ten in each group. By detecting the value of abdominal macrophage phagocytic index, serum hemolysin （HC50）, proliferation of lymphocytes and expression of related cytokines, interleukin （IL-2） and interferon γ, （INF-γ）, the effect of RIP on immunological function and its mechanism were studied. Results： RIP could improve the level of hemolysin in immunological function depressed mice. The results showed that the value of macrophage phagocytic index in the high dose RIP group increased from 1.11 ±0.13 to 1.42±0.26. The level of IL-2 and INF- γ, could be decreased by Cy to 38.12±6.88 ng/L and 139.23±29.87 ng/L, respectively, while RIP in high dose could increase the secretion of IL-2 and INF- γ, to 53.54 ± 14.43 ng/L and 189.91 ± 32.63 ng/L, respectively. Conclusion： RIP could enhance non-specific immunological function, humoral immunity and cellular immunity in mice.

Qualitative and quantitative analysis of glucosinolates and nucleosides in Radix Isatidis by HPLC and liquid chromatography tandem mass spectrometry

Multi-component fingerprinting and quantitation of the glucosinolates and nucleosides in samples of Radix Isatidis have been carried out using high-performance liquid chromatography with diode-array detection and electrospray ionization tandem mass spectrometry(HPLC–DAD–ESI/MS).Five nucleosides together with one glucosinolate were identified by comparing retention times,ultraviolet spectra,mass spectra and/or empirical molecular formulae of reference compounds.Quantitation of these six compounds was carried out simultaneously by HPLC on a Phenomenex Luna C18 column using gradient elution with methanol and water and detection at 254 nm.All calibration curves were linear(r40.9994)within test ranges.Limits of detection and quantitation were 0.33 ng and 2.50 ng on column,respectively.Intra-and inter-day precision(as relative standard deviation)for all analytes was o2.19%with recoveries in the range 99.6%–101.8%at three concentration levels.The validated method was successfully applied to fingerprinting and assay of 25 batches of Radix Isatidis sourced from different geographical regions of China.The method is simple and reliable and has potential value in the quality control of Radix Isatidis.

Actions of four organic acids in radix isatidis on endotoxin-neutralization investigated by kinetic turbidimetric assay

OBJECTIVE:To investigate anti-endotoxin action of four OAs reacted with endotoxin by the LAL assay with KTA.METHODS:Using a incubating kinetic tube reader and kinetic turbidimetric assay(KTA),the concentration-response time curve of endotoxin reacted with limulus amebocyte lysate(LAL) at 37℃ were obtained and the action of four organic acids(OAs) on it were investigated.The four OAs were benzoic acid,salicylic acid,syringic acid and 2-amino-benzoic acid from Radix isatidis.Meanwhile,the temperature variation caused by endotoxin with the four OAs was studied by the rabbit pyrogen test(RPT).RESULTS:It was showed that a low concentration(1 mg/mL) of the four OAs had a little effect of anti-endotoxin,and when the concentrations of the four OAs were 30 mg/mL,the endotoxin was neutralized completely.The relationships between the concentrations of endotoxin and the OAs were all linear with correlation coefficients of greater than 0.9995,indicating that the four OAs all had strong anti-endotoxin action,while syringic acid had the strongest action among the four OAs with IC50 of 12.84 mg/mL.CONCLUSION:The investigations of KTA agreed well with the results obtained by means of RPT.

The Influence of Polysaccharides Extracted from Radix Isatidis on Immunity

The Radix Isatidis polysaccharides (IIP) injected ip at the dose of 50mg/kg daily for seven days increased the spleen index. the total numbero of leukocytes and lymphocytes,the percentage of ANAE+ lymphocytes and the intensity of delayed type hyperoensitivity in normal mice as well as in immunosuppressed mice.Moreover,IIP could improve the phagocytic function of reticuloendothelial system,promote the yield of hemolysin,and increase the NK activity in norwal mice at the same dose.

Enzyme-Linked Immunosorbent Assay and Immunochromatographic Strip for Rapid Detection of Atrazine in Three Medicinal Herbal Roots

Objectives:An enzyme-linked immunosorbent assay(ELISA)and colloidal gold-based immunochromatographic(ICG)strip assay will be developed for the rapid and high-throughput detection of atrazine(ATZ)in medicinal herbs.Methods:A monoclonal antibody against ATZ was obtained after the immunization of mice,cell fusion,and hybridoma screening,and the antibody was used to develop direct competitive ELISA(dcELISA)and the ICG strip assay.Results:Both dcELISA and ICG strip methods were established,optimized,and validated for the detection of ATZ in Salviae miltiorrhizae radix et rhizome,Astragali radix,and Isatidis radix.dcELISA had a half-maximum inhibition concentration of 10.56 ng/m L(Salviae miltiorrhizae radix et rhizome),7.6 ng/m L(Astragali radix),and 8.15 ng/m L(Isatidis radix).The limit of detection(LOD)of the ICG strip assay was 12.5 ng/mL(Salviae miltiorrhizae radix et rhizome),12.5 ng/mL(Astragali radix),and 6.25 ng/mL(Isatidis radix)in different herb matrices.Due to the recognition characteristics of the monoclonal antibody for the pesticides ATZ,propazine,sebuthylazine,and prometryn,the detection results of real samples by the two immunoassays were confirmed by liquid chromatography–tandem mass spectrometry,which proved the accuracy and reliability of the established methods.Conclusion:The proposed dcELISA and ICG strip methods were suitable for the rapid,convenient,and high-throughput detection of ATZ in these medicinal herbs.