Evidence-Based Complementary and Alternative Medicine
Volume 2016 (2016), Article ID 9468087, 15 pages
http://dx.doi.org/10.1155/2016/9468087

Research Article

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 and Xiumei Gao¹

¹State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
²College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China

Received 30 January 2016; Accepted 2 March 2016

Academic Editor: Carmen Mannucci

Copyright © 2016 Xianghui Ma et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

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) OHNH ≤ 5; relative molecular mass MW ≤ 500; fat water partition coefficient ; and hydrogen bond acceptor (the number of O and N) ON ≤ 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 .

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.

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 acid
Neg
170.1207
C₇H₆O₅
RPR

15
2.199
Phenylalanine
Pos
165.1874
C₉H₁₁NO₂
FC

16
2.459
Danshensu
Neg
198.1701
C₉HO₅
RSM

17
2.606
Palmitic acid
Neg
256.3380
C₁₆H₃₂O₂
RAS, FC, RA, SCP

18
3.438
Senkyunolide B
Neg
204.2374
C₁₂HO₃
RCX

19
3.456
Senkyunolide C
Neg
204.2374
C₁₂HO₃
RCX

20
3.600
Protocatechuic aldehyde
Neg
138.1185
C₇H₆O₃
RSM, RC

21
3.974
Mulberroside A
Neg
568.5277
C₂₆H₃₂O₁₄
RM

22
4.122
Gallicin
Neg
184.1453
C₈H₈O₅
RPR

23
4.230
Hydroxysafflor yellow A
Pos/Neg
612.5364
CHO
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
CHO
CS

28
5.157
Catechin
Neg
290.2674
CHO
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
CHO
RA

32
5.970
Calycosin
Neg
284.2679
C₁₆H₁₂O₅
RA

33
5.988
Calycosin-7-O-glucoside
Pos
446.4075
CHO
RA

34
5.989
Ferulic acid
Neg
194.1815
C₁₀H₁₀O₄
RA, RCX, RAS, RAB

35
6.321
Paeoniflorin
Pos
480.466
C₂₃H₂₈O₁₁
RPR

36
6.358
Pentagalloylglucose
Neg
940.68
C₄₁H₃₂O₂₆
RPR

37
6.413
Kaempferol-3-O-rutinoside
Pos/Neg
594.5179
C₂₇H₃₀O₁₅
FC

38
6.654
3,5-Di-O-caffeoylquinic acid
Pos/Neg
516.4573
C₂₅H₂₄O₁₂
CS

39
6.987
Dicaffeoylquinic acid
Neg
516.1275
C₂₅H₂₄O₁₂
RCX

40
7.042
Z-Butylidenephthalide
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
CHO
RSM

44
7.688
Ononin
Pos
430.4107
CHO
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
CHO
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
Formononetin
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)-ligustilide
Pos
190.2109
C₁₂H₁₄O₂
RAS

70
13.483
(E)-ligustilide
Pos
190.2109
C₁₂H₁₄O₂
RAS

71
13.917
Trijuganone B
Pos
280.1107
C₁₈H₁₆O₃
RSM

72
16.098
Cryptotanshinone
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 acid
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

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

(a)

(b)

Figure 1: UPLC/Q-TOF-MS analysis of NXT. (a) Chromatograms of NXT in positive ion mode. (b) Chromatograms of NXT in negative ion mode.

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
ON
OHNH
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 acid
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 A
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
Calycosin
284.267
5
2
2.377
√

33
Calycosin-7-O-glucoside
446.408
10
5
0.59
√

34
Ferulic acid
194.186
4
2
1.249
√

35
Paeoniflorin
480.466
11
5
0.044
✕

36
Pentagalloylglucose
940.681
26
15
2.761
√

37
Kaempferol-3-O-rutinoside
594.522
15
9
−0.574
√

38
3,5-Di-O-caffeoylquinic acid
516.455
12
7
1.424
✕

39
Dicaffeoylquinic acid
516.46
12
7
1.21
✕

40
Z-Butylidenephthalide
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
Formononetin
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)-Ligustilide
190.242
2
0
2.927
√

70
(E)-Ligustilide
190.242
2
0
2.927
√

71
Trijuganone B
280.32
3
1
3.9
√

72
Cryptotanshinone
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 acid
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
✕

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: 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 (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 ().

Rank
Pathway
Count
-value
-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

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

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: 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: 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.”

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.

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).

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