3.1. Fingerprint and quantification of major components in EMW
HPLC fingerprinting was performed to identify the main chemical compounds in EMW. Seven chemical constituents of EMW were identified according to the spectrograms and retention times of the standard substances (Fig.2A). Fingerprints of EMW were identified from six batches, with satisfactory similarity (Fig.2B), which suggested product stability. The seven components of EMW were measured quantitatively using HPLC, and their chemical structures are shown in Fig.2C.
The concentrations of major EMW components were as follows: chlorogenic acid, 1.52 ± 0.48 mg/mL; phellodendrine, 7.62 ± 0.81 mg/mL; magnoflorine, 0.62 ± 0.34 mg/mL; jateorhizine, 0.42 ± 0.26 mg/mL; palmatine, 0.02 ± 0.004 mg/mL; berberine, 49.68 ± 1.38 mg/mL; and atractydin, 38.43 ± 0.92 mg/mL. The compounds present at high concentrations in EMW originated mainly from CP, including chlorogenic acid, phellodendrine, magnoflorine, jateorhizine, palmatine, and berberine, whereas atractydin originated from RA. Alkaloids constituted the highest composition, followed by organic acids. The seven compounds represented the major bioactive components of EMW, and were therefore used for subsequent network pharmacology research in this study.
3.2. Method validation
(1) The linear regression analysis results, correlation coefficient (R2> 0.991), and linear range indicated that the established calibration curves could be quantified (Table.1). (2) After six consecutive injections, the relative standard deviation (RSD) % of chlorogenic acid, phellodendrine, magnolamine, jatrorrhizine, palmatine, berberine, and atractylodin was 0.56%, 0.24%, 0.90%, 0.67%, 0.88%, 0.17%, and 0.19%, respectively. This shows that the instrument has good precision. (3) Repeated experiment results showed that the content of chlorogenic acid, phellodendrine, magnolamine, jatrorrhizine, palmatine, berberine, and atractylodin was 1.31, 7.74, 0.61, 0.40, 0.02, 49.37, and 38.24 mg/mL, respectively. The RSD% of the content was 0.63%, 0.28%, 0.75%, 0.54%, 0.86%, 0.19%, and 0.18%, respectively, indicating that this method was reproducible. (4) The stability experiment showed that the average content of chlorogenic acid, phellodendrine, magnolamine, jatrorrhizine, palmatine, berberine, and atractylodin was 1.32, 7.69, 0.60, 0.39, 0.02, 49.28, and 38.34 mg/mL, respectively. The RSD% of the content was 0.32%, 1.24%, 1.15%, 0.32%, 1.43%, 0.22%, and 0.50%, respectively. The above results show that the components of EMW were stable within 24 h. (5) The recovery range of each component in EMW was 95.0-105.0%, which indicates that the method has good accuracy (Table.2).
Table 1 The linear regression equation and linear range of compounds in the EMW
Compound
|
Linear Regression Equation
|
R2
|
Linear Gange (mg/mL)
|
Chlorogenic acid
|
Y=7030.2X-18.292
|
0.999
|
0.040-3.500
|
Phellodendrine
|
Y=4248X+2.7625
|
1.000
|
0.071-7.521
|
Magnolamine
|
Y=18208X+6.8479
|
0.999
|
0.050-1.312
|
Jatrorrhizine
|
Y=14844X-1.5417
|
0.995
|
0.040-0.094
|
Palmatine
|
Y=11545X-5.9875
|
0.992
|
0.002-0.051
|
Berberine
|
Y=16162X-58.504
|
0.998
|
0.502-100.210
|
Atractydin
|
Y=16813X-60.708
|
0.998
|
0.407-70.105
|
Table 2 The Recoveries of each component in EMW (n=6)
Components
|
Amount of Standard Substance (mg)
|
Recoveries ± SD %
|
RSD%
|
Chlorogenic acid
|
0.65
|
101.88 ± 1.70
|
1.82
|
Phellodendrine
|
3.35
|
100.71 ± 1.60
|
1.74
|
Magnolamine
|
0.30
|
102.00 ± 1.75
|
1.88
|
Jatrorrhizine
|
0.20
|
101.70 ± 1.75
|
1.88
|
Palmatine
|
0.01
|
100.61 ± 1.40
|
1.52
|
Berberine
|
24.71
|
100.82 ± 1.28
|
1.39
|
Atractydin
|
19.10
|
101.29 ± 0.74
|
0.80
|
3.3. PPI network
The chemical components and eczema-related targets were screened according to a previous study [16]. A total of 788 and 295 targets were acquired for eczema and EMW, respectively, and 57 common targets were screened (P < 0.001, according to Fisher's exact test) and identified as the potential targets of EMW in treating eczema (Fig.3A). A PPI network was constructed to scientifically summarize the interfaces of EMW targets associated with eczema treatment. The network showed 57 possible protein target nodes connected by 318 edges, with an average node degree of 11.2. and an average local clustering coefficient of 0.548. The P-value of PPI enrichment was < 1.0 e−16 (Fig.3B). These results suggest that the key proteins are closely related to each other. The top 10 predicted hub genes included EGFR, AKT1, PTGS2, STAT3, MMP9, ICAM1, MAPK8, JUN, MAPK1, and VCAM1. As shown in Fig.3B, the darker the color of the node, the larger the size, which indicates that the protein may be more crucial for the treatment of eczema.
3.4. Compound-target network
Based on the results of target interaction analysis, a compound-target network was built as described in Fig.4. The network showed that most inflammation-related targets and immune-related targets were interrelated, indicating that the action mechanism of EMW was associated with inflammation and immune mechanisms. Furthermore, in terms of the compound structures, alkaloids corresponded to more targets than other compounds.
3.5. GO and pathway enrichment analysis
GO analysis results revealed 76 terms of biological processes, and the core terms of EMW targets against eczema were mainly involved in the negative regulation of apoptotic process, positive regulation of vasoconstriction, positive regulation of nitric oxide biosynthetic process, positive regulation of smooth muscle cell proliferation, cellular response to mechanical stimulus, aging, response to drug, positive regulation of transcription from RNA polymerase II promoter, signal transduction, and regulation of sequence-specific DNA binding transcription factor activity (Fig.5A and 5 B and Table.3). The 71 enriched KEGG pathways (P<0.05) included the TNF signaling pathway, pathways in cancer, hepatitis B, pancreatic cancer, ErbB signaling pathway, estrogen signaling pathway, choline metabolism in cancer, Epstein-Barr virus infection, hepatitis C, and FoxO signaling pathways (Fig.5C and 5D and Table.4).
Table 3. Top 10 Biological Processes of EMW against Eczema
ID
|
Description
|
Gene symbol
|
Number of gene
|
P-Value
|
Term1
|
negative regulation of apoptotic process
|
MAPK8, STAT3, AKT1, MMP9, EGFR
|
5
|
6.02 e-5
|
Term2
|
positive regulation of vasoconstriction
|
AKT1, PTGS2, EGFR, ICAM1
|
4
|
6.24 e-07
|
Term3
|
positive regulation of nitric oxide biosynthetic process
|
AKT1, PTGS2, EGFR, ICAM1
|
4
|
1.30 e-06
|
Term4
|
positive regulation of smooth muscle cell proliferation
|
JUN, AKT1, PTGS2, EGFR
|
4
|
3.59 e-06
|
Term5
|
cellular response to mechanical stimulus
|
MAPK8, AKT1, PTGS2, EGFR
|
4
|
5.97 e-06
|
Term6
|
aging
|
JUN, VCAM1, STAT3, AKT1
|
4
|
7.49 e-05
|
Term7
|
response to drug
|
JUN, STAT3, PTGS2, ICAM1
|
4
|
4.55 e-04
|
Term8
|
positive regulation of transcription from RNA polymerase II promoter
|
JUN, STAT3, AKT1, EGFR
|
4
|
1.28 e-02
|
Term9
|
signal transduction
|
STAT3, MAPK1, AKT1, EGFR
|
4
|
2.02 e-02
|
Term10
|
regulation of sequence-specific DNA binding transcription factor activity
|
JUN, MAPK8, MAPK1
|
4
|
7.61 e -05
|
Table 4. Top 10 KEGG Pathway of EMW Against Eczema
ID
|
Description
|
Gene symbol
|
Number of gene
|
P-Value
|
Term1
|
TNF signaling pathway
|
JUN, MAPK8, VCAM1, MAPK1, AKT1, PTGS2, MMP9, ICAM1
|
8
|
6.35 e-12
|
Term2
|
Pathways in cancer
|
JUN, MAPK8, STAT3, MAPK1, AKT1, PTGS2, MMP9, EGFR
|
8
|
6.15 e-08
|
Term3
|
Hepatitis B
|
JUN, MAPK8, STAT3, MAPK1, AKT1, MMP9
|
6
|
4.57 e-07
|
Term4
|
Pancreatic cancer
|
MAPK8, STAT3, MAPK1, AKT1, EGFR
|
5
|
8.83 e-07
|
Term5
|
ErbB signaling pathway
|
JUN, MAPK8, MAPK1, AKT1, EGFR
|
5
|
2.87 e-06
|
Term6
|
Estrogen signaling pathway
|
JUN, MAPK1, AKT1, MMP9, EGFR
|
5
|
4.81 e-06
|
Term7
|
Choline metabolism in cancer
|
JUN, MAPK8, MAPK1, AKT1, EGFR
|
5
|
5.21 e-06
|
Term8
|
Epstein-Barr virus infection
|
JUN, MAPK8, STAT3, AKT1, ICAM1
|
5
|
1.11 e-05
|
Term9
|
Hepatitis C
|
MAPK8, STAT3, MAPK1, AKT1, EGFR
|
5
|
1.56 e-05
|
Term10
|
FoxO signaling pathway
|
MAPK8, STAT3, MAPK1, AKT1, EGFR
|
5
|
1.61 e -05
|
3.6. Compound-target-pathway network
On the basis of the compound-target network and pathway enrichment analysis results, we created a compound-target-pathway network consisting of the top 10 KEGG pathways (Fig.6A). The network showed that EMW and eczema shared 10 KEGG pathways, representing their combined anti-eczema targets. The ErbB signaling pathway, estrogen signaling pathway, and Epstein-Barr virus infection were the three pathways connected with the key targets and associated with the effect of EMW on eczema. Hence, their network relationships were isolated and further analyzed through annotation of the KEGG pathway. The network highlighted the hub genes EGFR, AKT1, MMP9, ICAM1, MAPK8, JUN, and MAPK1 (Fig.6B).
3.7. Molecular Docking
EMW components were chosen for molecular docking analysis based on their high content and network pharmacology results. Moreover, the importance of the top 10 hub genes in eczema treatment was validated through network pharmacology analysis. The docking results showed that EGFR, AKT1, and PTGS2 bound well with all components. Palmatine, phellodendrine, chlorogenic acid, and jatrorrhizine bound well with all docking targets (Fig.7 and Table.5). The Surflex-Dock scores (total scores) represent binding affinities. Generally, a docking score greater than 4.25 indicates certain binding activity, whereas a docking score greater than 7.00 indicates strong binding activity [17]. The comparable binding score between the compounds may be due to their similar molecular structures, which is consistent with a previous finding [18]. However, among the seven chemical components of EMW, atractylodin only exhibited 3 (33.33%) binding efficiencies with the docking targets. Among the nine targets, ICAM1 only exhibited 2 (28.57%) binding efficiencies with the components of EMW. These findings imply that not every component has a good binding efficiency with every target, but the interaction between each component and each target is consistent with the multi-component and multi-target characteristics of TCM [19].
Table.5 Total score of 7 chemical composition of EMW and hub genes molecule docking.
Compound
|
Palmatine
|
Phellodendrine
|
Chlorogenic acid
|
Magnoflorine
|
Berberine
|
Jateorhizine
|
Atractylodin
|
EGFR
|
5.8814
|
4.9264
|
6.6037
|
4.5828
|
6.6112
|
5.2378
|
4.4279
|
AKT1
|
5.1865
|
6.6481
|
6.4127
|
5.6517
|
5.6987
|
5.4850
|
4.2639
|
PTGS2
|
5.3339
|
4.9394
|
6.9139
|
4.4581
|
6.9586
|
6.3229
|
4.7931
|
STAT3
|
5.4003
|
4.9290
|
7.6658
|
4.6498
|
5.0630
|
5.0233
|
3.9572
|
MMP9
|
5.4053
|
4.8698
|
6.9478
|
4.7065
|
4.5353
|
5.1131
|
4.0299
|
ICAM1
|
5.0165
|
4.2205
|
4.6740
|
3.4962
|
3.9165
|
5.1307
|
3.3362
|
MAPK8
|
4.8426
|
5.5351
|
7.2720
|
4.4886
|
5.0827
|
5.3900
|
4.1246
|
JUN
|
6.4617
|
5.3219
|
5.1704
|
4.3920
|
5.9145
|
6.2663
|
3.6751
|
MAPK1
|
6.4342
|
6.8556
|
5.9204
|
4.5037
|
5.0003
|
6.2831
|
3.7213
|
The 3D mode and schematic 2D representation of chlorogenic acid in the active site of MAPK8 are shown in Fig.8A. Chlorogenic acid showed 6H-bond interactions with ARGs 69, 34, 109, 111, 156, and 153. Other interactions included P-π conjugation and π-π conjugation. The 3D mode and schematic 2D dimensional representation of chlorogenic acid in the active site of MMP9 are shown in Fig.8B. Chlorogenic acid showed 9H-bond interactions, three of which were amidogen with GLY 186, LEU 188, and ALA 189. ARG 424, GLN 402, HIS 401, and TRY 420. Other interactions, including Pi-Sigma and Pi-Anion, were connected to ALA 417 and HIS 411. The 3D mode and schematic 2D representation of chlorogenic acid in the active site of STAT3 are shown in Fig.8C. Chlorogenic acid showed 5H-bond interactions, which were GLU 883, SER 963, GLY 1020, GLU 996, and GLU 957. Other interactions included Pi-Sigma, Pi-Alkyl, and Pi-Pi. The 3D mode and schematic 2D dimensional representation of berberine in the active site of PTGS2 are shown in Fig.8D. Berberine showed 6H-bond interactions, including hydroxyls with ARGs 69, 34, 109, 111, 156, and 153. Other interactions included Pi-sigma and π-π conjugation.