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. 2016 Mar 31;2016:9468087. doi: 10.1155/2016/9468087

Identification of “Multiple Components-Multiple Targets-Multiple Pathways” Associated with Naoxintong Capsule in the Treatment of Heart Diseases Using UPLC/Q-TOF-MS and Network Pharmacology

Xianghui Ma 1, Bin Lv 1, Pan Li 1, Xiaoqing Jiang 1, Qian Zhou 1, Xiaoying Wang 1,2,*, Xiumei Gao 1
PMCID: PMC4830706  PMID: 27123036

Abstract

Naoxintong capsule (NXT) is a commercial medicinal product approved by the China Food and Drug Administration which is used in the treatment of stroke and coronary heart disease. However, the research on the composition and mechanism of NXT is still lacking. Our research aimed to identify the absorbable components, potential targets, and associated pathways of NXT with network pharmacology method. We explored the chemical compositions of NXT based on UPLC/Q-TOF-MS. Then, we used the five principles of drug absorption to identify absorbable ingredients. The databases of PharmMapper, Universal Protein, and the Molecule Annotation System were used to predict the main targets and related pathways. By the five principles of drug absorption as a judgment rule, we identified 63 compositions that could be absorbed in the blood in all 81 chemical compositions. Based on the constructed networks by the significant regulated 123 targets and 77 pathways, the main components that mediated the efficacy of NXT were organic acids, saponins, and tanshinones. Radix Astragali was the critical herbal medicine in NXT, which contained more active components than other herbs and regulated more targets and pathways. Our results showed that NXT had a therapeutic effect on heart diseases through the pattern “multiple components-multiple targets-multiple pathways.”

1. Introduction

Naoxintong capsule (NXT) is a commercial medicinal product approved by the China Food and Drug Administration which is widely used in the treatment of stroke and coronary heart disease. NXT contains 16 Chinese herbal medicines (Table 1). NXT exerts significant therapeutic effects and has high safety for stroke recovery in the clinical setting [1]. Recent studies showed that NXT could reduce the infarct size of acute myocardial infarction (AMI) patients by improving vascular endothelial function [2]. Long-term administration of NXT was also reported to alleviate inflammation, reduce the recurrence of angina pectoris, and decrease the incidence of ACS attack in borderline lesion coronary heart disease patients [3]. Some studies investigated the mechanisms of NXT in vitro or in vivo. NXT was reported to protect against atherosclerosis through its lipid-lowering activity [4] and to reduce the expression of iNOS mRNA and the NO level in the vessel wall to benefit the treatment of atherosclerosis [5]. NXT also protected cardiomyoblasts against H2O2-induced oxidative injury [6]. Although some mechanisms of NXT have been reported, existing studies on unilateral factors and single targets could not demonstrate the complex mechanisms of NXT, a herbal prescription with 16 medicines which is prescribed for the treatment of complex diseases like cardiovascular and cerebrovascular diseases.

Table 1.

Sixteen Chinese traditional medical herbs of NXT.

Abbreviation Medicinal herbs Original plants Content (g)∗
RA Radix Astragali Astragalus membranaceus (Fisch.) Beg. var. mongholicus (Bge.) Hsiao or A. membranaceus (Fisch.) Bge. 66
RPR Radix Paeoniae Rubra Paeonia lactiflora Pall. or P. veitchii Lynch 27
RSM Radix Salviae Miltiorrhizae Salvia miltiorrhiza Bge. 27
RAS Radix Angelicae Sinensis Angelica sinensis (Oliv) Diels. 27
RCX Rhizoma Chuanxiong Ligusticum chuanxiong Hort. 27
SP Semen Persicae Prunus persica (L.) Batsch or Prunusdavidiana (Carr.) Franch. 27
FC Flos Carthami Carthamus tinctorius L. 13
FK Frankincense Boswellia carterii Birdw. 13
MRH Myrrha Commiphora myrrha Engl. 13
CS Caulis Spatholobi Spatholobus suberectus Dunn 20
RAB Radix Achyranthis Bidentatae Achyranthes bidentata Bl. or Cyathula officinalis Kuan 27
RC Ramulus Cinnamomi Cinnamomum cassia Presl 20
RM Ramulus Mori Morus alba L. 27
PT Pheretima Pheretima aspergillum (E. Perrier) or Pheretima vulgaris Chen. or Pheretima guillelmi (Michaelsen) or Pheretima pectinifera Michaelsen 27
SCP Scorpio Buthus martensii Karsch 13
HRD Hirudo Whitmania pigra Whitman or Hirudo nipponica Whitman or Whitmania acranulata Whitman 27
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∗ Note. The content of 16 Chinese traditional medical herbs of NXT came from Chinese Pharmacopoeia 2015.

With the prominence of network pharmacology in system biology, this distinct and novel approach to the study of complicated analytical systems is becoming more widely known and more frequently used in the field of drug research. The functions of network pharmacology include uncovering the functions of traditional Chinese medicines (TCMs), providing deeper insights into and scientific evidence for TCMs, and identifying TCMs as scientifically proven. Here, we attempt to explore the mechanism of NXT using this method.

In the current study, based on the use of UPLC/Q-TOF-MS to investigate the involved components, we aimed to analyse the absorbable components of NXT, to identify potential targets and associated pathways using the network pharmacology method, and to systematically discuss the mechanism of NXT in the treatment of heart diseases.

2. Material and Methods

2.1. Prediction of Components

2.1.1. Sample Preparation

NXT was obtained from HezeBuchang Pharmaceutical Co., Ltd. (Heze, China). Deionized water was prepared from aqua distillate using a Milli-Q system (Millipore, Bedford, MA, USA). Analytical grade methanol was purchased from Merck (Darmstadt, Germany). We dissolved 1 g of NXT powder in 10 mL of 75% analytical grade methanol and subjected the mixture to ultrasonic extraction for 30 min. We then brought the solution to room temperature and obtained the supernatant as a capture reagent. The sample was filtered using a 0.22 μm microporous membrane before UPLC analysis.

2.1.2. UPLC/Q-TOF-MS

We used a Waters Acquity UPLC System (Waters Co., USA) furnished with a photodiode array detector for the analysis. The sample was diluted on a Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). UV detection was achieved at 190–400 nm. The system was controlled using the MassLynx version 4.1 software (Waters Co.). The gradient duration program for A (UPLC-grade acetonitrile) and B (water with 0.1% formic acid) was performed as follows: 2% A from 0 min to 3 min, 10% to 50% A from 3 min to 12 min, 50% to 63% A from 12 min to 18 min, 63% to 83% A from 18 min to 21 min, 83% to 84% A from 21 min to 22 min, 84% to 87% A from 22 min to 26 min, 87% to 90% A from 26 min to 28 min, 90% to 95% A from 28 min to 31 min, 95% to 100% A from 31 to 33 min, 100% to 100% A from 33 to 35 min, and 100% to 2% A from 35 min to 37 min. The flow rate was maintained at 0.4 mL/min, and the column temperature was maintained at 30°C.

The components of NXT were identified using a Waters Q-TOF Premier with an electrospray ionization (ESI) system (Waters MS Technologies, Manchester, UK). The ESI-MS spectra were acquired at both negative and positive ion voltages. The capillary voltage was set to 2.5 kV for the negative mode and to 3.0 kV for the positive mode. The sample cone voltage was set to 30 V, and the source temperature was 110°C. High-purity nitrogen was used as the nebulization and auxiliary gas. The nebulization gas was set to 600 L/h, the cone gas was set to 50 L/h, and the desolation temperature was 350°C. The Q-TOF Premier acquisition rate was 0.1 s, and there was a 0.02 s interscan delay. Argon, which was used as the collision gas, was maintained at a pressure of 5.3 × 10−5 Torr. The instrument was operated with the first resolving quadruple in a wide pass mode (100 Da–1500 Da). Leucineen kephalinamide acetate was used as the lock mass ([M − H]− = 553.2775, [M + H]+ = 555.2931).

2.2. Calculation and Prediction of Absorbable Components

First, we determined the structural formulas of the chemical components that were identified in compound NXT from the Chemical Book website and used the Chemdraw software to draw these formulas. Then, we imported these structural formulas into the Online SMILES Translator and Structure File Generator (http://cactus.nci.nih.gov/translate/) to obtain the smiles format. Finally, we input the smiles format of the chemical components into the Molispiration Smiles website (http://www.molinspiration.com/cgi-bin/properties) to calculate the prediction parameters of drug absorption. According to the five principles of drug absorption, if a component was subject to the following provisions of the corresponding parameters, it could be identified as an absorbable component: hydrogen bond donor (the number of hydrogen atoms attached to the O and N) nOHNH ≤ 5; relative molecular mass MW ≤ 500; fat water partition coefficient miLogP ≤ 5; and hydrogen bond acceptor (the number of O and N) nON ≤ 10.

2.3. Prediction and Screening of Targets

Using the software of Chembio3D Ultru12.0, we transformed the structure of the absorbed components into the sdf structure format. Then, to predict the possible targets, we imported the components into the public network server of the target database of the efficacy group PharmaMapper website (http://59.78.96.61/pharmmapper/) to perform reverse docking. We selected the top 10 targets for subsequent study.

2.4. Prediction and Screening of Pathways

We imported the obtained targets into the Bio database (http://bioinfo.capitalbio.com/mas3/) and then screened for pathways that met the criterion of P < 0.01.

2.5. Construction of Network

According to the screening pathways with their corresponding targets and components, we created a component-target-pathway illustration using Cytoscape. Then, according to the main selected targets, we drew a target-composition diagram.

3. Results

3.1. UPLC/Q-TOF-MS Analysis

We analysed the chemical components of NXT using ultraperformance liquid chromatography combined with quadrupole time-of-flight mass spectrometry. Because different chemical components had better responses in different modes, MS data were obtained in both positive ion mode (Figure 1(a)) and negative ion mode (Figure 1(b)). MS data in (+/−) ESI modes and the identification results for the constituents in NXT were presented in Table 2. In all 16 herbs from NXT, no related component in Myrrha and Hirudo was found.

Figure 1.

Figure 1

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UPLC/Q-TOF-MS analysis of NXT. (a) Chromatograms of NXT in positive ion mode. (b) Chromatograms of NXT in negative ion mode.

Table 2.

MS data in (+/−) ESI modes and the identification results for the constituents in NXT.

Peak number RT (min) Identification Mode MS (m/z) Composition Herbal source
1 0.647 Arginine Pos/Neg 174.2024 C6H14N4O2 PT
2 0.702 Valine Pos 117.1478 C5H11NO2 PT
3 0.721 Proline Pos 115.1331 C5H9NO2 PT
4 0.776 Malic acid Neg 134.0911 C4H6O5 RA
5 1.053 Citric acid Neg 192.1286 C6H8O7 RA
6 1.201 D-5-oxoproline Neg 129.1174 C5H7NO3 RAS
7 1.201 L-5-oxoproline Neg 129.1174 C5H7NO3 RAS
8 1.275 Succinic acid Neg 118.0910 C4H6O4 RAS, RAB, PT
9 1.294 ρ-Coumaric acid Pos 164.1601 C9H8O3 RAS
10 1.310 o-Phthalic acid Pos 166.1294 C8H6O4 RAS
11 1.312 Adenosine Pos 267.2403 C10H13N5O4 RAS, PT, RCX
12 1.331 Leucine Pos 131.1688 C6H13NO2 PT
13 1.460 Isoleucine Pos 131.1688 C6H13NO2 RAB
14 1.589 Gallic acida Neg 170.1207 C7H6O5 RPR
15 2.199 Phenylalanine Pos 165.1874 C9H11NO2 FC
16 2.459 Danshensu Neg 198.1701 C9H10O5 RSM
17 2.606 Palmitic acid Neg 256.3380 C16H32O2 RAS, FC, RA, SCP
18 3.438 Senkyunolide B Neg 204.2374 C12H12O3 RCX
19 3.456 Senkyunolide C Neg 204.2374 C12H12O3 RCX
20 3.600 Protocatechuic aldehyde Neg 138.1185 C7H6O3 RSM, RC
21 3.974 Mulberroside Aa Neg 568.5277 C26H32O14 RM
22 4.122 Gallicin Neg 184.1453 C8H8O5 RPR
23 4.230 Hydroxysafflor yellow A Pos/Neg 612.5364 C27H32O16 FC
24 4.232 7-Hydroxycoumarin Pos 162.1457 C9H6O3 RM
25 4.565 Vanillic acid Neg 168.1459 C8H8O4 RCX, RPR
26 4.694 Benzoic acid Neg 122.1209 C7H6O2 RPR
27 4.935 Epicatechin Neg 290.2674 C15H14O6 CS
28 5.157 Catechin Neg 290.2674 C15H14O6 RPR
29 5.212 Albiflorin Pos 480.4653 C23H28O11 RPR
30 5.730 Quercetin-7-O-glucoside Neg 464.3754 C21H20O12 FC
31 5.952 Rutin Neg 610.5203 C27H30O16 RA
32 5.970 Calycosina Neg 284.2679 C16H12O5 RA
33 5.988 Calycosin-7-O-glucoside Pos 446.4075 C22H22O10 RA
34 5.989 Ferulic acida Neg 194.1815 C10H10O4 RA, RCX, RAS, RAB
35 6.321 Paeoniflorina Pos 480.466 C23H28O11 RPR
36 6.358 Pentagalloylglucosea Neg 940.68 C41H32O26 RPR
37 6.413 Kaempferol-3-O-rutinosidea Pos/Neg 594.5179 C27H30O15 FC
38 6.654 3,5-Di-O-caffeoylquinic acida Pos/Neg 516.4573 C25H24O12 CS
39 6.987 Dicaffeoylquinic acid Neg 516.1275 C25H24O12 RCX
40 7.042 Z-Butylidenephthalidea Neg 188.2259 C12H12O2 RCX
41 7.210 Salvianolic acid A Neg 494.4578 C26H22O10 RSM
42 7.449 4-Hydroxyl-3-butylphthalide Pos 206.2346 C12H14O3 RCX
43 7.540 Salvianolic acid B Neg 718.6220 C36H30O16 RSM
44 7.688 Ononin Pos 430.4107 C22H22O9 CS
45 7.763 Senkyunolide F Pos 206.1017 C12H14O3 RCX, RAS
46 7.855 Salvianolic acid E Neg 718.1512 C36H30O16 RSM
47 8.243 Biochanin A Pos/Neg 284.2689 C16H12O5 CS
48 8.262 (6aR,11aR)-3-Hydroxy-9,10-dimethoxy pterocarpan Pos 300.3107 C17H16O5 RA
49 8.594 N1-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine Pos 583.2703 C34H37N3O6 FC
50 8.740 Benzoylpaeoniflorin Pos 584.5723 C30H32O12 RPR
51 9.518 Pratensein Neg 300.0679 C16H12O6 RA
52 9.611 Hydroxyl calendic acid Neg 294.4342 C18H30O3 SP
53 9.648 Trans-oxyresveratrol Pos 244.2435 C14H12O4 RM
54 10.240 Formononetina Pos/Neg 268.2580 C16H12O4 RA
55 10.405 Astragaloside IV Neg 784.4633 C41H68O14 RA
56 10.590 Senkyunolide H Neg 220.2305 C12H12O4 RCX
57 10.978 Astragaloside II Neg 826.4701 C43H70O15 RA
58 11.311 Soyasaponin I Neg 942.5145 C48H78O18 RA
59 11.422 Methyl tanshinonate Pos 338.1087 C20H18O5 RSM
60 11.588 Carnosic acid Neg 332.4311 C20H28O4 RSM
61 11.644 Kaempferol-3-O-glucoside Neg 448.3752 C21H20O11 FC
62 11.699 Hydroxytanshinone IIA Pos 310.1199 C19H18O4 RSM
63 11.792 3-Butylidene-7-hydroxyphalide Neg 204.2331 C12H12O3 RCX
64 11.921 Tanshinone II-B Pos 310.1187 C19H18O4 RSM
65 12.198 Senkyunolide A Pos 192.2516 C12H16O2 RCX
66 12.975 Salvianolic acid F Neg 314.0735 C17H14O6 RSM
67 13.196 Kumatakenin Neg 314.3359 C17H14O6 RA
68 13.233 3-n-Butylphthalide Pos 190.2356 C12H14O2 RCX
69 13.474 (Z)-ligustilidea Pos 190.2109 C12H14O2 RAS
70 13.483 (E)-ligustilidea Pos 190.2109 C12H14O2 RAS
71 13.917 Trijuganone B Pos 280.1107 C18H16O3 RSM
72 16.098 Cryptotanshinonea Neg 296.3642 C19H20O3 RSM
73 16.394 Senkyunolide M Pos 278.1565 C16H22O4 RCX
74 17.503 O-Phthalic anhydride Pos 148.0207 C8H4O3 FC
75 17.614 Chlorogenic acida Neg 354.3120 C16H18O9 CS
76 18.076 Tanshinone IIA Pos 294.3430 C19H18O3 RSM
77 18.205 Angelicide Pos 380.1917 C24H28O4 RCX
78 20.460 Carthamidin Pos 288.2575 C15H12O6 FC
79 22.078 Linoleic acid Neg 280.2387 C18H32O2 SP
80 22.659 Acetyl-11-keto-β-boswellic acid Pos/Neg 512.7458 C32H48O5 FK
81 22.881 Oleanolic acid Neg 456.3652 C30H48O3 RSM
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“a” refers to the component has been verified by standard substance.

3.2. Absorption Parameters of Components

Using a computer prediction method to calculate the identified compounds of NXT, we obtained absorption parameters that could determine whether the chemical compositions could be absorbed. Table 3 showed the specific absorption parameters of all of the components. The data indicated that there were a total of 63 chemical compositions (Figure 2) that met the five principles of drug absorption. As shown, 7 glycosides were identified. Although the relative molecular masses of those compounds were greater than 500, they could also be absorbed, because those compounds could be divided into two parts, including aglycones which mainly mediated drug efficacy and sugar chains in the body. So we could import these glycosides' aglycones into PharmMapper to obtain the relevant parameters. The results showed that both of these components were consistent with the five principles of drug absorption, so we considered that these 7 chemical compositions could be absorbed in the body.

Table 3.

Absorption parameters of the components.

Number Compounds MW nON nOHNH miLogP Results
1 Arginine 174.204 6 7 −3.632 ✕
2 Valine 117.15 3 3 −1.91 √
3 Proline 115.132 3 2 −1.723 √
4 Malic acid 134.087 5 3 −1.57 √
5 Citric acid 192.123 7 4 −1.983 √
6 D-5-oxoproline 129.115 4 2 −2.402 √
7 L-5-oxoproline 129.115 4 2 −2.402 √
8 Succinic acid 118.088 4 2 −0.655 √
9 ρ-Coumaric acid 164.160 3 2 1.43 √
10 o-Phthalic acid 166.132 4 2 1.034 √
11 Adenosine 267.245 9 5 −0.854 √
12 Leucine 131.175 3 3 −1.382 √
13 Isoleucine 131.175 3 3 −1.41 √
14 Gallic acida 170.120 5 4 0.589 √
15 Phenylalanine 165.192 3 3 −1.231 √
16 Danshensu 198.174 5 4 −0.251 √
17 Palmitic acid 256.43 2 1 7.059 ✕
18 Senkyunolide B 204.225 3 1 2.81 √
19 Senkyunolide C 204.225 3 1 2.574 √
20 Protocatechuic aldehyde 138.122 3 2 0.759 √
21 Mulberroside Aa 568.528 14 10 −0.852 √
22 Gallicin 184.147 5 3 0.848 √
23 Hydroxysafflor yellow A 612.54 16 12 −4.12 √
24 7-Hydroxycoumarin 162.144 3 1 1.511 √
25 Vanillic acid 168.148 4 2 1.187 √
26 Benzoic acid 122.123 2 1 1.848 √
27 Epicatechin 290.271 6 5 1.369 √
28 Catechin 290.271 6 5 1.369 √
29 Albiflorin 480.466 11 5 −1.636 ✕
30 Quercetin-7-O-glucoside 464.379 12 8 −0.104 ✕
31 Rutin 610.521 16 10 −1.063 √
32 Calycosina 284.267 5 2 2.377 √
33 Calycosin-7-O-glucoside 446.408 10 5 0.59 √
34 Ferulic acida 194.186 4 2 1.249 √
35 Paeoniflorina 480.466 11 5 0.044 ✕
36 Pentagalloylglucosea 940.681 26 15 2.761 √
37 Kaempferol-3-O-rutinosidea 594.522 15 9 −0.574 √
38 3,5-Di-O-caffeoylquinic acida 516.455 12 7 1.424 ✕
39 Dicaffeoylquinic acid 516.46 12 7 1.21 ✕
40 Z-Butylidenephthalidea 188.226 2 0 3.077 √
41 Salvianolic acid A 494.452 10 7 3.014 ✕
42 4-Hydroxyl-3-butylphthalide 206.241 3 1 3.42 √
43 Salvianolic acid B 718.620 16 9 1.615 ✕
44 Ononin 430.409 9 4 1.307 √
45 Senkyunolide F 206.24 3 1 1.72 √
46 Salvianolic acid E 718.62 16 10 2.83 ✕
47 Biochanin A 284.267 5 2 2.804 √
48 (6aR,11aR)-3-Hydroxy-9,10-dimethoxy pterocarpan 300.31 5 1 2.546 √
49 N1-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine 538.68 9 5 4.3 ✕
50 Benzoylpaeoniflorin 584.574 12 4 2.472 ✕
51 Pratensein 300.27 6 3 2.09 √
52 Hydroxyl calendic acid 294.435 3 2 4.93 √
53 Trans-Oxyresveratrol 244.246 4 4 2.723 √
54 Formononetina 268.268 4 1 3.095 √
55 Astragaloside IV 784.98 14 9 1.21 √
56 Senkyunolide H 220.224 4 2 2.314 √
57 Astragaloside II 827.02 15 8 1.91 √
58 Soyasaponin I 943.13 18 11 1.7 ✕
59 Methyl tanshinonate 338.36 5 0 0.93 √
60 Carnosic acid 332.440 4 3 4.603 √
61 Kaempferol-3-O-glucoside 448.380 11 7 0.125 ✕
62 Hydroxytanshinone IIA 310.35 4 1 3.24 √
63 3-Butylidene-7-hydroxyphthalide 204.225 3 1 2.81 √
64 Tanshinone II-B 310.35 4 1 2.97 √
65 Senkyunolide A 192.258 2 0 3.521 √
66 Salvianolic acid F 314.29 6 5 2.33 √
67 Kumatakenin 314.29 6 2 2.98 √
68 3-n-Butylphthalide 190.242 2 0 3.483 √
69 (Z)-Ligustilidea 190.242 2 0 2.927 √
70 (E)-Ligustilidea 190.242 2 0 2.927 √
71 Trijuganone B 280.32 3 1 3.9 √
72 Cryptotanshinonea 296.366 3 0 3.83 √
73 Senkyunolide M 278.35 4 1 2.55 √
74 O-Phthalic anhydride 148.12 3 0 0.93 √
75 Chlorogenic acida 354.311 9 6 −0.453 ✕
76 Tanshinone IIA 294.350 3 0 4.158 √
77 Angelicide 380.48 4 0 5.73 ✕
78 Carthamidin 288.255 6 4 1.649 √
79 Linoleic acid 280.45 2 1 6.86 ✕
80 Acetyl-11-keto-β-boswellic acid 512.73 5 1 6.39 ✕
81 Oleanolic acid 456.71 3 2 6.72 ✕
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Note. “√” means that component could be absorbed; “✕” means that component could not be absorbed.

“a” refers to the component has been verified by standard substance.

Figure 2.

Figure 2

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Structures of 63 absorbable components.

3.3. Potential Targets and Pathways

By importing 63 chemical compositions that were predicted to be absorbable into the PharmMapper database for directional docking, we obtained a total of 123 targets. We then imported these targets into the Molecule Annotation System and obtained 77 pathways regulated by NXT with highly significant differences, from which we chose the top 40 pathways that met the criterion of P < 0.01 (Table 4). A total of 34 targets were related to these top 40 pathways, and HRAS, MAP2K1, and MAPK14 were associated with most of these pathways, so we considered these factors to be the main targets. As shown in Table 4, NFAT and hypertrophy of the heart (transcription in the broken heart) ranked first among these pathways.

Table 4.

Top 40 Biocarta pathways regulated by NXT (P < 0.01).

Rank Pathway Count P-value q-value Gene
1 NFAT and hypertrophy of the heart (transcription in the broken heart) 6 5.75E − 10 3.58E − 09 HRAS; GSK3B; MAPK14; FKBP1A; F2; MAP2K1
2 Phosphoinositides and their downstream targets 5 1.39E − 09 8.47E − 09 GSK3B; PDPK1; BTK; RAB5A; EEA1
3 Intrinsic Prothrombin Activation Pathway 4 8.50E − 08 2.82E − 07 F10; FGG; F11; F2
4 Bioactive Peptide Induced Signaling Pathway 4 4.08E − 07 9.30E − 07 HRAS; MAPK14; F2; MAP2K1
5 BCR Signaling Pathway 4 4.88E − 07 1.08E − 06 HRAS; MAPK14; BTK; MAP2K1
6 Estrogen-responsive protein Efp controls cell cycle and breast tumors growth 3 6.40E − 07 1.34E − 06 CDK2; ESR1; CDK6
7 Nuclear receptors in lipid metabolism and toxicity 4 8.02E − 07 1.58E − 06 CYP2C9; VDR; NR1H3; PPARA
8 Map kinase inactivation of SMRT corepressor 3 1.53E − 06 2.48E − 06 THRB; MAPK14; MAP2K1
9 MAP Kinase Signaling Pathway 5 2.09E − 06 3.05E − 06 HRAS; MAPK10; MAPK14; TGFBR1; MAP2K1
10 Extrinsic Prothrombin Activation Pathway 3 2.99E − 06 4.05E − 06 F10; FGG; F2
11 amiPathway 3 5.17E − 06 6.40E − 06 F10; FGG; F2
12 Roles of β-arrestin-dependent recruitment of Src kinases in GPCR signaling 3 6.57E − 06 7.86E − 06 HRAS; HCK; MAP2K1
13 Aspirin blocks signaling pathway involved in platelet activation 3 8.19E − 06 9.49E − 06 HRAS; F2; MAP2K1
14 Insulin Signaling Pathway 3 2.03E − 05 2.03E − 05 HRAS; INSR; MAP2K1
15 IL-2 Signaling Pathway 3 2.37E − 05 2.29E − 05 HRAS; MAP2K1; LCK
16 Role of ERBB2 in signal transduction and oncology 3 2.37E − 05 2.29E − 05 HRAS; ESR1; MAP2K1
17 Links between Pyk2 and MAP kinases 3 2.74E − 05 2.45E − 05 HRAS; MAPK14; MAP2K1
18 NF-κB activation by nontypeable Hemophilus influenzae 3 2.74E − 05 2.45E − 05 MAPK14; TGFBR1; NR3C1
19 Influence of Ras and Rho proteins on G1 to S transition 3 3.14E − 05 2.82E − 05 HRAS; CDK2; CDK6
20 fMLP induced chemokine gene expression in HMC-1 cells 3 3.14E − 05 2.82E − 05 HRAS; MAPK14; MAP2K1
21 Growth Hormone Signaling Pathway 3 3.14E − 05 2.82E − 05 HRAS; INSR; MAP2K1
22 Cell cycle: G1/S checkpoint 3 4.06E − 05 3.37E − 05 CDK2; GSK3B; CDK6
23 Fc epsilon receptor I signaling in mast cells 3 4.58E − 05 3.70E − 05 HRAS; BTK; MAP2K1
24 Signaling of hepatocyte growth factor receptor 3 6.40E − 05 4.89E − 05 HRAS; MET; MAP2K1
25 p38 MAPK signaling pathway 3 7.85E − 05 5.76E − 05 HRAS; MAPK14; TGFBR1
26 Keratinocyte differentiation 3 1.13E − 04 7.81E − 05 HRAS; MAPK14; MAP2K1
27 T cell receptor signaling pathway 3 1.13E − 04 7.81E − 05 HRAS; MAP2K1; LCK
28 TSP-1 induced apoptosis in microvascular endothelial cell 2 1.46E − 04 9.59E − 05 CASP3; MAPK14
29 The role of FYVE-finger proteins in vesicle transport 2 1.46E − 04 9.59E − 05 RAB5A; EEA1
30 Mechanism of gene regulation by peroxisome proliferators via PPARa(alpha) 3 1.82E − 04 1.15E − 04 HSP90AA1; NR1H3; PPARA
31 Visceral fat deposits and the metabolic syndrome 2 1.95E − 04 1.21E − 04 HSD11B1; NR3C1
32 RB tumor suppressor/checkpoint signaling in response to DNA damage 2 2.50E − 04 1.44E − 04 CDK2; CHEK1
33 Platelet Amyloid Precursor Protein Pathway 2 2.50E − 04 1.44E − 04 F11; F2
34 Fibrinolysis Pathway 2 3.12E − 04 1.77E − 04 FGG; F2
35 Corticosteroids and cardioprotection 2 3.12E − 04 1.77E − 04 HSP90AA1; NR3C1
36 Phosphorylation of MEK1 by cdk5/p35 downregulates the MAP kinase pathway 2 3.81E − 04 2.09E − 04 HRAS; MAP2K1
37 VEGF, hypoxia, and angiogenesis 2 5.38E − 04 2.79E − 04 HRAS; KDR
38 How progesterone initiates oocyte membrane 2 6.27E − 04 3.17E − 04 HRAS; PGR
39 IL-3 Signaling Pathway 2 6.27E − 04 3.17E − 04 HRAS; MAP2K1
40 Sprouty regulation of tyrosine kinase signals 2 6.27E − 04 3.17E − 04 HRAS; MAP2K1
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In Table 5, these top 40 pathways were classified into 5 categories, which included pathways associated with heart diseases and blood vessels, metabolism, cell cycle (with proliferation and apoptosis), immunity, and other pathways. By classifying these pathways, we accessed and marked the corresponding medicinal materials of NXT (Table 5). In the pathways associated with heart diseases and blood vessels, RCX, RSM, and FC were the most important. In the regulation of metabolism, RA, RSM, and RCX showed diametrical effect. All the herbs except Semen Persicae (SP) were related metabolism pathways due to the current research. RA, RSM, RCX, and FC could regulate the pathways about cell cycle, proliferation, and apoptosis. Some other important pathways were also affected by some herbs like RA, RSM, and RCX, for example, Insulin Signaling Pathway and p38 MAPK Signaling Pathway.

Table 5.

The herbs of NXT involved in the top 40 pathways.

Category Pathway NXT RA RPR RSM RAS RCX SP FC CS RAB RC RM PT
Pathway associated with heart diseases and blood vessels NFAT and hypertrophy of the heart (transcription in the broken heart) 1 1 1 1 1 1 0 1 1 1 1 1 1
Intrinsic Prothrombin Activation Pathway 1 0 1 1 1 1 0 1 0 0 0 1 0
Extrinsic Prothrombin Activation Pathway 1 0 1 1 1 1 0 1 0 0 0 0 0
Aspirin blocks signaling pathway involved in platelet activation 1 1 1 1 1 1 0 1 1 1 1 1 1
TSP-1 induced apoptosis in microvascular endothelial cell 1 1 1 1 1 1 0 1 0 0 0 0 0
Platelet Amyloid Precursor Protein Pathway 1 0 0 0 0 1 0 1 0 0 0 1 0
Fibrinolysis Pathway 1 0 1 1 0 1 0 1 0 0 1 0 0
Corticosteroids and cardioprotection 1 1 0 1 0 1 0 0 0 0 0 1 0
VEGF, hypoxia, and angiogenesis 1 1 1 1 1 1 1 1 1 1 1 1 1
Pathway associated with metabolism Nuclear receptors in lipid metabolism and toxicity 1 1 1 1 0 1 1 1 0 1 0 1 0
Growth Hormone Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
Visceral fat deposits and the metabolic syndrome 1 1 0 1 1 1 0 0 0 0 0 0 0
Pathway associated with immunity BCR Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
IL-2 Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
fMLP induced chemokine gene expression in HMC-1 cells 1 1 1 1 1 1 0 1 1 1 1 1 1
T Cell Receptor Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
Pathway associated with cell cycle, proliferation, and apoptosis Phosphoinositides and their downstream targets 1 0 0 1 0 1 0 1 1 0 0 0 0
Estrogen-responsive protein Efp controls cell cycle and breast tumors growth 1 1 1 1 1 1 0 0 1 0 0 0 0
Map kinase inactivation of SMRT corepressor 1 1 1 1 1 1 0 1 0 0 0 0 0
MAP Kinase Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
Roles of β-arrestin-dependent recruitment of Src kinases in GPCR signaling 1 1 1 1 1 1 0 1 1 1 1 1 1
Role of ERBB2 in signal transduction and oncology 1 1 1 1 1 1 0 1 1 1 1 1 1
Links between Pyk2 and MAP kinases 1 1 1 1 1 1 0 1 1 1 1 1 1
NF-κB activation by nontypeable Hemophilus influenzae 1 1 0 1 1 1 0 1 0 0 0 0 0
Influence of Ras and Rho proteins on G1 to S transition 1 1 1 1 1 1 0 1 1 1 1 1 1
Cell cycle: G1/S checkpoint 1 1 1 1 1 1 0 0 1 0 0 0 0
Fc epsilon receptor I signaling in mast cells 1 1 1 1 1 1 0 1 1 1 1 1 1
Signaling of hepatocyte growth factor receptor 1 1 1 1 1 1 0 1 1 1 1 1 1
Keratinocyte differentiation 1 1 1 1 1 1 0 1 1 1 1 1 1
RB tumor Suppressor/checkpoint signaling in response to DNA damage 1 1 1 1 1 1 0 0 1 0 0 0 0
IL-3 Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
Sprouty regulation of tyrosine kinase signals 1 1 1 1 1 1 0 1 1 1 1 1 1
Other pathways Bioactive Peptide Induced Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
amiPathway 1 0 1 1 1 1 0 1 0 0 0 0 0
Insulin Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
p38 MAPK Signaling Pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
The role of FYVE-finger proteins in vesicle transport 1 0 0 0 0 0 0 1 0 0 0 0 0
Mechanism of gene regulation by peroxisome proliferators via PPARα 1 1 0 1 0 1 0 0 0 0 0 1 0
Phosphorylation of MEK1 by cdk5/p35 downregulates the MAP kinase pathway 1 1 1 1 1 1 0 1 1 1 1 1 1
How progesterone initiates oocyte membrane 1 1 1 1 1 1 1 1 1 1 1 1 1
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Note. “1” means that the Chinese herbal medicine acts on the pathway while “0” means it does not. The pathways in each category are sorted by the significant differences in P value.

3.4. Pharmacology Network of NXT

Using the Cytoscape software, we constructed a pharmacology network of NXT (Figure 3), which showed us the relationships of the top 40 pathways, targets, and chemical components. We obtained preliminary understanding of the mechanism of NXT through this network.

Figure 3.

Figure 3

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Pharmacology network of the “components-targets-pathways” regulated by NXT.

In this research, we found three major targets of NXT: HRAS, MAP2K1, and MAPK14, which were involved in most regulated pathways. By Figure 4, based on illustration of the main targets with their corresponding compounds, we found the most effective ingredients of NXT were organic acids, saponins, and tanshinones. The main sources of organic acids were RA, RCX, RAS, and RAB. The saponins were mainly derived from RA. Meanwhile, tanshinones were mainly concentrated in RSM.

Figure 4.

Figure 4

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Network of major targets of NXT with corresponding compounds.

4. Discussion

The burden of cardiovascular and circulatory disease is becoming more and more serious, with cerebrovascular disease (CBD) and ischemic heart disease being the most serious [7]. As the causes of cardiovascular disease (CVD) and CBD are complicated, the symptoms of these diseases are also very diverse. NXT is commonly used during clinical treatment of CVD and CBD, and the effect of this drug is remarkable. Although complex traditional Chinese medicine has great significance for the treatment of complex diseases, some questions such as the material basis and the potential mechanisms remain unanswered.

Our study successfully predicted absorbable chemical compositions of NXT. These constituents primarily included ferulic acid, succinic acid, astragaloside IV, and tanshinone IIA. Ferulic acid, which is derived primarily from RA, RCX, RAS, and RAB, is reported to act as an angiogenic agent that augments angiogenesis, which is critical in ischemic diseases, such as myocardial infarction and stroke [8]. Succinic acid has been demonstrated to activate Akt phosphorylation to inhibit apoptosis and necrosis caused by cardiomyocyte hypoxia/reoxygenation [9]. Previous studies demonstrated that astragaloside IV could protect the heart through NO-dependent mechanism [10]. NO has been confirmed to prevent the mitochondrial permeability transition pore from opening [11]. During early reperfusion, it can prevent the heart from reperfusion injury by inhibiting the opening of the mitochondrial permeability transition pore [12]. Tanshinone IIA also has cardioprotective effects, such as protection of cardiomyocytes from oxidative stress-triggered damage [13]. These reports were consistent with our results.

In addition to active ingredients, we also successfully predicted drug targets of NXT. The major targets were HRAS, MAP2K1, and MAPK14. The HRAS gene encodes the GTPase HRas, which is an enzyme known as transforming protein p21 [14]. With the ability to increase the effects of growth factor, HRas plays an important role in regulating the growth, differentiation, and death of endothelial cells [15]. The MAP2K1 gene encodes an enzyme named dual specificity mitogen-activated protein kinase kinase 1, and MAPK14 encodes p38-α. Both of these factors are closely related to inflammation and p38-α is also associated with cardiac hypertrophy via p38 MAPK activity in the heart. In addition, p38-α has been recognized as an isoenzyme of cardiovascular importance [16].

Among the numerous identified pathways, NFAT and hypertrophy of the heart (transcription in the broken heart) were ranked first. Nuclear factor of activated T-cells (NFAT) transcription factors, which have four different isoforms, plays crucial roles in the regulation of gene expression during heart development [17]. The isoforms NFATc3 and NFATc4 are involved in hypertrophic development, while NFATc1 plays a key role in cardiac development [18]. The dephosphorylation of NFATs can promote calcineurin regulating immune response genes [19]. Via compensatory hypertrophy, the heart adapts to persistent stress conditions, but, over time, dysfunction and myocardial failure evolve [20]. Like NFAT and hypertrophy of the heart (transcription in the broken heart), most of these pathways are involved in the formation and regulation of cardiovascular disease, such as nuclear receptors in lipid metabolism and toxicity. Nuclear receptors include a superfamily of ligand-dependent transcription factors that regulate genetic networks that control cell growth, development, and metabolism. Regulating nuclear receptors is beneficial for patients with metabolic diseases, such as cardiovascular disease, due to the requirement for balance among a number of pathways for normal metabolic control [21]. These studies confirmed the validity of our study.

From the above results, we also found the different significances of the total of 16 herbs in NXT. According to Chinese Pharmacopoeia 2015, the content of RA in NXT is 66 g, which is 2-3 times the content of any other herb in the whole prescription. It was reported that RA was the monarch drug of NXT and played a key role in improving the immune system, invigorating blood circulation, and the condition of myocardial ischemia and hypoxia [22]. Our study found that RA contained a lot of effective components, organic acids, and saponins and was critical source of the main active components of NXT. Through the comparison of the herbs involved in the top 40 pathways, RA was also proved to be the most important. In the top 40 pathways regulated by NXT, RA was involved in 33 pathways. Some other herbs, such as RSM, RCX, FC, and RAS, were also the important contents in the whole prescription of NXT.

The network pharmacology method used in this study is a novel methodology based on the construction of multilayer networks of disease phenotype-gene-drug to predict drug targets in a holistic manner and promote efficient drug discovery [23]. This method represents a breakthrough in comparison to the traditional herbal medicine research pattern “gene-target-disease” and initiates the new pattern “multiple genes-multiple targets-complex diseases” [24]. By this method, we proved that RA was the critical ingredient mainly involved in the regulation of metabolism and immunity in NXT. RAS was a major herb that regulated cell growth. RSM, RCX, and FC also played important roles in regulation of heart disease, blood vessels, and others. The results indicated that NXT, a complex prescription in the treatment of complex diseases, played a therapeutic effect through multiple targets and multiple pathways. This was the first study to investigate the mechanism of NXT using this method, and we successfully predicted the main targets and pathways, providing a foundation for further research. This method has important value for the study of complex drugs and should be applied in future studies.

5. Conclusion

The main components that mediated the efficacy of NXT were organic acids, saponins, and tanshinones. Radix Astragali was the critical herbal medicine in NXT, which contained more active components than others and regulated more targets and pathways. NXT had a therapeutic effect on the treatment of heart diseases through the pattern “multiple components-multiple targets-multiple pathways.”

Acknowledgments

This work was supported by the National Program for Key Basic Research Projects (2012CB518404), the NSFC (81202850 and 81125024), the Ministry of Education of PRC “Program for Innovative Research Team in University” (no. IRT1276), and the Program of International S&T Cooperation Project of China (2015DFA30430).

Abbreviations

ACS:

Acute coronary syndrome

AMI:

Acute myocardial infarction

CBD:

Cerebrovascular disease

CVD:

Cardiovascular disease

ESI:

Electrospray ionization

HRAS:

Harvey rat sarcoma viral oncogene homolog

MAP2K1:

Mitogen-activated protein kinase kinase 1

MAPK14:

Mitogen-activated protein kinase 14

NFAT:

Nuclear factor of activated T cells

NXT:

Naoxintong capsule

TCMs:

Traditional Chinese medicines

UPLC/Q-TOF-MS:

Ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Competing Interests

The authors have no conflicting financial interests.

Authors' Contributions

Xianghui Ma and Bin Lv contributed equally to this work.

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