Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand.
Analyst. 2011 Mar 7;136(5):1013-7. doi: 10.1039/c0an00868k. Epub 2010 Dec 22.
Asiaticoside (AS), the major active component of Centella asiatica (L.) Urban, is used as a memory enhancer and for wound healing. We have successfully prepared monoclonal antibodies (MAbs) against AS, and developed an enzyme-linked immunosorbent assay (ELISA) system for its determination. AS was conjugated to the carrier protein bovine serum albumin (BSA), which acted as an immunogen. In order to confirm its immunogenicity, the ratio of hapten in the AS-BSA conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting MAbs against AS were produced by fusing splenocytes with the mouse myeloma cell line, SP2/0-Ag14. After the screening, anti-asiaticoside MAb 2B4 was obtained. Weak cross-reactivities occurred with madecassoside (7.08%), but no cross-reactivities were observed with other related triterpenoid glycosides (<0.01%). The assay was suitable for quantitating AS in the range of 0.78 to 50 µg mL(-1). A good correlation of AS concentrations in crude extracts of C. asiatica between ELISA and HPLC methods was obtained (r(2) = 0.999). The contents of AS in various cultivated C. asiatica samples were assayed by the newly established ELISA. The recovery rates of AS in the samples were in the range of 95-103% with coefficients of variation of <10%. The intra- and inter-assay variations were 3.9 and 4.5%, respectively. The ELISA method described should prove useful as an analytical tool for quality control and standardization of medicinal plants and pharmaceutical products containing AS.
积雪草苷（AS）是积雪草（Centella asiatica（L.）Urban）的主要活性成分，用作记忆增强剂和伤口愈合剂。我们已经成功地制备了针对 AS 的单克隆抗体（MAbs），并开发了用于其测定的酶联免疫吸附测定（ELISA）系统。AS 与载体蛋白牛血清白蛋白（BSA）缀合，作为免疫原。为了确认其免疫原性，通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）测定 AS-BSA 缀合物中半抗原的比例。免疫后，通过将脾细胞与小鼠骨髓瘤细胞系 SP2/0-Ag14 融合来产生分泌针对 AS 的 MAbs 的杂交瘤。筛选后，获得了针对积雪草酸的单克隆抗体 2B4。与积雪草苷（7.08％）发生弱交叉反应，但与其他相关三萜糖苷（<0.01％）没有交叉反应。该测定法适用于定量分析 0.78 至 50 µg mL（-1）范围内的 AS。ELISA 和 HPLC 方法在测定粗提取物中的 AS 浓度方面具有良好的相关性（r（2）= 0.999）。通过新建立的 ELISA 测定了各种栽培的积雪草中的 AS 含量。样品中 AS 的回收率在 95-103％的范围内，变异系数<10％。内部和之间的测定变异分别为 3.9％和 4.5％。所描述的 ELISA 方法应证明可作为含有 AS 的药用植物和药物产品的质量控制和标准化的分析工具。
