A new highly selective and specific anti-puerarin polyclonal antibody for determination of puerarin using a mannich reaction hapten conjugate

Orapin Udomsin; Supaluk Krittanai; Tharita Kitisripanya; Hiroyuki Tanaka; Waraporn Putalun

Articles

Abstract

Pharmacognosy Magazine,2017,13,52s,s845-s851.

DOI:10.4103/0973-1296.224331

Published:January 2018

Type:Original Article

Authors:

Author(s) affiliations:

Orapin Udomsin¹, Supaluk Krittanai¹, Tharita Kitisripanya¹, Hiroyuki Tanaka², Waraporn Putalun¹
¹Department of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University; Research Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology, National Research University, Khon Kaen University, Khon Kaen 40002, Thailand
²Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Abstract:

Background: Puerarin (PUE) is a phytoestrogen found in Pueraria candollei and Pueraria lobata. These plants are substantial for traditional medicine in various Asian countries. PUE is a key marker that can be found only in the Pueraria species. Objective: To establish the method for determination of PUE content which is required for quality control of pharmaceutical products. Materials and Methods: PUE-cationized bovine serum albumin conjugate was created via Mannich reaction. After the rabbit immunization, the obtain anti-PUE polyclonal antibody (PAb) was used to develop an enzyme-linked immunosorbent assay (ELISA). Results: An anti-PUE PAb possess a great sensitivity and specificity. The cross-reactivity analysis shows no cross-reaction of an established antibody against other substances. In addition, we successfully developed an indirect competitive ELISA (icELISA) for the quantitative analysis of PUE. The result of method validation conforms to acceptance criteria and correlates with high-performance liquid chromatography, the reference method. The icELISA was applied to determine PUE content in Pueraria spp. plant samples and its derived pharmaceutical products. Conclusion: This highly specific immunogen was created from the Mannich reaction. An icELISA can also be applied to other research propose in the further studies.