Collection and screening of compounds
A total of 657 compounds were obtained from TCMSP database and related literature search. Among them, there are 143 compounds from Scutellaria baicalensis, 48 compounds from Coptis chinensis, 140 compounds from Phellodendri Chinrnsis Cortex, and 98 compounds from Gardenia jasminoides. After screening with OB ≥ 30% and DL ≥ 0.18 as screening criteria, 143 compounds were obtained. After removing duplicates, there were 126 main compounds in HLJDD. They are listed in Table-1 below.
Table-1
Ingredients of Huanglian Jiedu Decoction.
Herbs
|
TCMSP number
|
Compound name
|
Relative molecular mass
|
OB (%)
|
DL
|
Scutellaria baicalensis
|
MOL001689
|
Acacetin
|
284.28
|
34.97
|
0.24
|
Scutellaria baicalensis
|
MOL000173
|
Wogonin
|
284.28
|
30.68
|
0.23
|
Scutellaria baicalensis
|
MOL000228
|
(2R)-7-hydroxy-5-methoxy-2-phenylchroman-4-one
|
270.30
|
55.23
|
0.20
|
Scutellaria baicalensis
|
MOL002714
|
Baicalein
|
270.25
|
33.52
|
0.21
|
Scutellaria baicalensis
|
MOL002908
|
5,8,2'-Trihydroxy-7-methoxyflavone
|
300.28
|
37.01
|
0.27
|
Scutellaria baicalensis
|
MOL002909
|
5,7,2,5-tetrahydroxy-8,6-dimethoxyflavone
|
376.34
|
33.82
|
0.45
|
Scutellaria baicalensis
|
MOL002910
|
Carthamidin
|
288.27
|
41.15
|
0.24
|
Scutellaria baicalensis
|
MOL002911
|
2,6,2',4'-tetrahydroxy-6'-methoxychaleone
|
302.30
|
69.04
|
0.22
|
Scutellaria baicalensis
|
MOL002913
|
Dihydrobaicalin_qt
|
272.27
|
40.04
|
0.21
|
Scutellaria baicalensis
|
MOL002914
|
Eriodyctiol (flavanone)
|
288.27
|
41.35
|
0.24
|
Scutellaria baicalensis
|
MOL002915
|
Salvigenin
|
328.34
|
49.07
|
0.33
|
Scutellaria baicalensis
|
MOL002917
|
5,2',6'-Trihydroxy-7,8-dimethoxyflavone
|
330.31
|
45.05
|
0.33
|
Scutellaria baicalensis
|
MOL002925
|
5,7,2',6'-Tetrahydroxyflavone
|
286.25
|
37.01
|
0.24
|
Scutellaria baicalensis
|
MOL002926
|
dihydrooroxylin A
|
286.30
|
38.72
|
0.23
|
Scutellaria baicalensis
|
MOL002927
|
Skullcapflavone II
|
374.37
|
69.51
|
0.44
|
Scutellaria baicalensis
|
MOL002928
|
oroxylin a
|
284.28
|
41.37
|
0.23
|
Scutellaria baicalensis
|
MOL002932
|
Panicolin
|
314.31
|
76.26
|
0.29
|
Scutellaria baicalensis
|
MOL002933
|
5,7,4'-Trihydroxy-8-methoxyflavone
|
300.28
|
36.56
|
0.27
|
Scutellaria baicalensis
|
MOL002934
|
NEOBAICALEIN
|
374.37
|
104.34
|
0.44
|
Scutellaria baicalensis
|
MOL002937
|
DIHYDROOROXYLIN
|
286.30
|
66.06
|
0.23
|
Scutellaria baicalensis
|
MOL000358
|
beta-sitosterol
|
414.79
|
36.91
|
0.75
|
Scutellaria baicalensis
|
MOL000359
|
Sitosterol
|
414.79
|
36.91
|
0.75
|
Scutellaria baicalensis
|
MOL000525
|
Norwogonin
|
270.25
|
39.40
|
0.21
|
Scutellaria baicalensis
|
MOL000552
|
5,2'-Dihydroxy-6,7,8-trimethoxyflavone
|
344.34
|
31.71
|
0.35
|
Scutellaria baicalensis
|
MOL000073
|
ent-Epicatechin
|
290.29
|
48.96
|
0.24
|
Scutellaria baicalensis
|
MOL000449
|
Stigmasterol
|
412.77
|
43.83
|
0.76
|
Scutellaria baicalensis
|
MOL001458
|
Coptisine
|
320.34
|
30.67
|
0.86
|
Scutellaria baicalensis
|
MOL001490
|
bis[(2S)-2-ethylhexyl] benzene-1,2-dicarboxylate
|
390.62
|
43.59
|
0.35
|
Scutellaria baicalensis
|
MOL001506
|
Supraene
|
410.80
|
33.55
|
0.42
|
Scutellaria baicalensis
|
MOL002879
|
Diop
|
390.62
|
43.59
|
0.39
|
Scutellaria baicalensis
|
MOL002897
|
Epiberberine
|
336.39
|
43.09
|
0.78
|
Scutellaria baicalensis
|
MOL008206
|
Moslosooflavone
|
298.31
|
44.09
|
0.25
|
Scutellaria baicalensis
|
MOL010415
|
11,13-Eicosadienoic acid, methyl ester
|
322.59
|
39.28
|
0.23
|
Scutellaria baicalensis
|
MOL012245
|
5,7,4'-trihydroxy-6-methoxyflavanone
|
302.30
|
36.63
|
0.27
|
Scutellaria baicalensis
|
MOL012246
|
5,7,4'-trihydroxy-8-methoxyflavanone
|
302.30
|
74.24
|
0.26
|
Scutellaria baicalensis
|
MOL012266
|
Rivularin
|
344.34
|
37.94
|
0.37
|
Coptis chinensis
|
MOL001454
|
Berberine
|
336.39
|
36.86
|
0.78
|
Coptis chinensis
|
MOL013352
|
Obacunone
|
454.56
|
43.29
|
0.77
|
Coptis chinensis
|
MOL002894
|
berberrubine
|
322.36
|
35.74
|
0.73
|
Coptis chinensis
|
MOL002897
|
epiberberine
|
336.39
|
43.09
|
0.78
|
Coptis chinensis
|
MOL002903
|
(R)-Canadine
|
339.42
|
55.37
|
0.77
|
Coptis chinensis
|
MOL002904
|
Berlambine
|
351.38
|
36.68
|
0.82
|
Coptis chinensis
|
MOL002907
|
Corchoroside A_qt
|
404.55
|
104.95
|
0.78
|
Coptis chinensis
|
MOL000622
|
Magnograndiolide
|
266.37
|
63.71
|
0.19
|
Coptis chinensis
|
MOL000762
|
Palmidin A
|
510.52
|
35.36
|
0.65
|
Coptis chinensis
|
MOL000785
|
palmatine
|
352.44
|
64.60
|
0.65
|
Coptis chinensis
|
MOL000098
|
quercetin
|
302.25
|
46.43
|
0.28
|
Coptis chinensis
|
MOL001458
|
coptisine
|
320.34
|
30.67
|
0.86
|
Coptis chinensis
|
MOL002668
|
Worenine
|
334.37
|
45.83
|
0.87
|
Coptis chinensis
|
MOL008647
|
Moupinamide
|
313.38
|
86.71
|
0.26
|
Phellodendri Chinrnsis Cortex
|
MOL001454
|
berberine
|
336.39
|
36.86
|
0.78
|
Phellodendri Chinrnsis Cortex
|
MOL001458
|
coptisine
|
320.34
|
30.67
|
0.86
|
Phellodendri Chinrnsis Cortex
|
MOL002636
|
Kihadalactone A
|
512.70
|
34.21
|
0.82
|
Phellodendri Chinrnsis Cortex
|
MOL013352
|
Obacunone
|
454.56
|
43.29
|
0.77
|
Phellodendri Chinrnsis Cortex
|
MOL002641
|
Phellavin_qt
|
374.42
|
35.86
|
0.44
|
Phellodendri Chinrnsis Cortex
|
MOL002643
|
delta 7-stigmastenol
|
414.79
|
37.42
|
0.75
|
Phellodendri Chinrnsis Cortex
|
MOL002644
|
Phellopterin
|
300.33
|
40.19
|
0.28
|
Phellodendri Chinrnsis Cortex
|
MOL002651
|
Dehydrotanshinone II A
|
292.35
|
43.76
|
0.40
|
Phellodendri Chinrnsis Cortex
|
MOL002652
|
delta7-Dehydrosophoramine
|
242.35
|
54.45
|
0.25
|
Phellodendri Chinrnsis Cortex
|
MOL002656
|
dihydroniloticin
|
458.80
|
36.43
|
0.81
|
Phellodendri Chinrnsis Cortex
|
MOL002659
|
kihadanin A
|
486.56
|
31.60
|
0.70
|
Phellodendri Chinrnsis Cortex
|
MOL002660
|
Niloticin
|
456.78
|
41.41
|
0.82
|
Phellodendri Chinrnsis Cortex
|
MOL002662
|
rutaecarpine
|
287.34
|
40.30
|
0.60
|
Phellodendri Chinrnsis Cortex
|
MOL002663
|
Skimmianin
|
259.28
|
40.14
|
0.20
|
Phellodendri Chinrnsis Cortex
|
MOL002666
|
Chelerythrine
|
332.37
|
34.18
|
0.78
|
Phellodendri Chinrnsis Cortex
|
MOL000449
|
Stigmasterol
|
412.77
|
43.83
|
0.76
|
Phellodendri Chinrnsis Cortex
|
MOL002668
|
Worenine
|
334.37
|
45.83
|
0.87
|
Phellodendri Chinrnsis Cortex
|
MOL002670
|
Cavidine
|
353.45
|
35.64
|
0.81
|
Phellodendri Chinrnsis Cortex
|
MOL002671
|
Candletoxin A
|
608.79
|
31.81
|
0.69
|
Phellodendri Chinrnsis Cortex
|
MOL002672
|
Hericenone H
|
580.88
|
39.00
|
0.63
|
Phellodendri Chinrnsis Cortex
|
MOL002673
|
Hispidone
|
472.78
|
36.18
|
0.83
|
Phellodendri Chinrnsis Cortex
|
MOL000358
|
beta-sitosterol
|
414.79
|
36.91
|
0.75
|
Phellodendri Chinrnsis Cortex
|
MOL000622
|
Magnograndiolide
|
266.37
|
63.71
|
0.19
|
Phellodendri Chinrnsis Cortex
|
MOL000762
|
Palmidin A
|
510.52
|
35.36
|
0.65
|
Phellodendri Chinrnsis Cortex
|
MOL000785
|
palmatine
|
352.44
|
64.60
|
0.65
|
Phellodendri Chinrnsis Cortex
|
MOL000787
|
Fumarine
|
353.40
|
59.26
|
0.83
|
Phellodendri Chinrnsis Cortex
|
MOL000790
|
Isocorypalmine
|
341.44
|
35.77
|
0.59
|
Phellodendri Chinrnsis Cortex
|
MOL000098
|
quercetin
|
302.25
|
46.43
|
0.28
|
Phellodendri Chinrnsis Cortex
|
MOL001131
|
phellamurin_qt
|
356.40
|
56.60
|
0.39
|
Phellodendri Chinrnsis Cortex
|
MOL001455
|
(S)-Canadine
|
339.42
|
53.83
|
0.77
|
Phellodendri Chinrnsis Cortex
|
MOL001771
|
poriferast-5-en-3beta-ol
|
414.79
|
36.91
|
0.75
|
Phellodendri Chinrnsis Cortex
|
MOL002894
|
berberrubine
|
322.36
|
35.74
|
0.73
|
Phellodendri Chinrnsis Cortex
|
MOL005438
|
campesterol
|
400.76
|
37.58
|
0.71
|
Phellodendri Chinrnsis Cortex
|
MOL006392
|
dihydroniloticin
|
458.80
|
36.43
|
0.82
|
Phellodendri Chinrnsis Cortex
|
MOL006401
|
melianone
|
470.76
|
40.53
|
0.78
|
Phellodendri Chinrnsis Cortex
|
MOL006413
|
phellochin
|
488.83
|
35.41
|
0.82
|
Phellodendri Chinrnsis Cortex
|
MOL006422
|
thalifendine
|
322.36
|
44.41
|
0.73
|
Gardenia jasminoides
|
MOL001663
|
(4aS,6aR,6aS,6bR,8aR,10R,12aR,14bS)-10-hydroxy-2,2,6a,6b,9,9,12a-heptamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid
|
456.78
|
32.03
|
0.76
|
Gardenia jasminoides
|
MOL002883
|
Ethyl oleate (NF)
|
310.58
|
32.40
|
0.19
|
Gardenia jasminoides
|
MOL001506
|
Supraene
|
410.80
|
33.55
|
0.42
|
Gardenia jasminoides
|
MOL001941
|
Ammidin
|
270.30
|
34.55
|
0.22
|
Gardenia jasminoides
|
MOL001406
|
Crocetin
|
328.44
|
35.30
|
0.26
|
Gardenia jasminoides
|
MOL000358
|
beta-sitosterol
|
414.79
|
36.91
|
0.75
|
Gardenia jasminoides
|
MOL000422
|
kaempferol
|
286.25
|
41.88
|
0.24
|
Gardenia jasminoides
|
MOL001494
|
Mandenol
|
308.56
|
42.00
|
0.19
|
Gardenia jasminoides
|
MOL000449
|
Stigmasterol
|
412.77
|
43.83
|
0.76
|
Gardenia jasminoides
|
MOL001942
|
isoimperatorin
|
270.30
|
45.46
|
0.23
|
Gardenia jasminoides
|
MOL009038
|
GBGB
|
550.57
|
45.58
|
0.83
|
Gardenia jasminoides
|
MOL000098
|
quercetin
|
302.25
|
46.43
|
0.28
|
Gardenia jasminoides
|
MOL003095
|
5-hydroxy-7-methoxy-2-(3,4,5-trimethoxyphenyl)chromone
|
358.37
|
51.96
|
0.41
|
Gardenia jasminoides
|
MOL007245
|
3-Methylkempferol
|
300.28
|
60.16
|
0.26
|
Gardenia jasminoides
|
MOL004561
|
Sudan III
|
352.42
|
84.07
|
0.59
|
Component-target network of HLJDD
The corresponding targets of the main compounds of HLJDD were collected in the TCMSP database, and the results were imported into Cytoscape 3.7.2. The active ingredient-prediction target network was constructed, and 425 nodes (84 active ingredient nodes and 341 predicted target nodes) and 1801 interaction relationships were obtained, as shown in Figure 2. Among them, yellow represents the compound of Scutellaria baicalensis, green represents the compound of Coptis chinensis , red represents the compound of Phellodendron amurense, and blue represents the compound of Gardenia jasminoides.
Intersecting compounds in HLJDD
From the result of "3.2", it can be known that the same compound exists in different medicinal materials in HLJDD. It can be obtained from Funrich's Venn diagram (Figure 3). Coptis chinensis and Phellodendron amurense all have MOL001454-Berberine and MOL002894- Berberrubine; MOL000358-Beta-sitosterol is common to Scutellaria baicalensis, Phellodendron amurense and Gardenia jasminoides; MOL001458-Coptisine is common to Coptis chinensis , Scutellaria baicalensis and Phellodendron amurense; MOL002897-Epiberberine is common to Coptis chinensis and Scutellaria baicalensis;MOL000622-Magnograndiolide, MOL013352-Obacunone, MOL000785-Palmatine, MOL000762-Palmidin-A is common to Coptis chinensis and Phellodendron amurense; MOL000098-Quercetin is common to Coptis chinensis , Phellodendron amurense and Gardenia jasminoides; Scutellaria baicalensis, Phellodendron amurense and Gardenia jasminoides all have MOL000449-Stigmasterol; Scutellaria baicalensis and Gardenia jasminoides both have MOL001506-Supraene; Coptis chinensis and Phellodendron amurense both have MOL002668-Worenine.
GO, KEGG and Tissue enrichment analysis
The GO database was used to annotate the GO functional annotation of HLJDD and the Pathway analysis of reactome. GO function annotation is to annotate and classify genes through biological processes (BP), cell components (CC), and molecular function (MF), as shown in Figure 4. For each biological process, cellular components and molecular functional categories are represented by red, blue, and green bars, respectively. The height of the bar graph represents the number of genes in which the annotated genes overlap. Biological regulation, stress response, and metabolic processes are highly relevant in biological processes, mainly including nitrogen utilization regulation, cell response to vitamin E, negative regulation of chronic inflammatory responses, regulation of fibroblast differentiation, and smooth muscle cell-matrix adhesion regulation, inflammatory apoptotic process and blood pressure regulation, etc. The proportion of cell membrane, nucleus, and cytoplasmic matrix in the cell components is relatively high, and protein binding, ion binding, and molecular sensor activity have a great impact on molecular functions.
KEGG pathway enrichment screen obtained 306 signal pathways (P <0.05), as shown in Figures 5 and 6, including nuclear receptor transcription pathway, SREBF (SREBP) activation gene expression, SREBP (SREBF) regulation of cholesterol biosynthesis, interleukin 4 And interleukin 13 signaling, RNA polymerase II transcription, universal transcription pathway, cytokine signaling in the immune system, interleukin signaling, gene expression (transcription), nuclear receptor signaling, extranuclear estrogen signaling, ESR-mediated signaling Conduction, cell senescence, estrogen-dependent nuclear events downstream of ESR membrane signals, interleukin 10 signals, intrinsic pathways of apoptosis, steroid metabolism, immune system, amine ligand binding receptors and VEGF signal transduction, etc. The immune system involved 175 related genes such as IL6, NOS2, IL10, TGFB1, AKT1, IL2, IL4, FOS, CCL2, CASP3, CXCL8, CXCL2, SOD1, ALOX5, STAT1, CXCL10, MYC, MAPK10 and MAPK14.
Further carry out tissue enrichment analysis on targets. As shown in Figure 7, tissue enrichment revealed that targets expression sites are mainly distributed in lung tissue, liver, and placenta, and involve a variety of immune cells, such as T cells and B cells. It shows that the key targets of the active ingredients of HLJDD are mainly expressed in lung tissues and immune cells.
Component-target molecular docking
It is generally believed that the lower the energy is, the more stable conformation of the ligand-receptor binding is, the more likely the interaction is. The results of molecular docking showed that the affinity of the core active compounds in HLJDD to SARS-COV-2 3CL hydrolase, ACE2 and TMPRSS2 was less than zero, which indicated that the core active compounds in HLJDD could spontaneously bind to SARS-COV-2 3CL hydrolase, ACE2, TMPRSS2. The results are shown in table 2.The result of molecular docking showed that the binding energy of ACE2 with stigmasterol was -9.1KJ/Mol.And the binding energy of SARS-COV-2 3CL hydrolase and TMPRSS2 with beta sitosterol was -7.5KJ/Mol and 8.4KJ/Mol respectively. The results show that the conformation of the Ligand and its receptor is the most stable. The binding ability of the major components in HLJDD to the SARS-COV-2 3CL hydrolase, ACE2, TMPRSS2 is lower than that of the effective drugs reported so far, which indicates that the major components in HLJDD bind to the SARS-COV-2 3CL hydrolase, ACE2, TMPRSS2 more stable and have more possibility to play a role. The docking results are shown in Figure 8-1,2,3.
Table 2
The binding energy values of the core compounds and SARS-CoV-2 3CL hydrolase in HLJDD and effective drugs reported clinically
Receptor
|
ligand
|
Formula
|
MW(g/mol)
|
binding energy(kJ/mol)
|
SARS-CoV-2 3CL hydrolase
|
Stigmasterol
|
C29H48O
|
412.70
|
-4.13
|
SARS-CoV-2 3CL hydrolase
|
beta_sitosterol
|
C29H50O
|
414.70
|
-3.85
|
SARS-CoV-2 3CL hydrolase
|
abidol
|
C30H40O6
|
496.60
|
-3.14
|
SARS-CoV-2 3CL hydrolase
|
kaempferol
|
C15H10O6
|
286.24
|
-3.11
|
SARS-CoV-2 3CL hydrolase
|
quercetin
|
C15H10O7
|
302.23
|
-3.08
|
SARS-CoV-2 3CL hydrolase
|
lopinavir
|
C37H48N4O5
|
628.80
|
-1.53
|
SARS-CoV-2 3CL hydrolase
|
daruinavir
|
C27H37N3O7S
|
547.66
|
-1.39
|
SARS-CoV-2 3CL hydrolase
|
supraene
|
C30H50
|
410.72
|
-0.85
|
SARS-CoV-2 3CL hydrolase
|
ritonavir
|
C37H48N6O5S2
|
720.94
|
0.05
|