Protective Effects of Baicalin on Peritoneal Tight Junctions in Piglets Challenged with Glaesserella parasuis

Glaesserella parasuis (G. parasuis) causes inflammation and damage to piglets. Whether polyserositis caused by G. parasuis is due to tight junctions damage and the protective effect of baicalin on it have not been examined. Therefore, this study aims to investigate the effects of baicalin on peritoneal tight junctions of piglets challenged with G. parasuis and its underlying molecular mechanisms. Piglets were challenged with G. parasuis and treated with or without baicalin. RT-PCR was performed to examine the expression of peritoneal tight junctions genes. Immunofluorescence was carried out to detect the distribution patterns of tight junctions proteins. Western blot assays were carried out to determine the involved signaling pathways. Our data showed that G. parasuis infection can down-regulate the tight junctions expression and disrupt the distribution of tight junctions proteins. Baicalin can alleviate the down-regulation of tight junctions mRNA in peritoneum, prevent the abnormalities and maintain the continuous organization of tight junctions. Our results provide novel evidence to support that baicalin has the capacity to protect peritoneal tight junctions from G. parasuis-induced inflammation. The protective mechanisms of baicalin could be associated with inhibition of the activation of PKC and MLCK/MLC signaling pathway. Taken together, these data demonstrated that baicalin is a promising natural agent for the prevention and treatment of G. parasuis infection.     

黄芩苷与铝离子配合物的电喷雾质谱研究

利用电喷雾多级串联质谱(ESI-MS^n)研究了黄芩苷与铝离子在不同浓度配比时形成的络合物,并通过质谱碎裂规律对其结构进行了初步确认。研究结果表明,黄芩苷与铝离子主要形成比较稳定的1：1和1：2配合物,分别为[AIR^1R^2L]^ 和[AlL2]^ ,其中L=[M-H]^-,R^1=CH3OH,R^2=CH3O。     

Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin’s impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.     

Determination of rhein, baicalin and berberine in traditional Chinese medicinal preparations by capillary electrophoresis with two-marker technique

A CZE method for the identification and determination of three bioactive components, rhein, baicalin and berberine, was developed, with 10 mM borate at pH 9.20 as background electrolyte and direct UV detection at 254 nm. The two-marker (glycyrrhizin acid and cefalexin) technique was used to improve the repeatability of analysis. When the migration indices were used, the repeatability was greatly enhanced compared with migration times. The apparent dissociation contents of rhein and baicalin were also obtained. This method has been successfully applied to the simultaneous analysis of the three components with the recoveries from 96.7% to 104.6%.     

Simultaneous determination of baicalin,oroxylin A‐7‐O‐glucuronide and wogonoside in rat plasma by UPLC‐DAD and its application in pharmacokinetics of pure baicalin,Radix Scutellariae and Yinhuang granule

A novel UPLC‐DAD method was developed and validated for the simultaneous determination of baicalin (baicalein‐7‐glucuronide, BG), oroxylin A‐7‐O‐glucuronide (OAG) and wogonoside (WG) in rat plasma using rutin as the internal standard. Plasma samples were precipitated using acetonitrile containing 0.1% formic acid. Separation was performed on an Agilent Eclipse Plus C₁₈ column (2.1 × 50 mm, 1.8 µm) using gradient acetonitrile and 0.2% formic acid water solution as mobile phase. The flow‐rate was set at 0.4 mL/min and the eluate was detected at 275 nm. The method was linear over the ranges of 0.075–17.50, 0.050–12.60 and 0.056–14.10 µg/mL for BG, OAG and WG, respectively. The intra‐ and inter‐day precisions were respectively <4.8% and 6.4%. All of the limits of detection of three analytes in rat plasma were 0.01 µg/mL, whereas the limits of quantification were, respectively, 0.035, 0.025 and, 0.025 µg/mL. This assay has been successfully applied to pharmacokinetics of BG, OAG and WG in rats after oral administration of Yinhuang granule (YHG) and comparative pharmacokinetics of BG in rats following oral administration of the pure BG, Radix Scutellariae (RS) or YHG. We speculate that some co‐existing ingredients in RS or YHG may increase the absorption and elimination of BG in rat. This work may be helpful for the quality control of Yinhuang granule. Copyright © 2015 John Wiley & Sons, Ltd.     

Assessment of baicalin in mouse blood by monoclonal antibody‐based icELISA

An indirect competitive enzyme‐linked immunosorbent assay (icELISA) based on monoclonal antibaodies (MAb) was recently developed. This new method displays high sensitivity and accuracy, and is especially suitable for pharmacokinetic studies in small laboratory animals. This study aimed to develop an icELISA procedure for baicalin (BAL) quantitation in blood. We successfully developed the icELISA and applied in pharmacokinetic assays of Gegen Qinlian Decoction in mice. A linear correlation was obtained for BAL concentrations in the range from 34.69 to 2220.00 µg/L. The regression equation was y = 1.5557 ? 0.4028log(C) with a correlation coefficient of 0.9936. Precision and accuracy of the icELISA method were evaluated by the variations between replicates from well to well (intra‐assay) and plate to plate (inter‐assay). The values obtained for these parameters were within the normal range (<15%). The recovery rates ranged from 98.93 to 126.78%, meeting the requirements for biological samples. Stability studies showed that BAL sample solutions were intact for 1 h, enough time for UV detection. However, long‐term storage and especially freeze–thaw procedures were detrimental to BAL. The pharmacokinetic parameters derived from mouse experiments were as follows: area under the curves from time 0 to 48 h, 1876.15 ± 1108.14 mg h/L; mean maximum blood concentrations, 101.09 ± 31.53 mg/L; time of maximum concentration, 3.58 ± 2.88 h; mean residence time, 79.30 ± 61.21 h. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

6,7-二乙酰氧基黄芩素的一步合成、选择性水解及甲醚化

以中药黄芩中的主要活性成分黄芩苷为原料,在醋酸钾、吡啶催化回流条件下,用醋酐为乙酰化试剂,以67.6%的收率一步实现了6,7-二乙酰氧基黄芩素的合成,6,7-二乙酰氧基黄芩素经K2CO3/丙酮-水条件下的水解反应或Me2SO4/K2CO3/丙酮中的甲醚化反应以良好收率及高选择性得到6-乙酰氧基黄芩素或7-甲氧基-6-乙酰氧基黄芩素.     

黄芩素与黄芩苷抗氧化活性差异的结构原因

黄芩素是黄芩苷的初级代谢产物,二者的分子结构差别仅在于7-位取代基,前者为酚羟基而后者为糖苷.本文通过稳态吸收光谱和循环伏安实验,以及量化计算等方法考察了黄芩素和黄芩苷的酸离解常数(pKa)、脂水分配系数、氧化-还原电位、分子偶极矩等基本物理化学性质,以及清除ABTS.+自由基活性(TEAC)的差异.结果表明,黄芩素酚羟基7-OH的酸性较强(pKa=5.4);在生理pH下黄芩素的氧化还原电位(0.32 Vvs.NHE)略低于黄芩苷,且TEAC值约为黄芩苷的1.8倍.量化计算结果表明,7-取代基对黄酮分子骨架的构型、物理化学性质以及自由基清除活性有显著影响.文中探讨了黄芩素和黄芩苷的微观和宏观分子属性与抗氧化活性之间的关系,得出酚羟基数量及其pKa值、黄酮分子骨架构型以及电子结构是影响类黄酮化合物抗氧化活性主要因素的结论.研究结果可为深入研究类黄酮化合物的抗氧化结构-活性关系提供依据.