Screening and Identification of Cardioprotective Compounds From Wenxin Keli by Activity Index Approach and in vivo Zebrafish Model

Abstract

Wenxin Keli (WXKL) is a widely used Chinese botanical drug for the treatment of arrhythmia, which is consisted of four herbs and amber. In the present study, we analyzed the chemical composition of WXKL using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) to tentatively identify 71 compounds. Through typical separate procession, the total extract of WXKL was divided into fractions for further bioassays. Cardiomyocytes and zebrafish larvae were applied for assessment. In vivo arrhythmia model in Cmlc2-GFP transgenic zebrafish was induced by terfenadine, which exhibited obvious reduction of heart rate and occurrence of atrioventricular block. Dynamic beating of heart was recorded by fluorescent microscope and sensitive camera to automatically recognize the rhythm of heartbeat in zebrafish larvae. By integrating the chemical information of WXKL and corresponding bioactivities of these fractions, activity index (AI) of each identified compound was calculated to screen potential active compounds. The results showed that dozens of compounds including ginsenoside Rg₁, ginsenoside Re, notoginsenoside R₁, lobetyolin, and lobetyolinin were contributed to cardioprotective effects of WXKL. The anti-arrhythmic activities of five compounds were further validated in larvae model and mature zebrafish by measuring electrocardiogram (ECG). Our findings provide a successful example for rapid discovery of bioactive compounds from traditional Chinese medicine (TCM) by activity index based approach coupled with in vivo zebrafish model.

Keywords: Wenxin Keli; arrhythmia; cardioprotection; drug screen; zebrafish.

Figure 1

Scheme of active index calculation based active compounds screening from WXKL.

Figure 2

Negative ion model HPLC–MS base peak chromatograms of WXKL.

Figure 3

Cardioprotection of Wenxin Keli and fractions on zebrafish model. (A) Represent images of heart structures in zebrafish under bright-field and fluorescence. (B) Represent images of heartbeat rhythm of different groups exhibited by Matlab. (C) Normalized heart rate of zebrafish larvae influenced by WXKL fractions. n ≥ 8. ^##P < 0.01 vs. control, *P < 0.05, **P < 0.01 vs. model.

Figure 4

Identification of active compounds by coefficient ranking. (A) Heatmap for relative content of identified compounds in each WXKL fractions. (B) Bio-active map was converted from content map and corresponding bioactivity coefficient in each fraction, and active indexes of identified compounds were calculated and showed on the right. (C) Compounds were plot by cell protection and bio-active scores and mass spectrum of several compounds on top. X axis means the contribution of compounds to the cardiomyocytes protection. Y axis means the AI of compounds.

Figure 5

(A) Normalized heart rate of zebrafish larvae treated with compounds. n = 7. ^#P < 0.05 vs. control, ^##P < 0.01 vs. control, *P < 0.05, **P < 0.01 vs. model. (B) The schematic of ECG measurement, and the result of different group. n = 3. (C) Represent electrocardiograms of adult zebrafish treated in different groups.