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Production of Polyclonal Antibody Against Madecassoside and Development of Immunoassay Methods for Analysis of Triterpene Glycosides in Centella asiatica

Authors

  • Patcharin Tassanawat,

    1. Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand
    2. Research Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology (PANPB), National Research University–Khon Kaen University, Khon Kaen, Thailand
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  • Waraporn Putalun,

    Corresponding author
    1. Research Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology (PANPB), National Research University–Khon Kaen University, Khon Kaen, Thailand
    • Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand
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  • Gorawit Yusakul,

    1. Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand
    2. Research Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology (PANPB), National Research University–Khon Kaen University, Khon Kaen, Thailand
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  • Boonchoo Sritularak,

    1. Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand
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  • Thaweesak Juengwatanatrakul,

    1. Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, Thailand
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  • Hiroyuki Tanaka

    1. Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
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Correspondence to: Waraporn Putalun, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand. E-mail: waraporn@kku.ac.th

ABSTRACT

Introduction

Centella asiatica (L.) Urban consists of two major triterpene glycosides, asiaticoside (AS) and madecassoside (MA), as active components used for wound healing and enhancing memory.

Objective

To produce a polyclonal antibody against madecassoside (MA-PAb) and develop enzyme-linked immunosorbent assay (ELISA) and Eastern blotting methods for quantitative analysis of triterpene glycosides in Centella asiatica.

Methods

An ELISA method was developed using polyclonal antibody against MA. An Eastern blotting method on the PES membrane was established for determination of MA and AS. The immunoassays were validated for sensitivity, precision, specificity and accuracy.

Results

The prepared MA-PAb shows specificity to MA and AS. The measuring range of triterpene glycosides was 0.39–50 µg/mL using the ELISA method. An Eastern blotting method was developed for determining individual MA and AS, which could be detected in the range of 62.5–500 ng. The limit of detection for MA and AS was 31.25 ng. The two methods developed showed good specificity, precision, and accuracy, and also correlated with high-performance liquid chromatography.

Conclusion

These immunoassays have several advantages that include high sensitivity as well as being rapid and facile for determination of the triterpene glycosides in C. asiatica. Copyright © 2012 John Wiley & Sons, Ltd.

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