Development of Eastern Blotting Technique for Sennoside A and Sennoside B using Anti-Sennoside A and Anti-Sennoside B Monoclonal Antibodies
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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DOI：
10.1002/pca.1111
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发表时间：
2009-03-01
影响因子：
3.3
通讯作者：
Shoyama, Yukihiro
中科院分区：
文献类型：
--
作者：
Morinaga, Osamu;Uto, Takuhiro;Shoyama, Yukihiro
大黄、番泻叶和含番泻叶皂苷的制剂目前被广泛用作泻药。这些药物的主要活性成分是sennoside A （SA）和sennoside B （SB）。目的：建立一种快速测定植物提取物中SA和SB含量的东部印迹技术，用于大黄和泻泻草的标准化和鉴定。方法：SA和SB经薄层色谱分离，转移到PVDF膜上，用1-乙基-3-（3'-二甲氨基丙基）-盐酸碳二亚胺溶液处理，最后用牛血清白蛋白（BSA）处理。得到的膜结合SA-BSA和SB-BSA偶联物分别与抗sa和抗sb单克隆抗体（mab）连接，然后与过氧化酶标记的二抗连接。通过过氧化物酶反应产物的可视化检测SA和SB。结果-两种皂苷的东部印迹检测限均为62.5 ng。将该方法应用于新鲜大黄根中SA的免疫组织化学定位。韧皮部和辐射木材中SA的含量高于其他组织（髓和芽），这与ELISA法的结果一致。韧皮部、辐射材、髓和芽中SA含量分别为64.4、48.1、15.0和1.8 ng/mg鲜重。结论-所描述的技术允许使用免疫染色技术可视化已结合到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在含有大量杂质的背景中鉴定SA和SB的有效方法。版权所有John Wiley & Sons， Ltd. 2009
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. Copyright (C) 2009 John Wiley & Sons, Ltd.