Wenxin Keli was obtained from Shanxi Buchang Pharmaceutical Co., Ltd (Shanxi, China). Ginsenoside Rg₁, Ginsenoside Re, Notoginsenoside R₁ were purchased from Winherb Medical Tech. Co., Ltd (Shanghai, China). Lobetyolin was obtained from Push Bio-Tech (Chengdu, China). Extract of codonopsis glycosides was obtained from Dasfbio (Nanjing, China).

HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Formic acid (HPLC grade) was purchased from Roe Scientific (Newark, DE, USA). Ethanol was purchased from Zhejiang Changqing Chemical (Hangzhou, China). Deionized water was prepared with an Elga PURELAB flex system (ELGA LabWater, UK).

High-glucose Dulbecco's modified Eagle's medium, fetal bovine serum, trypsin-EDTA and antibiotics (100 U/ml penicillin G and 100 g/mL streptomycin) were obtained from Gibico BRL (Grand Island, NY, USA). Tert-Butyl hydroperoxide solution, Thiazolyl Blue Tereazolium Bromide, Terfenadine, DMSO, N-Phenylthiourea (PTU), and Tricaine were acquired from Sigma–Aldrich (St. Louis, MO, USA).

Tecan infinite M1000 system (Tecan, Zurich, Switzerland). AB TripleTOF 5600^plus System (AB SCIEX, Framingham, USA), coupled to a Waters ACQUITY UPLC™ system (Waters, MA, USA). Finnigan LCQ DecaXP^plus mass spectrometer equipped with an ESI source (Thermo, MA, USA) coupled to Agilent 1100 liquid chromatography (Agilent, Waldbronn, Germany). Agilent 1200 preparative performance liquid chromatography (Agilent, Waldbronn, Germany). Leica DMI 3000B Fluorescence Inversion Microscope System (Leica Microsystems Inc., USA), Andor Zyla 5.5 sCMOS Cameras (Oxford Instruments plc, Tubney Woods, Abingdon, UK). IX-100F Zebrafish system (iWorx Systems, Inc., USA).

Chemical composition of WXKL was characterized by AB TripleTOF 5600^plus System coupled to a Waters Acquity UPLC system. The MS conditions: scan range m/z 100–2,000. Negative ion mode: source voltage was−4.5 kV, and the source temperature was 550°C. Positive ion mode: source voltage was +5.5 kV, and the source temperature was 600°C. The pressure of Gas 1 (Air) and Gas 2 (Air) were set to 50 psi. The pressure of Curtain Gas (N₂) was set to 35 psi. Maximum allowed error was set to ±5 ppm. Declustering potential (DP), 100 V; collision energy (CE), 10 V. For MS/MS acquisition mode, the parameters were almost the same except that the collision energy (CE) was set at 50 ± 20 V, ion release delay (IRD) at 67, ion release width (IRW) at 25. The acquisition parameters for Finnigan LCQ DecaXP^plus mass spectrometer were as follows: nebulizing gas, high purity nitrogen (N₂); collusion gas, high-purity helium (He); ion spray voltage:−3 kV; capillary temperature: 350°C; capillary voltage:−15 V; mass range: m/z 100–1,500. Chromatographic separation was carried out on an Agilent Zorbax SB-C18 analytical column (4.6 × 250 mm I.D., 5 μm; Agilent Technologies, USA). The mobile phase consisted of water (A) and acetonitrile (B) both containing 0.05% v/v formic acid. A gradient program was used as follows: 0–5 min, 10% B; 5–15 min, 10–25% B; 15–35 min, 25–35% B; 35–40 min, 35–40% B; 40–45 min, 40–70% B; 45–55 min, 70–95% B; 55–65 min: 95% B. The flow rate was 0.5 mL/min, the column temperature was 30°C, and the injection volume was 20 μL.

H9c2 cell were obtained from Cell Bank of the Chinese Academy of Science (Shanghai, China) and cultured in high glucose Dulbecco's modified Eagle's medium supplemented containing 10% fetal bovine serum (FBS) and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin). The cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂. The anti-oxidation activity of each fraction was determined by tetrazolium based colorimetric assay (MTT assay). Briefly, cells (5 × 10⁴ cell/mL) were seeded to 96-well plates for 24 h and then treated with fractions of WXKL for another 24 h prior to 150 μM H₂O₂ exposure in fresh medium for 3 h, After that, 100 μL 0.5 mg/mL MTT in fresh medium replaced the former medium for 4 h at 37°C. Then, the medium was replaced by 100 μL DMSO and vibrated for 10 min. The cell viabilities of tested fractions were determined by measuring the optimal densities (ODs) of untreated cells (control), the cells exposed to H₂O₂ (model), and the cells pre-incubated with components (tested). The activities of the components were calculated using the following formula: Survival rate% = OD of tested/OD of control. Protection rate% = (OD of model – OD of tested)/(OD of model – OD of control).

Heterozygotes and homozygote transgenic Cmlc2-GFP zebrafish expressing green fluorescent protein (GFP) exclusively in myocardium were provided by Zebrafish Resource Center, Zhejiang University School of Medicine (Hangzhou, China) and maintained according to established standard procedures. Two parent zebrafish were placed separately in a mating box equipped with a separator to protect the eggs from being eaten. Spawning was induced in the morning and embryos from each box were collected and rinsed with system fish water (containing 0.3% Instant Ocean Salt in deionized water with final pH 6.9–7.2, conductivity 450–550 μs/cm, and hardness of about 90 mg/L NaHCO₃). The embryos were maintained in the Petri dish with system fish water and transferred to the incubator and incubated at 28°C. This study was granted by the Institutional Animal Care and Use Committee of the Laboratory Animal Center, Zhejiang University. We followed the relevant guidelines from the Laboratory Animal Center of Zhejiang University.

In 24 hpf, larvae with fluorescence were picked under fluorescent microscope and membranes of these larvae were ruptured artificially. Larvae were distributed into a 24-well plate and 8–10 larvae in each well with system fish water added with 0.2 mM N-Phenylthiourea (PTU) and 6 nM methylene blue for treatment. Set groups by wells, including Control, Model, and Treat. Terfenadine was stocked in DMSO at 100 mM, fractions of WXKL was stocked in DMSO at 100 mg/mL and fish water was used to dilute the stock to appropriate concentration. The model group was only given terfenadine, and the treat groups were given terfenadine and corresponding fractions. In 48 hpf incubating, the previous medium was discarded, and added fractions and terfenadine working solution, according to the groups, and filled to 2 mL with fish water medium in each well. The final concentrations of terfenadine was 6 μM. The fractions were diluted to appropriate concentration, mostly 50 μg/mL and some were 25, 12.5, 6.25 μg/mL, depending on the toxicity refer to cell assay.

In 72 hpf, the beating of zebrafish heart was recorded under florescence with Leica DMI 3000B Fluorescence Inversion Microscope System (Leica). The readout speed of the sCMOS camera was set at 10 frames or 20 frames per second with 4 × 4 pixel binning. L5 filter cube (excitation wave length of 480 nm and emission wave length of 527 nm). One hundred continuous dynamic images were captured by Zyla 5.5 sCMOS Cameras (Andor), subsequently were recognized by Matlab. The area of heart in each picture was measured. The area change with time was supposed to exhibit the heart rhythm. The heartbeats were also recorded manually for accuracy. We calculated the heartbeat of ventricle uniformly.

The recovery rate (R_i) of the components were calculated using the following formula: Ri=Bi-BMBC-BM×100%

R_i: normalized heart rate recovery rate of fraction i; B_i: beats of larvae treated by fraction i; B_M: beats of larvae treated by terfenadine; B_C: beats of larvae treated in control group.

The peak area of each compound was normalized according to the following formula: Aj=Ai,j∑i=1mAi,j

A_j: normalized values of peak area of constituent j in fraction i; A_{i, j}: peak area of compound j in fraction i; m: the numbers of fractions obtained from whole extract.

The activity indexes of compounds were given by the following formula: AIj=∑(Ri×Aj)

AI_j: activity index of compound j.

The ECG of zebrafish was measured by IX-100F Zebrafish system (iWorx Systems, Inc., USA). Zebrafish was anesthetic at first and positioned on its back on a fish-bed. Use a paper to gently remove excess water and ensure that the fins are not crossing the belly of the fish. Place the fish-bed with the fish, head to the right, in the chamber and position it under Ag/AgCl surface electrodes. The two electrodes were placed axially along the center-line of the fish's belly and the forward electrode should be placed close to the gills. ECG was recorded by LabScribe v3 software (iWorx System Inc., USA).

The data are expressed as mean ± standard deviation (SD). Parameter comparisons between groups were made with one-way ANOVA analysis of variance. GraphPad prism 7 software (GraphPad Software, USA) was used to carry out statistical analysis. P < 0.05 was considered statistically significant.

The main compounds of WXKL include sugar, glycosides, lignans, polyynes, saponins, iridoid glycosides, detailed information is listed in Table 1. The negative ion model base peak LC-MS chromatograms of WXKL was showed as Figure 2. We collected 71 compounds information of MS/MS and identified 53 compounds primarily, including saponins, phenylpropanoids, polyacetylene, triterpenoid, and others. Twenty-seven compounds of them belong to Notoginseng, including notoginsenoside R₁, ginsenoside Rg₁, ginsenoside Re, ginsenoside Ra₃, ginsenoside Rb₁, notoginsenoside R₂, ginsenoside Rc, ginsenoside Rd and so on. Sixteen compounds belong to Codonopsis, including tangshenoside V, lobetyolinin, lobetyolin, atractylenolide III, gentisic acid β-D-glucoside, syringin, hexyl 6-O-β-D-glucopyranosyl-β-D-glucopyranoside, hexyl 2-O-β-D-glucopyranosyl-beta-D-glucopyranoside and others. Besides, 5 were identified from Polygonatum, and 2 were from Nard (Table 1).

We first performed standard isolation by preparative chromatography to obtain fractions, which were analyzed by Finnigan LCQ DecaXP^plus mass spectrometer. The mass spectrums of every fractions were shown in Supplementary Material.

Oxidative stress plays a key role in the pathogenesis of various diseases (Furukawa et al., 2004). These fractions were then evaluated the protective activity on H9c2 cell damaged by H₂O₂. The toxicity of all fractions were tested at first, and the safe concentration of most fraction were 50 μg/mL, and some were 25/12.5/6.25 μg/mL to insure no toxicity. The cell viability with these fractions treated were shown in Supplementary Material.

Zebrafish (D. rerio) has been an ideal model for drug screening (Goldsmith, 2004). There have been many applications of zebrafish as a high-throughput screening model and cardiotoxicity risk assessment of drug candidates (Wen et al., 2012; Zhu et al., 2014). Here we used Cmlc2-GFP Tg zebrafish as the base. This transgenic line expressing GFP exclusively in myocardium driven by promoter cmlc2 (cardiac myosin light chain 2 gene) (Huang et al., 2003). Terfenadine has been reported can induce QT prolongation in zebrafish and guinea pig (Milan et al., 2006; Lu et al., 2012; Chaudhari et al., 2013), which is associated with ventricular tachyarrhythmia (Gowda et al., 2004). Terfenadine causes QT prolongation in adult zebrafish, also demonstrate in zebrafish embryos (Langheinrich et al., 2003).

As shown in Figure 3, terfenadine had less effect on the structure of the heart (Figure 3A) but influenced the rhythm of beat obviously (Figure 3B). The rhythm of heartbeat was exhibited through the area change of heart analyzed by Matlab. It's obvious that the control groups performed fast and regular rhythm (about 180 beats/min), and in the model groups, the heart rate were down to 80–100 beats/min with irregular heartbeats after incubated with terfenadine for 24 h, and some showed typically atrioventricular block (Peal et al., 2011), while co-incubated with WXKL stabilized the rhythm (Figure 3B, Supplementary Videos). And WXKL and its fractions performed varying affection on heart rate. Fraction 1 and Fraction 2 and former part of Fraction 3 showed beneficial effect on heart rate, while the remaining parts of Fraction 3 lowered the rate even more (Figure 3C).

Activity index (AI) of each compound was calculated according to mathematical formulae proposed in our previous study. It was assumed that the compounds with positive activity index might be active and has contribution to the activity of whole formula to some extent (Wang et al., 2014). The relative intensities of the identified compounds in each fraction were visually presented in a heatmap (Figure 4A). After multiply corresponding bioactivity coefficient (i.e., heart rate recovery rate) of each fraction, the heatmap was converted into a bio-active map, and the red and gray color represent good or bad effect, respectively. The calculated scores were exhibited as histogram on the right (Figure 4B). The detailed scores were listed in Supplementary Material. We plot compounds with the effect on cardiomyocytes and the heart rate of zebrafish (Figure 4C), the compounds in upper right region represented a better activity.

According to the scores, Ginsenoside Rg₁, Ginsenoside Re, Notoginsenoside R₁, Lobetyolin, Lobetyolinin were selected to validate activity, considering the available. Lobetyolinin was prepared and enriched by ourselves from commercial codonopsis glycosides. Their toxicity was confirmed before. We increased the dosage of terfenadine and shortened the incubation time as an acute injury model to improve significance when validating the active of pure compounds by reason of the pure compounds were not strong enough to exhibit activity in original method. After pre-treated with compounds (50 μM) for 24 h, the larvae were treated with 15 μM terfenadine for 2 h and recorded heartbeat under fluorescent. As the consequence, the heart rate of larvae was recovered in varying degree (Figure 5A). Ginsenoside Rg₁ and lobetyolinin exhibited better activities. Meanwhile, ECG of adult zebrafish treated with compound was measured. The heart rate of normal zebrafish was around 100 beats/min, and after treated with 25 μM terfenadine for 1 h, the heart rate was down and occurred irregular rhythm (Figure 5B). Lobetyolinin pre-treated for 6 h recovered the heart rate and represent electrocardiograms were showed as Figure 5C.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.