Ultrasensitive Time-Resolved Fluoroimmunoassay for Saikosaponin a in Chaihu (Bupleuri Radix).

Zhi Chao,Yukihiro Shoyama,Weiping Zeng,Qian Cui,Xuan Cai,Enwei Tian,Hiroyuki Tanaka,Xiaolei Li,Yingsong Wu,Saikat Dewanjee

doi:10.1371/journal.pone.0151032

Zhi Chao, Yukihiro Shoyama + Show 8 more

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https://doi.org/10.1371/journal.pone.0151032

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Abstract

The aim of this study is to establish a time-resolved fluoroimmunoassay (TRFIA) system for quantitative analysis of saikosaponin a (SSa) in the crude drug of Chaihu (Bupleuri Radix). A 96-well microplate coated with rabbit anti-mouse IgG was incubated with the methanol extracts of Chaihu samples and a mouse anti-SSa monoclonal antibody, and a Eu3+-labeled SSa-human serum albumin conjugate was used as the tracer. The established competitive TRFIA showed a good fourth order polynomial fitting from 0.01 to 10.0 μg/mL for standard SSa sample with a detection limit of 0.006 μg/mL. The intra- and inter-assay coefficients of variation of the assay were 7.3% and 8.9%, respectively, and the average SSa recovery was 119.2%. For samples of Chaihu extract, the results of this assay showed a good correlation with those by enzyme-linked immunosorbent assay established previously. This TRFIA system is ultrasensitive for detecting SSa with a wide detection range and a good stability and represents the first attempt of using TRFIA for quality evaluation of the crude drug of Chaihu.

Highlights

Chaihu (Bupleuri Radix), a common traditional Chinese medicinal herb derived from the dried roots of Bupleurum chinense DC. or B. scorzonerifolium Willd., has been used for medical purposes in China for more than 2000 years
Compared with the conventional enzyme-linked immunosorbent assay (ELISA), which had a detection range of saikosaponin a (SSa) from 0.026 to 1.5 μg/mL [27] (0.16– 2.5 μg/ml in another case [28]), the time-resolved fluoroimmunoassay (TRFIA) system showed a sensitivity about 3–15 times greater and a much wider detection range
SSa contents determined by TRFIA were similar to those by ELISA

Summary

Introduction

Chaihu (Bupleuri Radix), a common traditional Chinese medicinal herb derived from the dried roots of Bupleurum chinense DC. or B. scorzonerifolium Willd., has been used for medical purposes in China for more than 2000 years. Saikosaponins, which have a typical oleanan-type skeleton as the aglycon, have been identified by modern techniques as the major biological active constituents in Chihu. Of these saikosaponins, saikosaponin a (SSa) (Fig 1), a major saponin, has been shown to possess versatile bioactivities to suppress inflammation [3] and oxidation, protect liver function [4], induce tumor cell apoptosis, inhibit carcinogenesis [5,6,7,8,9], and induce cell differentiation [10]; research evidence. Because of the uneven quality of Chaihu in the market [2], quantification of SSa is critical to ensure the effectiveness of the crude drug [1], an accurate, sensitive, and convenient method for determination of SSa in Chaihu is essential

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