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.

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.

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.

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.

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.

Mechanism of Radix isatidis in influencing pulmonary inflammation in tuberculosis rats by TLR4-NF-κB pathway

Objective:To investigate the mechanism of the effects of Radix isatidis on lung inflammation in tuberculosis rats through TLR4-NF-κB pathway.Methods:Forty-five SD rats were randomly divided into three groups(n=15 in each group):control group,model group,and isatis root polysaccharide group.Through the tail vein injection of H37RV Mycobacterium tuberculosis model,the rats in the isatis root polysaccharide group received the intervention of Radix isatidis polysaccharides on the second day of infection.The serum inflammatory factors and the expression levels of TLR4-NF-κB pathway and inflammatory factors in lung tissue of each group were detected and compared.Results:A large number of Mycobacterium tuberculosis infection in the lung tissue of the model group,and clustered outside the cell.In the isatis root polysaccharide group,the CFU level was significantly lower compared with the model group,and the number of Mycobacterium tuberculosis was less than that of the model group(P<0.05),mainly distributed in tuberculous nodules and giant cells.The level of serum inflammatory factors in the model group was significantly higher than that in the control group(P<0.05).The levels of IL-1β,IL-6 and IFN-γin the serum of the isatis root polysaccharide group were significantly lower than those in the model group and significantly higher than those in the control group(P<0.05).The TLR4-NF-κB pathway and inflammatory factor mRNA levels in the lung tissue of the model group were significantly higher than those of the control group(P<0.05).The levels of TLR4,NF-κB,IL-1β,IL-6 and IFN-γin the lung tissue of the isatis root polysaccharide group were significantly lower than those of the model group and significantly higher than those of the control group(P<0.05).The expression level of TLR4-NF-κB pathway was significantly up-regulated in the lung tissue of model rats(P<0.05).The levels of TLR4 and NF-κB in the lung tissue of the isatis root polysaccharide group were significantly lower than those of the model group and significantly higher than those of the control group(P<0.05).Conclusion:Radix isatidis can alleviate lung inflammation caused by Mycobacterium tuberculosis infection by regulating TLR4-NF-κB pathway.

载中药聚乳酸多孔纳米纤维医用敷料

为构筑人体可吸收、抗菌消炎且轻薄柔软的可用于伤口愈合的医用敷料,以聚乳酸为原料,掺杂板蓝根(RI)、柴胡(BC)和茶多酚(GTP)等中药成分,采用氯仿与丙酮二溶剂体系,借助静电纺技术制备聚乳酸(PLA)多孔纳米纤维载中药敷料。通过扫描电镜、红外光谱仪、X射线衍射仪(XRD)和接触角测试仪等手段分析了载中药敷料的微观结构、润湿性能、保水吸液性、抗菌性以及体外降解性能等。结果表明:采用氯仿和丙酮二溶剂体系制备的载中药敷料其纤维具有多孔特征,纤维直径为747~1 550 nm。敷料中所载板蓝根、柴胡、茶多酚与聚乳酸间并未发生负面改性的化学反应。载板蓝根、柴胡、茶多酚的敷料在载药量为3%时对金黄色葡萄球菌抑菌率分别为79.2%、82.5%、75.7%,对大肠杆菌抑菌率分别为79.1%、80.2%、73.9%。同时,较纯聚乳酸敷料,载中药敷料具有较好的透气性和体外降解性,其保水吸液性均提高20%以上,适合用作伤口敷料。

板蓝根多糖结构修饰及抗氧化、抗炎活性研究

目的:以板蓝根为原料,优化板蓝根多糖乙酸酐法修饰工艺,制得乙酰化板蓝根多糖,并研究其修饰前后体外抗氧化和抗炎活性。方法:采用乙酸酐法修饰制备乙酰化板蓝根多糖,以乙酰基取代度为评价指标,结合单因素和响应面方法对其乙酰化修饰工艺条件进行优化,采用体外化学模型法评估多糖对DPPH、ABTS^(+)、羟基自由基的清除能力,并通过细胞活力实验检测其抗炎能力。结果:通过红外光谱分析结果,显示乙酰化板蓝根多糖修饰成功,并未改变多糖基本结构骨架。其最佳工艺条件为反应温度39℃,反应时间5.6 h,料液比1:19 g·mL^(-1),取代度可达到0.3889。在抗氧化实验中发现,板蓝根多糖乙酰化修饰前后对DPPH自由基、ABTS^(+)自由基和羟基自由基均具有较好的清除能力,并呈现出剂量依赖关系,其中乙酰化板蓝根多糖抗氧化活性更强,同时可以有效降低炎症细胞的活力,显示出较显著的抗炎活性。结论:板蓝根多糖经乙酰化修饰后,可显著提高体外抗氧化和抗炎活性,为板蓝根多糖的进一步开发与利用提供理论依据和数据参考。

板蓝根多糖酶法脱蛋白工艺、组成分析与免疫调节活性研究

目的优化板蓝根多糖脱蛋白工艺,并进一步探讨其免疫调节活性,为板蓝根多糖的开发利用提供科学依据。方法通过单因素结合Box-Behnken响应面法优化酶法脱蛋白的最佳工艺条件;利用紫外可见光谱、傅里叶变换红外光谱、高效凝胶渗透色谱、高效液相色谱和扫描电镜等方法对板蓝根多糖化学组成与结构特征进行分析;采用斑马鱼免疫低下模型探讨脱蛋白板蓝根多糖对斑马鱼体内中性粒细胞、巨噬细胞、IL-1β和IL-6含量的影响。结果酶法脱蛋白最佳工艺为:胰蛋白酶500 U·mL^(-1)、pH 8.0、酶解时间5 h、酶解温度37℃,脱蛋白率为(86.39±0.07)%,综合评分(91.15±0.37)%。紫外、红外光谱扫描和电镜扫描显示酶法可以除去粗多糖中含有的蛋白质,脱蛋白后相对分子量在5.82~60.26 kDa之间,单糖摩尔组成为甘露糖∶鼠李糖∶半乳糖醛酸∶葡萄糖∶半乳糖∶阿拉伯糖=2.17∶0.96∶2.90∶83.25∶4.88∶5.84。免疫活性评价结果表明,脱蛋白后的板蓝根多糖浓度在50~300μg·mL^(-1)时,能显著增加斑马鱼免疫细胞密度,增加巨噬细胞增殖,降低免疫低下斑马鱼体内IL-1β和IL-6含量,从而发挥免疫调节作用。结论酶法可以有效去除板蓝根粗多糖中的蛋白质,脱蛋白后的板蓝根多糖具有一定的免疫调节作用。

基于板蓝根药渣氮掺杂碳点的制备及缓蚀性能研究

目的 为提高板蓝根药渣资源回收利用率及减缓碳钢腐蚀所造成的环境污染和经济损失等一系列问题,制备一种环境友好型碳点缓蚀剂。方法 采用一步水热法制备了氮掺杂板蓝根渣碳点(N-CD_(I))缓蚀剂。利用红外光谱(FTIR)、紫外光谱(UV-Vis)、X射线光电子能谱(XPS)等手段对N-CD_(I)的结构、粒径及荧光性能进行表征,并通过失重法和电化学测试,研究了N-CD_(I)对Q235碳钢在1 mol/L HCl溶液中的缓蚀性能与机理。结果 303 K时,在1 mol/L HCl溶液中N-CD_(I)投加量仅为15 mg/L时,缓蚀率可达91.6%,较未掺杂前有明显提高。测试结果表明,N-CD_(I)是以抑制阳极铁的溶解为主的混合型抑制剂,在碳钢表面的吸附符合Langmuir吸附等温模型,SEM断面图也进一步证实N-CD_(I)在碳钢表面形成有效的保护层。结论 证实了中药渣碳点在酸性介质中可以有效抑制Q235碳钢的腐蚀,且具有低投加量、高效、热稳定性好等优势,拥有广阔的应用前景。

根肿菌侵染下菘蓝生物碱合成机制

为探究根肿菌胁迫对菘蓝生物碱及其合成关键酶基因表达的影响,该研究对根肿菌侵染后0、7、14、21 d的菘蓝进行病情形态分级、组织学观察、生理生化指标测定以及转录组学和代谢组学分析。结果表明:(1)接菌后0、7、14、21 d菘蓝根部分别发展为0级、1级、3级、5级的肿根,并且7 d是皮层入侵的关键时间点。(2)接种根肿菌14 d后,菘蓝叶内可溶性蛋白、丙二醛的含量,超氧化物歧化酶、过氧化物酶、多酚氧化酶、过氧化氢酶的活性与同时间对照组比较显著提高,并随着接菌时间的延长呈增加的趋势。(3)代谢组学一共检测到161种生物碱,其中吲哚类生物碱数量较多;与未接菌相比,菘蓝接菌后7、14、21 d分别存在16种、17种、39种差异代谢物且各组差异代谢物多富集在生物碱和氨基酸代谢通路。(4)转录组测序结果显示,与未接菌相比,菘蓝接菌后7、14、21 d分别存在2439个、256个、6437个差异表达基因,这3组共同富集到11个生物碱相关的代谢通路;与未接菌相比,接菌后7、14、21 d有9个基因(编码4种酶THS、TAT、YUCCA、ALDH)表达量均上升。该研究结果揭示了芸薹根肿菌与菘蓝之间的互作机制,探究了芸薹根肿菌对吲哚生物碱合成及其关键酶基因的影响,为后期菘蓝根肿病抗性基因及生物碱次生代谢途径的研究奠定了基础。

双贝糖浆和君药板蓝根的入血成分及抗炎活性成分

为了研究双贝糖浆的抗炎物质基础,利用UHPLC-Q-Orbitrap HRMS识别鉴定双贝糖浆与组方君药板蓝根的主要化学成分及入血成分。UHPLC-Q-Orbitrap HRMS结果显示,板蓝根提取物中含71个主要的化学成分、其中有46个成分可入血,以上化学成分在双贝糖浆中被检测到66个、其中42个成分可入血。进一步通过网络药理学探讨其君药板蓝根的抗炎活性成分及作用机制,通过Griess法结合MTT法验证双贝糖浆和板蓝根二者共同入血成分的抗炎活性。网络药理学分析结果表明板蓝根抗炎的关键化学成分有金合欢素、异牡荆素、半齿泽兰素等。以LPS诱导RAW 264.7细胞建立体外炎症模型,5-羟甲基糠醛、胞苷、异牡荆素、丁香酸在100μmol/L时,能够显著抑制炎症介质NO的释放。在大鼠灌胃双贝糖浆与板蓝根提取物的血清样品中均能够检测到异牡荆素,抗炎活性实验结果表明,其对炎症介质NO的抑制作用最强,为双贝糖浆及其君药板蓝根的关键抗炎活性成分。

板蓝根配方颗粒喷雾干燥工艺优化研究

目的基于质量源于设计(QbD)理念,优化板蓝根配方颗粒喷雾干燥工艺。方法结合信息熵赋值法,以喷干粉得粉率及尿苷、腺苷、鸟苷和(R,S)-告依春含量作为关键质量属性(CQAs),利用Plackett-Burman试验设计对进风温度、雾化压力、药液温度、进液速度和药液相对密度进行考察,筛选出关键工艺参数(CPPs),再运用中心点复合设计(Center Point Composite design,CCD)试验对筛出的CPPs进行优化,在二次多项式回归模型的基础上建立板蓝根配方颗粒喷雾干燥工艺设计空间,并加以验证。结果Plackett-Burman试验结果显示,药液相对密度和进液速度为该研究的关键工艺参数。CCD试验方差分析结果显示构建的模型具有显著性差异(P<0.01),失拟项不显著(P>0.05),表明模型预测能力良好。构建的CPPs优化设计空间为药液相对密度1.05-1.08,进液速度30%-40%。结论基于QbD理念建立的板蓝根配方颗粒喷雾干燥工艺设计空间,可提高其工艺的稳定性,有利于保障产品质量的一致性。

板蓝根提取物对肺炎支原体感染小鼠血清白介素-6、肿瘤坏死因子-α和白介素-17A的影响研究

目的探讨板蓝根提取物对肺炎支原体感染小鼠血清细胞因子白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白介素-17A(IL-17A)水平的影响。方法选取40只BALB/c小鼠并随机分为对照组、模型组、三种不同浓度(250、125、62.5 mg/ml)板蓝根提取物组,每组8只。采用肺炎支原体标准菌株M19滴鼻法建立肺炎支原体感染小鼠模型。建模成功后灌胃不同浓度的板蓝根提取物,对照组和模型组灌胃等量的生理盐水。苏木精-伊红染色法观察肺组织病理变化,流式法检测小鼠血清IL-6、TNF-α、IL-17A的水平。结果模型组血清IL-6、TNF-α、IL-17A水平高于对照组;板蓝根低、中、高浓度组IL-6和IL-17A水平均低于模型组,板蓝根中、高浓度组TNF-α水平均低于模型组,差异有统计学意义(P<0.05)。结论板蓝根能缓解肺炎支原体感染引起的肺组织炎性病变及过量炎症因子IL-6、TNF-α、IL-17A的释放,抑制过度免疫反应,减轻免疫病理损伤。

喷施槲皮素对惠水县国储林下发展林下种植作物的影响分析

分析喷施槲皮素对惠水县国储林下发展林下种植作物的影响,旨在为惠水县国储林及林下经济的进一步发展提供借鉴和参考。本研究在惠水县国储林项目示范基地进行,对林下作物喷施槲皮素,分析喷施槲皮素对林下南板蓝根以及林下羊肚菌种植作物的影响。结果证实,喷施槲皮素可促进国储林林下南板蓝根作物的生长,Z3处理的南板蓝根株高较CK处理增加10.27%,分枝条数较CK处理增加25%;Z1、Z2和Z3处理的林下南板蓝根作物的叶片总数较CK处理呈递增趋势;喷施槲皮素可促进国储林林下羊肚菌作物的生长,各个处理的氨基酸、蛋白质、粗多糖、脂肪排序为:T2>T3>T1>CK;T2处理的DPPH自由基清除率、ABTS自由基清除率、羟自由基清除率高于其他处理,整体而言,T2处理下的羊肚菌作物抗氧化性更强。因此得出,喷施槲皮素对惠水县国储林下发展林下种植作物均有不同程度的影响,喷施900μmol/L槲皮素促进南板蓝根作物生长,喷施600μmol/L槲皮素促进羊肚菌作物的生长,具体喷施剂量需结合林下种植作物的类型合理选择。