*Corresponding Authors:
Y. Q. Xu
Department of Oncology, the Second Affiliated Hospital of Nanjing University of Chinese Medicine, No. 23, Nanhu Road, Nanjing, Jiangsu 210017, China

L. Q. Xie
Department of Traditional Chinese Medicine, The First Affiliated Hospital of Nanjing Medical University, No. 300, Guangzhou Road, Nanjing, Jiangsu 210029, China
E-mail: [email protected]
This article was originally published in a special issue, “Biomedical applications in Pharmaceutical Sciences”
Indian J Pharm Sci 2020:82(2) Spl issue 3;135-141

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms

Abstract

This investigation dealt with the analysis of the active ingredients of Yiyi-Fuzi-Baijiang powder in the treatment of colorectal cancer using computer network pharmacology technology to predict the mechanism of action. The chemical constituents and targets of Yiyi-Fuzi-Baijiang powder were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and the common targets in the treatment of colorectal cancer were obtained from Online Mendelian Inheritance in Man, Therapeutic Target Database and PharmGkb database. Excel was used to screen the molecular target and Cytoscape software was used to establish the network of Chinese medicine components target of Yiyi-Fuzi-Baijiang powder. The gene function and metabolic pathway were analyzed using the biological information annotation database. Nineteen components of Yiyi-Fuzi-Baijiang powder were found to interact with 121 target proteins of colorectal cancer, including progesterone receptor, prostaglandin G/H synthase 2, gamma-aminobutyric acid receptor subunit alpha-1, prostaglandin G/H synthase 1, nuclear receptor co-activator 2, beta-2 adrenergic receptor, sodium channel protein type 5 subunit alpha, alpha-1B adrenergic receptor. It is the target of Chaihushugansan in the treatment of post-stroke depression, mainly involving in neuroactive ligand-receptor interaction, endocrine regulated calcium reabsorption, TGF-β signaling pathway, Hedgehog signaling pathway, inflammatory mediator regulation transient receptor potential channels, cholinergic synapse, through the regulation of the nervous system, cell cycle, apoptosis, inflammatory regulation, cell communication and other biological processes to treat colorectal cancer. The possible mechanism of Yiyi-Fuzi-Baijiang powder in treating colorectal cancer was revealed by network pharmacological study, which laid a foundation for further elucidation of its action target.

Keywords

Coix seed, Monkshood, Patrinia villosa, Colorectal cancer, Network pharmacology

Colorectal cancer, including colorectal and rectal cancer, is the third most common cause of cancer death in the world. Its 5 y survival rate is only 30-65 %[1]. In recent years, the incidence of colorectal cancer has increased gradually in developing countries. The incidence and mortality of colorectal cancer in China ranks fifth and sixth, respectively among all the malignant tumours in the urban population, which has seriously threatened the health of people in China[2]. Its prevention and treatment has become a hot research topic[3-5]. Colorectal cancer in traditional Chinese clinical medicine clinical was grouped under intestinal Qin, accumulation, locking anal hemorrhoids, visceral toxin and scattered in enteropathy, intestinal wind, diarrhoea, colon Yin, intestinal wind blood and other diseases. The treatment outcomes of traditional Chinese medicine is significant[6-8]. The early clinical research and animal experiment of the research group confirmed that Yiyi-Fuzi-Baijiang powder showed good curative effect in the treatment of colorectal cancer[9]. However, due to the regulation of multi-component and multi-target of traditional Chinese medicine, the mechanism of Yiyi-Fuzi-Baijiang powder in the treatment of colorectal cancer is not clear.

Network pharmacology is a new method to study the mechanism of traditional Chinese medicinal compounds[10]. The purpose of this study is to explore the potential effective components and possible mechanism of Yiyi-Fuzi-Baijiang powder in the treatment of colorectal cancer and to systematically explore the overall regulatory effect of the multi-component and multi-target drug on colorectal cancer at the molecular level, so as to provide a basis for the further research and development of compounds in traditional Chinese medicine.

Materials and Methods

Traditional Chinese medicine systems pharmacology (TCMSP) is a platform integrating pharmacokinetics, pharmaceutical chemistry and drug target protein network disease network[11]. Based on the TCMSP database, the chemical constituents of Coix, aconite and Patrinia villosa were searched. All the chemical components were screened by Excel and the component data were obtained.

Research tools

TCMSP; UniProt Database to obtain gene information of components, targets, interrelations and targets of traditional Chinese Medicine; Online Frontal Analysis Mendelian Inheritance in Man (OMIM)[12,13], Drug target database, Databases/TTD/TTD.asp and PharmGkb database acquisition of target proteins for stroke and depression; Cytoscape 3.2.1 software building a network of compound target interaction[12-16].

Screening of active compounds of Yiyi-Fuzi-Baijiang powder

The target organs and tissues for traditional Chinese medicine should be achieved through the process of absorption, distribution, metabolism and excretion (ADME). Oral bioavailability (OB), drug likeness (DL), blood-brain barrier (BBB) and half-life (HL) are the key parameters of ADME. In this study, TCMSP data platform was used to evaluate OB, DL, BBB and HL of each active component of Yiyi-Fuzi-Baijiang powder and selected chemical components that simultaneously meet the requirements of OB ≥30 %, DL ≥0.18, BBB ≥0.3 and HL ≥4 h as the candidate active components.

Target selection of Yiyi-Fuzi-Baijiang powder in the treatment of colorectal cancer

GADCC, therapeutic target database (TTD) and PharmGKB databases were used to search the coding genes of colorectal cancer to determine the disease target. Based on the molecular docking technology, the effective components and disease targets of Yiyi-Fuzi-Baijiang powder were screened and the target of Yiyi-Fuzi-Baijiang powder in the treatment of colorectal cancer was obtained.

Biological process and metabolic pathway analysis of target

Database for annotation, visualization and integrated discovery (DAVID) can provide annotated information of biological functions and find the most significant biological annotation. By inputting the list of target gene names and defining the species as human, and correcting all the target gene names to their official names, the enrichment analysis of go biological process and the metabolism of Kyoto Encyclopaedia of genes and genomes (KEGG) pathway analysis were carried out pathway enrichment analysis.

Results and Discussion

The results showed that there were 19 active molecules in baizao powder that included 6, sitosterol a1, mandenol, (2R)-2,3-dihydroxypropyl(z)-octadec-9-enoate, sitosterol, stigmasterol and  CLR; 6,  11,14-eicosadienoic acid, deltoin, karakoline, karanjin, neonadsuranic acid B, sitosterol and 7 of Patrinia villosa L, morrianine C, asperglauccide, acacetin, sinoacutine, b-sitosterol, sitosterol, and stigmasterol (Table 1). Through UniProt database, the target points of the above ingredients are transformed into gene names, and the composition target map of traditional Chinese medicine is constructed (fig. 1).

Number Component OB/% DL BBB HL Medicinal Materials
MOL001323 Sitosterol a1 43.28 0.78 0.97 5.64 Coix seed
MOL001494 Mandenol 42 0.19 1.14 5.39 Coix seed
MOL002882 (2R)-2,3-dihydroxypropyl (Z)-octadec-9-enoate 34.13 0.3 -0.22 5.19 Coix seed
MOL000359 Sitosterol 36.91 0.75 0.87 5.37 Coix seed
MOL000449 Stigmasterol 43.83 0.76 1 5.57 Coix seed
MOL000953 CLR 37.87 0.68 1.13 4.52 Coix seed
MOL002211 11,14-eicosadienoic acid 39.99 0.2 0.76 5.6 monkshood
MOL002392 Deltoin 46.69 0.37 -0.12 7.7 monkshood
MOL002397 Karakoline 51.73 0.73 -0.03 11.1 monkshood
MOL002398 Karanjin 69.56 0.34 0.62 13.15 monkshood
MOL002401 Neokadsuranic acid B 43.1 0.85 -0.01 12.05 monkshood
MOL000359 Sitosterol 36.91 0.75 0.87 5.37 monkshood
MOL001676 Vilmorrianine C 33.96 0.22 0.14 21.71 Patrinia villosa
MOL001677 Asperglaucide 58.02 0.52 -0.22 6.88 Patrinia villosa
MOL001689 Acacetin 34.97 0.24 -0.05 17.25 Patrinia villosa
MOL001697 Sinoacutine 63.39 0.53 0.36 4.9 Patrinia villosa
MOL000358 b-sitosterol 36.91 0.75 0.99 5.36 Patrinia villosa
MOL000359 Sitosterol 36.91 0.75 0.87 5.37 Patrinia villosa
MOL000449 Stigmasterol 43.83 0.76 1 5.57 Patrinia villosa

Table 1: Candidate Active Components and Pharmacokinetic Parameters of Yiyi-Fuzi-Baijiang Powder

IJPS-Composition

Figure 1: Composition target map of Yiyi-Fuzi-Baijiang powder

Using GADCC, TTD and PharmGKB database to search the coding genes of colorectal cancer, there are 121 targets as disease targets (Table 2). Based on the molecular docking technology, 19 active ingredients and 121 disease targets of Coix Fuzi baizao powder were screened, and 8 well docking targets were obtained as the targets of Coix Fuzi baizao powder in the treatment of colorectal cancer (Table 3), respectively progesterone receptor, prostaglandin G/H synthase 2, g-aminobutyric acid receptor subunit a-1, prostaglandin G/H synthase 1, nuclear receptor coactivator 2, b-2 adrenergic receptor, sodium channel protein type 5 subunit a, a-1B adrenergic receptor.

1 Vascular endothelial growth factor receptor 1 65 PTGS1
2 Vascular endothelial growth factor receptor 2 66 PTGS2
3 Potassium-transporting ATPase a chain 1 67 ADRB2
4 Retinoic acid receptor a 68 ADH1B
5 DNA topoisomerase I 69 FH
6 DNA topoisomerase II 70 ZFHX3
7 FL cytokine receptor 71 MXI1
8 Histone deacetylase 1 72 ADRA1B
9 Tumor necrosis factor 73 EIF4G1
10 Calcium channel 74 OPCML
11 MLH3 75 NKX2-1
12 AXIN2 76 GABRA-1
13 MCC 77 PTPRJ
14 PIK3CA 78 HMMR
15 AKT1 79 SMAD4
16 FGFR3 80 ATR
17 MSH6 81 ACVR1B
18 CHEK2 82 BARD1
19 TP53 83 NCOA-2
20 MUTYH 84 STK11
21 DCC 85 PRKN
22 BRAF 86 FOXE1
23 PMS2 87 MAD1L1
24 DLC1 88 PHB
25 PDGFRL 89 RAD51C
26 CTNNB1 90 FGFR2
27 MSH2 91 RAD51D
28 APC 92 AURKA
29 MLH1 93 PPP2R1B
30 TGFBR2 94 RAD54L
31 NRAS 95 HABP2
32 CCND1 96 RAD54B
33 PLA2G2A 97 SCN5A
34 BAX 98 ELAC2
35 POLE 99 RAD51
36 POLD1 100 BRIP1
37 BUB1 101 RNASEL
38 EP300 102 IL1RN
39 BUB1B 103 IL1B
40 SMAD7 104 SRGAP1
41 TLR2 105 RB1CC1
42 FLCN 106 MPO
43 GALNT12 107 ATM
44 EPCAM 108 SASH1
45 BRCA2 109 PALLD
46 KRAS 110 ERCC6
47 CDH1 111 EHBP1
48 BRCA1 112 CHRNA3
49 EGFR 113 CHRNA5
50 ERBB2 114 RNF43
51 CDKN2A 115 SDCCAG8
52 EPHB2 116 CYP2A6
53 PTEN 117 RHBDF2
54 CASP8 118 MSMB
55 KLF6 119 HRAS
56 SLC22A18 120 SRC
57 SLC22A18 121 NQO1
58 PPM1D    
59 IRF1    
60 AR    
61 PALB2    
62 RB1    
63 MAP3K8    
64 PGR    

Table 2: Disease Targets

Number Target protein Gene name
MOL000359 Progesterone receptor PGR
MOL000449 Prostaglandin G/H synthase 2 PTGS2
MOL000449 Gamma-aminobutyric acid receptor subunit alpha-1 GABRA1
MOL000449 Prostaglandin G/H synthase 1 PTGS1
MOL000359 Nuclear receptor coactivator 2 NCoA-2
MOL000449 Beta-2 adrenergic receptor ADRB2
MOL000449 Sodium channel protein type 5 subunit alpha SCN5A
MOL000449 Alpha-1B adrenergic receptor ADRA1B

Table 3: Potential Target of Yiyi-Fuzi-Baijiang Powder in the Treatment of Colorectal Cancer

The results showed that the 8 predicted targets were related to biological processes such as neural regulation, cell cycle, apoptosis, inflammatory regulation, cell communication and so on (Table 4). It is suggested that Yiyi-Fuzi-Baijiang powder may play an anticancer role by improving these biological processes. The results of enrichment analysis of KEGG signal pathway of Yiyi-Fuzi-Baijiang powder 's anticolorectal cancer target (Table 5) showed that 8 therapeutic targets participate in 4 categories and 6 signal pathways, including neural regulation, apoptosis, cell cycle and inflammation regulation, as shown in Table 5, mainly including neuroactive ligand-receptor interaction, endocrine regulated calcium reabsorption, TGF-β signaling pathway, Hedgehog signaling pathway, inflammatory mediator regulation TRP channels, cholinergic synapse. The results showed that the main active components of Yiyi-Fuzi-Baijiang powder were distributed in different metabolic pathways, and the possible mechanism of action was to coordinate and regulate each other.

Biological Process Related Targets P Benjamini
Neural regulation PTGS2, ADRB2 0.00575 0.21
Cell cycle PGR, SCN5A, ADRA1B 0.01532 0.43
Apoptosis PTGS1, GABRA1, SCN5A 0.00784 0.20
Inflammation regulation PTGS2, ADRB2, NCoA-2 0.01074 0.38
Cell communication PTGS1, GABRA, SCN5A 0.00058 0.04

Table 4: Go Biological Process Enrichment Analysis of Target

Metabolic pathway Related targets P Benjamini
Neuroactive ligand-receptor interaction PGR, NCoA-2, SCN5A 0.032 0.45
Endocrine regulated calcium reabsorption NCoA-2, ADRA1B, SCN5A 0.045 0.65
TGF-β signaling pathway PTGS2, ADRB2 0.061 0.72
Hedgehog signaling pathway SCN5A, ADRA1B 0.015 0.33
Inflammatory mediator regulation TRP channels PTGS1, GABRA1, SCN5A 0.050 0.24
Cholinergic synapse PTGS2, ADRB2 0.012 0.46

Table 5: Enrichment Analysis of Target Kegg Metabolic Pathway

Yiyi-Fuzi-Baijiang powder comes from one of the 4 classics of traditional Chinese medicine, synopsis of the Golden Chamber ulceration, carbuncle, intestinal abscess, soakage, pulse syndrome and treatment of the 18th: the intestine carbuncle is a disease where the body is wrong, the abdomen is urgent, according to it, if it is swollen, the abdomen is not accumulated, the body is not hot, the pulse number, this is the intestine has carbuncle pus, Yiyi-Fuzi-Baijiang powder main. This prescription is composed of Coix seed, aconite and Patrinia villosa. It was first recorded in the treatment of intestinal carbuncle. It is mainly used for the treatment of intestinal carbuncle and pus[17]. It has the function of eliminating carbuncle and removing pus, and strengthening yang qi. In modern times, it is used in intestinal, skin, gynecology, andrology and other diseases, as well as dry skin, abnormal keratosis and other diseases[18-20]. It is most commonly used in the treatment of abscess and purulent inflammation[18]. In the prescription, Coix seed is used to eliminate carbuncle and swelling, Patrinia villosa is used to clear heat, detoxify and discharge pus, while Fuzi Wenyang powder is used to relieve cold and pain. Modern pharmacology research showed that many monomers in Yiyi-Fuzi-Baijiang powder have anticancer effect.

In this investigation it was found that sitosterol a-1, mandenol, (2R)-2,3-dihydroxypropyl (Z)-octadec-9-enoate, sitosterol, stigmasterol, CLR, 11,14-eicosadienoic acid, deltoin, karakoline, karanjin, neokadsuranic acid B, sitosterol, vilmorrianine C, asperglaucide, acacetin, sinoacutine, b-sitosterol, sitosterol, stigmasterol as 19 main active ingredients in Coix, aconite and Patrinia, acting on progesterone receptor, prostaglandin G/H synthase 2, g-aminobutyric acid receptor subunit a-1, prostaglandin G/H synthase 1, nuclear receptor coactivator 2, b-2 adrenergic receptor, sodium channel protein type 5 subunit a, a-1B adrenergic receptor several potential targets. These targets may play an important role in the antitumor network. The enrichment analysis of go biological process showed that the target genes of Yiyi-Fuzi-Baijiang powder were involved in the biological processes of neural regulation, cell cycle, apoptosis, inflammation regulation, cell communication and so on. These biological processes are closely related to the development of nervous system and cancer cells and may be involved in the pathophysiological process of colorectal cancer. Some targets are related to cell cycle, apoptosis and inflammation control, which proved that the alkaloid extract of Yiyi-Fuzi-Baijiang powder has good analgesic and antiinflammatory effect, could regulate the central nervous system and has a certain central nervous inhibitory effect[21-23]. Therefore, Yiyi-Fuzi-Baijiang powder can produce antitumor activity by inhibiting tumor growth and migration, regulating energy, regulating neural system multi phenotype intervention network mode, and has a certain relieving effect on cancer pain.

The results of KEGG metabolism pathway analysis showed that the related targets of the active components in the powder were, progesterone receptor, prostaglandin G/H synthase 2, g-aminobutyric acid receptor subunit a-1, prostaglandin G/H synthase 1, nuclear receptor coactivator 2, b-2 adrenergic receptor, sodium channel protein type 5 subunit alpha, a-1B adrenergic receptor participating in neuroactive ligand-receptor interaction, endocrine regulated calcium reabsorption, TGF-β signalling pathway, Hedgehog signaling pathway, inflammatory mediator regulation TRP channels, cholinergic synapse signal pathway. Neuroactive ligand-receptor interaction[24], endocrine regulated calcium reabsorption[25], cholinergic synapse[26], Hedgehog signaling pathway[27] are mainly related to the inflammatory reaction and the occurrence and development of cancer cells and participates in the process of cell cycle change and apoptosis. TGF-β signaling pathway[28] can affect PTGS2 and ADRB2 gene to mediate brain activity, thus affecting the transmission of neural information. Inflammatory mediator regulation TRP channels[29] mainly involved in cell communication and related to the growth and development of cancer cells. In this study, the target of Coix Fuzi-Baijiang powder in the treatment of colorectal cancer was involved as neuroactive ligand-receptor interaction (3 targets), endocrine regulated calcium reabsorption (3 targets), TGF-β signalling pathway (2 targets), Hedgehog signalling pathway (2 targets), inflammatory mediator regulation TRP channels (3 targets) and cholinergic synapse (2 targets). It is suggested that the active components of Yiyi-Fuzi-Baijiang powder may play an antiinflammatory and analgesic role by participating in the above pathway, thereby reducing the inflammatory response and regulating the nervous system.

In conclusion, this study described the reticular relationship among the main active components, targets and pathways of Yiyi-Fuzi-Baijiang powder. It was found that Yiyi-Fuzi-Baijiang powder not only has neuromodulatory effect, but also has multi-dimensional pharmacological effects such as anticancer and antiinflammatory. However, network pharmacology research is inseparable from the integrity and practicability of related databases and there are some differences between the technology of molecular simulation docking and the environment in vivo. Therefore, the relevant targets and molecular mechanisms of the research still need further experimental verification.

Acknowledgements

The authors thank the training program for core talents in TCM innovation of China and the 4th China National TCM Excellent Talents Training Projects 2017 supporting our study.

Funding

This work was supported by the Natural Science Foundation of Jiangsu Province (BK20171096).

Conflict of Interest

All authors report no conflicts of interest in this work.

References