The distribution of active compounds of traditional Chinese medicine
Cardiovascular disease (CVD), which is defined as a set of diseases and conditions including coronary heart disease (CHD), cerebrovascular disease, and heart failure, has been the leading cause of mortality across the world [
Traditional Chinese medicine (TCM) is a valuable asset for preventing and treating disease. In China, TCM as a complementary therapy has been widely used for CVD. Recent reviews have also suggested that TCM may be beneficial to patients with CVD. The TCM-Danshen is the dry root and rhizome of
Mapping and quantifying the distribution of drugs in vivo are critical to elucidating their mechanisms of action. The distributions of drugs and their quantities at target sites are closely related to their efficacy and safety. However, analysis of TCMs and their metabolites remains challenging because of the diversity of their compositions, the complexity of biological matrices, and the presence of trace amounts of components and metabolites [
In the present study, the distribution of four salvianolic acids (DSS, CAA, RA, and SAA) of Danshen in myocardial ischemic pig tissues was determined using a liquid extraction surface analysis coupled with tandem mass spectrometry (LESA-MS/MS) method which is a fully automated, chip-based method with the characteristics of simplicity and efficiency. An attempt was made to explore the relative amounts and spatial distributions of the target compounds in vivo and the therapeutic effect of Danshen on CHD.
Standards of CAA (lot no. LL90Q26, 99% purity), DSS sodium salt (lot no. LIA0Q 80, 99% purity), RA (lot no. L970N70, 99% purity), and SAA (lot no. L4B0P55, 99% purity) were purchased from J&K Scientific Ltd (Beijing, China) (Figure
Chemical structures of the compounds investigated in this study.
Methanol (CAS no.67-56-1, batch no. 150162, Fisher Scientific UK, Loughborough, UK), ammonium hydroxide (Beijing Chemical Plant, Beijing, China), and high-performance liquid chromatography (HPLC) grade water (CAS no. 7732-18-5, batch no. F8CJ21, DUKSAN, Ansan-si, Korea) were used.
All crude drugs were purchased from Beijing Tongrentang Pharmaceutical Co., Ltd. (Beijing, China), including
15 male Bama miniature pigs (25 ± 2 kg, 6–10 months, lot no. SCXK2015-0002) were purchased from Tianjin Bainong Laboratory Animal Breeding Technology Co., Ltd. (Tianjin, China). Pigs were housed under standard laboratory conditions, fed twice a day, and given tap water ad libitum. All the animal care and experimental procedures were performed in accordance with the China Physiological Society’s “Guiding Principles in the Care and Use of Animals” with the approval from the Animal Care Committee of Beijing Anzhen Hospital, Capital Medical University (no. 0000353).
After one week of adaptive feeding, inspection, and quarantine, eleven animals that met test standards were retained. Eight animals were randomly selected to a model building which underwent thoracotomy with an Ameroid constrictor (ø 2.75 mm, Research Instrument SW, USA) placing on the proximal left circumflex artery [
Study design and procedures.
Echocardiography was assessed four weeks after surgery and four weeks after administration for each animal, and ejection fraction (EF) and fractional shortening (FS) were obtained by the software.
After four weeks of administration, tissues including the kidney, spleen, lung, heart, and liver were harvested from each animal. After washing with PBS, tissues were cut into suitable pieces: kidney (10
12 kidney tissue sections were randomly selected from the −80°C freezer after being frozen for 12 h and then were preprocessed in four conditions as described below. Each condition used three sections to detect.
For normal experimental conditions, three tissue sections were rewarmed at room temperature for 30 min. For a repeated rewarming stability investigation, three tissue sections were rewarmed at room temperature for 30 min, then refrozen at −80°C for 12 h, and rewarmed again. This procedure was repeated three times. The third three tissue sections were used in a long-term frozen stability investigation. Sections were stored in a −80°C freezer for 14 days and rewarmed at room temperature for 30 min. The final three tissue sections were used in a stability study of short-term placement at room temperature for 24 h.
Tissue sections were analysed on a TriVersa NanoMate (Advion Inc., Ithaca, NY, USA) with a LESA instrument coupled to a 5500 QTRAP MS (AB Sciex, Concord, Ontario). Three points on the preprocessed tissue section were selected for LESA-MS/MS analysis, and the average signal intensity of these points was taken as the detection result. Tissue sections were fixed on the LESA universal adapter plate and scanned by an Epson Perfection V370 scanner. The pictures were processed further by LESA Points software to generate sampling locations and automatic injection. A conductive pipette tip was picked up by the robotic arm of the TriVersa NanoMate to aspirate 1.7
ESI flow rate was estimated to be 400–500 nL/min. A spray voltage of 1.7 kV and a gas pressure of 0.7 psi were applied in all experiments. Multiple reaction monitoring (MRM) in negative ion mode was used for transitions at the following
Distribution analysis was performed in the kidney, spleen, lung, heart (ischemic marginal zone of the myocardium), and liver. Three sections of each tissue from each animal were randomly selected for the detection process. The detection process was consistent with the stability study. The experiment was repeated in three animals in each group, and the average signal intensity was taken as the final detection result.
Data were collected using Analyst 1.6.2 software (AB Sciex), and statistical analysis was performed with SPSS version 20.0. All data are presented as the mean ± standard deviation. Statistical analysis was carried out on three or more groups using one-way analysis of variance and Dunnett’s test. Statistical analysis of data from repeats was performed by repeated-measures analysis. Values of
Four weeks after surgery, coronary angiography showed that the rate of coronary artery stenosis in eight miniature pigs was 100% (see Figure
Four weeks after surgery, the EF and FS of pigs in the model-blank group and the model-dose group were significantly lower than those in the normal-dose group (
Echocardiography results at different time points between groups. (a) Ejection fractions at different time points between groups. (b) Fractional shortening at different time points between groups. EF: ejection fractions; FS: fractional shortening; Normal-dose: normal-dose group; Model-blank: model-blank group; Model-dose: model-dose group.
A sphericity test was performed before analysing the correlation between repeated data. There was no correlation between repeated data in this experiment (
Comparison of the signal intensities for the four compounds under different storage conditions. Normal: normal experimental conditions; Repeat: after repeated rewarming for three times; Long-term: after long-term stored in a −80°C freezer; Short-term: after short-term placement at room temperature.
The CAA signal intensities in samples from the heart, spleen, kidney, lung, and liver were analysed. Both the model-dose group and normal-dose group showed significantly higher CAA signal intensities than the model-blank group in the heart, spleen, kidney, and lung (
Signal intensities of four salvianolic acids in different tissues and groups. (a) Signal intensities of caffeic acid in different tissues and groups. (b) Signal intensities of Danshensu in different tissues and groups. (c) Signal intensities of rosmarinic acid in different tissues and groups. (d) Signal intensities of salvianolic acid A in different tissues and groups. Model-blank: model-blank group; Normal-dose: normal-dose group; Model-dose: model-dose group.
Kidney, lung, liver, and heart samples from the model-dose group and kidney, liver, spleen samples from the normal-dose group showed higher DSS signal intensities than those from the model-blank group (
Compared with the model-blank group, both the normal-dose group and model-dose group had higher RA signal intensities in the liver, spleen, kidney, heart, and lung (
Compared with the model-blank group, liver, kidney, and spleen samples from the normal-dose group and heart, kidney, and spleen samples from the model-dose group showed higher SAA signal intensities (
The Danshen decoction used in the present study has been reported to commonly use in promoting blood circulation and removing blood stasis [
Studies have shown that DSS could greatly improve blood rheology, lower lipid levels, inhibit lipid peroxidation, and have antitumour and other pharmacological activities [
In the present study, after four weeks of administration of Danshen decoction, EF and FS were improved obviously in the model-dose group, which confirmed that Danshen decoction could improve the ischemic condition in a chronic myocardial ischemia model.
In addition, an HPLC-MS/MS method had been established in our previous study to determine the concentration of sodium Danshensu, protocatechualdehyde, caffeic acid, rosmarinic acid, and salvianolic acid A in rat plasma [
LESA-MS/MS was applied to analyse the distributions of four salvianolic acids. A stability investigation showed that DSS, CAA, and SAA were stable under four experimental conditions, but RA had a poor stability when placed at room temperature for 24 h. However, the samples were usually tested within 2 h of removal from the −80°C freezer. This short time out of the freezer might have little effect on the samples, and the results would be reliable for the distribution study.
Subsequently, distribution analysis showed that the signal intensities of DSS in the liver and kidney and SAA in the heart were higher in the model-dose group than in the normal-dose group. The reason for this might be that DSS has strong water solubility, and the liver and kidney are the main metabolic organs for DSS. It is reported that there is obvious inflammatory cell infiltration and an abnormal increase of vascular permeability in the ischemic zone of the myocardium [
Recently, several studies have analysed salvianolic acids using diverse analytical methods acids including liquid chromatography with ultraviolet detection (LC-UV), online solid-phase extraction coupled in series to liquid chromatography-tandem mass spectrometry (SPE-LC-MS), high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-MS [
As a fully automated, chip-based multichannel MS method, LESA-MS/MS combines microliquid extraction from a solid surface and nano-ESI analysis to obtain information from tissue sections of interest [
Danshen decoction has the effect of improving the ischemic condition in a chronic myocardial ischemia model and is basically distributed in the heart, liver, and kidney, which is worth further clinical study. A LESA-MS/MS method was applied for the simultaneous determination of four salvianolic acids (CAA, DSS, RA, and SAA) in animal tissues. The method has characteristics in simple pretreatment of samples, sensitivity, and stability, which showed to be worthy of further application on drug distribution research studies.
Traditional Chinese medicine
Liquid extraction surface analysis coupled with tandem mass spectrometry
Danshensu
Caffeic acid
Rosmarinic acid
Salvianolic acid A
Ejection fraction
Fractional shortening
High-performance liquid chromatography
Optimal cutting temperature
Multiple reaction monitoring
Liquid chromatography with ultraviolet detection
Solid-phase extraction coupled in series to liquid chromatography-tandem mass spectrometry
High-performance liquid chromatography-diode array detection
High-speed countercurrent chromatography
Mass spectrometry imaging
Chloroquine
Liquid chromatography coupled with mass spectrometry.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Qi Qiu and Yang Lin conceived and designed the experiments. Qi Qiu, Jinglin Cao, Yu Mu, and Yunnan Zhang performed the experiments. Jinglin Cao and Jing Li analysed the data. Xiujin Shi checked the results. Jinglin Cao, Yu Mu, Qi Qiu, and Yang Lin revised the manuscript. Qi Qiu, Jinglin Cao, Yu Mu, and Yang Lin are joint first authors and contributed equally to this article.
This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Thirteenth Five-year Plan Period (grant number 2017ZX09304017), the National Natural Science Foundation of China (grant numbers 81403200 and 81541169), and Beijing Hospitals Authority “Qing Miao” Program (grant number QML20150603). The authors are grateful to Yi Tian, from the Department of Nuclear Medicine of Beijing Anzhen Hospital, for establishing animal models in the present study, Mei-Juan Yang, from Beijing University of Chinese Medicine, for preparing the frozen tissue section, and Gabrielle David, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
Supplemental Figure 1: the linear graph of salvianolic acids. Supplemental Figure 2: HPLC chromatogram of caffeic acid. Supplemental Figure 3: HPLC chromatogram of rosmarinic acid. Supplemental Figure 4: HPLC chromatogram of salvianolic acid A. Supplemental Figure 5: HPLC chromatogram of Danshensu. Supplemental Figure 6: coronary angiography results four weeks after surgery. Supplemental Figure 7: echocardiography results at different time points. (A) Before left anterior descending ligation. (B) Four weeks after left anterior descending ligation, before administration. (C) Eight weeks after left anterior descending ligation, four weeks after administration.