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首页> 外文期刊>Phytochemical Analysis >Development of eastern blotting technique for sennoside A and sennoside B using anti-sennoside A and anti-sennoside B monoclonal antibodies
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Development of eastern blotting technique for sennoside A and sennoside B using anti-sennoside A and anti-sennoside B monoclonal antibodies

机译：使用抗SENNOSIDE A和抗SENNOSIDE B单克隆抗体开发了SENNOSIDE A和SENNOSIDE B的东方印迹技术

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INTRODUCTION: Rhubarb, senna and sennoside-containing preparations are currently widely employed as purgatives. The major active components of these medications are sennoside A (SA) and sennoside B (SB). OBJECTIVE: To develop an eastern blotting technique for the specific visualisation and easy determination of SA and SB in plant extracts for application in the standardisation and authentication of rhubarb and senna. METHODOLOGY: SA and SB were separated by TLC, transferred to a PVDF membrane, treated with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide hydrochloride solution and finally treated with bovine serum albumin (BSA). The resulting membrane-bound SA-BSA and SB-BSA conjugates were linked to anti-SA and anti-SB monoclonal antibodies (MAbs) and then to secondary antibodies labelled with peroxidase. SA and SB were detected by visualisation of the peroxidase reaction products. RESULTS: The limit of detection of the eastern blotting was 62.5 ng for both sennosides. The method was applied to the immunohistochemical localisation of SA in fresh rhubarb root. Phloem and radiate wood were found to contain higher concentrations of SA compared with other tissues (pith and bud) in agreement with results obtained by ELISA. The concentrations of SA in the phloem, radiate wood, pith and bud were 64.4, 48.1, 15.0 and 1.8 ng/mg fresh weight, respectively. CONCLUSION: The technique described permitted the visualisation of small molecular weight compounds that had been bound to a membrane, using immunostaining. Owing to the specificity of the MAbs, the eastern blotting may prove to be a useful method for the identification of SA and SB in a background containing large amount of impurities.
机译：简介：目前，大黄，番泻叶和番木瓜苷制剂被广泛用作泻药。这些药物的主要活性成分是番泻苷A（SA）和番泻苷B（SB）。目的：开发一种东方印迹技术，用于植物提取物中的SA和SB的特异性可视化和简便测定，以用于大黄和番泻叶的标准化和鉴定。方法：通过薄层色谱分离SA和SB，转移到PVDF膜上，用1-乙基-3-（3'-二甲基氨基丙基）-碳二亚胺盐酸盐溶液处理，最后用牛血清白蛋白（BSA）处理。将所得的结合膜的SA-BSA和SB-BSA偶联物与抗SA和抗SB单克隆抗体（MAb）相连，然后与用过氧化物酶标记的二级抗体相连。通过过氧化物酶反应产物的可视化来检测SA和SB。结果：两种森诺甙的东部印迹检测限均为62.5 ng。该方法用于新鲜大黄根中SA的免疫组织化学定位。发现韧皮部和辐射木与其他组织（小菜和芽）相比，SA中的SA含量更高，这与ELISA的结果一致。韧皮部，辐射木材，髓和芽中的SA浓度分别为鲜重64.4、48.1、15.0和1.8 ng / mg。结论：所描述的技术允许使用免疫染色技术观察结合在膜上的小分子量化合物。由于单克隆抗体的特异性，东部印迹法可能被证明是用于在含有大量杂质的背景中鉴定SA和SB的有用方法。

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