Development of Indirect Competitive ELISA for Lithospermic Acid B of Salvia miltiorrhiza with Its Specific Antibodies Generated via Artificial Oil Bodies.

Yu-En Shih,Jason T.C Tzen,Chao-Hsiang Chen,Nan-Hei Lin

doi:10.3390/molecules24101952

Yu-En Shih, Jason T.C Tzen + Show 2 more

Open Access

https://doi.org/10.3390/molecules24101952

Copy DOI

Abstract

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.

Highlights

Salvia miltiorrhiza, named Danshen in Chinese, is a traditional medicinal herb used to treat a diversity of illnesses, especially cardiac and vascular disorders [1,2]
The content of Lithospermic acid B (LSB) in Danshen is regularly detected as an index for the quality control of this herb
According to the protocol developed in our previous work [22], recombinant caleosin, Cal-K

Summary

Introduction

Named Danshen in Chinese, is a traditional medicinal herb used to treat a diversity of illnesses, especially cardiac and vascular disorders [1,2]. In the past three decades, lipid-soluble constituents of Danshen known as tanshinones were actively investigated and found to possess anti-inflammatory and immunomodulatory activities [3,4]. Water-soluble compounds of Danshen, lithospermic acid B (LSB) and its magnesium complex, have attracted more and more attention, and were found to possess several biological activities, such as attenuating atherosclerosis, improving blood circulation and treating coronary heart disease [5,6,7,8]. LSB is present as the most abundant phenolic compound in the water extract of Danshen.

Objectives

Methods

Results