Can Yin-Chai-Xiao-Du decoction be useful of COVID-19? the mechanism research based on network pharmacology

ARTICLE
TMR | July 2020 | vol. 5 | no. 4 | 188
doi: 10.12032/TMR20200601185
Submit a manuscript: https://www.tmrjournals.com/tmr
Traditional Chinese Medicine
Can Yin-Chai-Xiao-Du decoction be useful of COVID-19? the
mechanism research based on network pharmacology
Lu Yang1#
, Ning Li2#
, Hai-Bo Hu3
, Bin Yin3
, Guo-Jing Zhao3
, Feng-Chan Wang3
, Xu-Hui Wang4
, Hong-Wu Wang5
, Xue-Chao
Lu3*
, Huan-Tian Cui6*
1
Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China; 2
First Clinical Hospital,
Shandong University of Traditional Chinese Medicine, Jinan 250000, China; 3
Department of Pulmonary Disease in
Qingdao Hospital of Traditional Chinese Medicine, Qingdao 266700, China; 4
College of Traditional Chinese Medicine,
Shandong University of Traditional Chinese Medicine, Jinan 250000, China; 5
College of Traditional Chinese Medicine,
Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China; 6
Shandong Provincial Key Laboratory of Animal
Cell and Developmental Biology, School of Life Sciences, Shandong University, Qingdao 250100, China.
#
These authors are co-first authors on this work.
*Corresponding to: Xue-Chao Lu. Department of Pulmonary Disease in Qingdao Hospital of Traditional Chinese Medicine,
No.4 Renmin Road, North District, Qingdao 266700, China. E-mail: hospitalbreathing@163.com; Huan-Tian Cui.
Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences, Shandong
University, No.72 Binhai Road, Jimo District, Qingdao 250100, China. E-mail: 1762316411@qq.com.
Highlights
Our current study explored the potential mechanisms of Yin-Chai-Xiao-Du decoction, an empirical formula
of traditional Chinese medicine derived from the classic ancient prescription Gan-Lu-Xiao-Du decoction,
on COVID-19 and found its core herbs.
Traditionality
The empirical formula Yin-Chai-Xiao-Du decoction has evolved based on the classic ancient prescription of
Gan-Lu-Xiao-Du decoction, which was first mentioned in Wei Zhixiu’s Xu Mingyi Lei'an (Supplement to
Classified Case Records of Celebrated Physicians) in the Qing Dynasty of China that was released in
approximately 1770 C.E. Gan-Lu-Xiao-Du decoction could alleviate fever, cough and fatigue, which were
the major clinical outcomes of COVID-19. However, the mechanisms of action of Yin-Chai-Xiao-Du
decoction in the treatment of COVID-19 remain unclear.

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Abstract
Background: In this study, we preliminarily investigated the mechanism of Yin-Chai-Xiao-Du decoction for the
treatment of COVID-19 by the method of network pharmacology. Methods: The potential targets and pathways of
Yin-Chai-Xiao-Du decoction for the treatment of COVID-19 were examined using network pharmacology; the
ingredient and active targets of Yin-Chai-Xiao-Du decoction were collected from the Traditional Chinese Medicine
Systems Pharmacology Database and Analysis Platform and PharmMapper databases; the COVID-19-related
targets were obtained from the online Mendelian inheritance in man, GeneCards, and GeneMANIA databases; the
STRING database and Cytoscape were used to build a protein-protein interaction network, and a Network Analyzer
tool was used to perform topology analysis to screen for the key ingredients and targets; the ClueGO and KOBAS
3.0 databases were for the enrichment analysis of gene function (Gene Oncology) and gene pathway (Kyoto
Encyclopedia of Genes and Genomes); the herb-ingredient-target-pathway network diagram was constructed by
Cytoscape. Results: The core herbs screened by the network pharmacological analysis were Jinyinhua (Lonicerae
japonicae flos), Lianqiao (Forsythia suspensa), Chaihu (Bupleuri radix), Huangqin (Scutellariae radix), Yinchen
(Herba Artemisiae Scopariae), Guanghuoxiang (Pogostemonis herba), Roudoukou (Semen myristicae) and
Qinghao (Artemisiae annuae herba). A total of 293 active ingredients were screened by Traditional Chinese
Medicine Systems Pharmacology Database and Analysis Platform, and the key ingredients were quercetin,
kaempferol, isorhamnetin, stigmasterol, beta-sitosterol, and luteolin. Yin-Chai-Xiao-Du decoction has 138
COVID-19-related targets, and the key targets were mitogen-activated protein kinase 3, interleukin-6, tumor
necrosis factor, vascular endothelial growth factor A, and CC motif ligand 2. Kyoto Encyclopedia of Genes and
Genomes analysis revealed 120 enriched gene pathways, and the key pathways were signaling by interleukins,
immune system, cytokine signaling in the immune system, and the signaling pathways of interleukin-17, tumor
necrosis factor, and relaxin. Conclusion: The core herbs of Yin-Chai-Xiao-Du decoction are Jinyinhua (Lonicerae
japonicae flos), Lianqiao (Forsythia suspensa), Chaihu (Bupleuri radix), Huangqin (Scutellariae radix), Yinchen
(Herba Artemisiae Scopariae), Guanghuoxiang (Pogostemonis herba), Roudoukou (Semen myristicae) and
Qinghao (Artemisiae annuae herba). The key ingredients are quercetin, kaempferol, isorhamnetin, stigmasterol,
and beta-sitosterol; the critical targets are luteolin, interleukin-6, mitogen-activated protein kinase 3, tumor necrosis
factor, and CC motif ligand 2; and the core signaling pathways are those mediated by interleukin-17, tumor
necrosis factor, and relaxin.
Keywords: Coronavirus disease 2019, Bupleuri radix, Scutellariae radix, Artemisiae annuae herba, Cytokine
Storm, Tumor necrosis factor, Interleukin-6
Author contributions:
Lu Yang and Ning Li contributed to study concept; Hai-Bo Hu and Bin Yin contributed to study design and
performance; Guo-Jing Zhao, Feng-Chan Wang and Xu-Hui Wang contributed to analysis of data; Hong-Wu
Wang, Lu Yang and Ning Li contributed to drafting of the paper; Xue-Chao Lu and Huan-Tian Cui contributed to
study supervision.
Competing interests:
The authors declare no conflicts of interest.
Acknowledgments:
This study was supported by the National Key Research and Development Program (No. 2018YFC1704800) and
the Chinese Medicine Standardization Project of State Administration of Traditional Chinese Medicine (No.
SATCM-2015-BZ125).
Abbreviations:
COVID-19, coronavirus disease 2019; TNF, tumor necrosis factor; OB, oral bioavailability; DL, drug-like; GO,
Gene Oncology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK3, mitogen-activated protein kinase
3; ACE2, angiotensin I converting enzyme 2; IL, interleukin; PPI, protein-protein interaction; CCL2, CC motif
ligand 2.
Citation:
Lu Yang, Ning Li, Hai-Bo Hu, et al. Can Yin-Chai-Xiao-Du decoction be useful of COVID-19? the mechanism
research based on network pharmacology. Traditional Medicine Research 2020, 5 (4): 188–200.
Executive editor: Rui-Wang Zhao.
Submitted: 13 May 2020, Accepted: 28 May 2020, Online: 09 June 2020.

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Background
In February 2020, the World Health Organization
announced the coronavirus disease 2019 (COVID-19)
as a public health emergency of international concern
[1], and its main manifestations are fever, dry cough,
fatigue, and pharyngalgia. More so, pneumonia, acute
respiratory distress syndrome, and septic shock
characterized some of the severe cases [2]. According
to the latest real-time statistics of the World Health
Organization on April 28, 2020, there were 2,924,722
confirmed COVID-19 cases in total globally, with
more than 200,000 cumulative death cases. Sadly,
symptomatic and supportive treatments are the main
methods for the disease as there are no specific drugs
currently and the vaccines are under trials [3]. Many
scholars have pinned their hopes on antiviral drugs
such as redoxivir and chloroquine. Wang et al. showed
that redoxivir and chloroquine have considerable
antiviral effects in the COVID-19-infected African
green monkey kidney cell line VeroE6 in vitro [4].
However, in a randomized clinical trial published on
April 20, 2020, in Lancet, redoxivir did not improve
the recovery of the COVID-19 patients or decrease
mortality compared with the placebo control group [5].
In addition, there is no sufficient clinical evidence for
the anti-COVID-19 effect of chloroquine. Moreover,
both medications have some adverse effects.
Multi-ingredient, multi-target, and multi-path
characterized the traditional Chinese medicine
compound. Network pharmacology is a regular method
for investigating the mechanisms of traditional Chinese
medicine for treating COVID-19. We can find the
relationship between the traditional Chinese medicine
decoction and diseases by constructing the
ingredient-target-pathway-disease network, identifying
the key targets and submodules, and collectively
analyzing the biological functions of the key targets
and submodules in the network. He [7] showed that the
empirical formula Xuebijing injection can treat
COVID-19 by regulating the key targets of tumor
necrosis factor (TNF), PRKCB, and RELA, and
controlling the signaling pathways of NF-κB and
hypoxia inducible factor 1. The main active ingredients
of the Xiaochaihu decoction can also regulate
mitogen-activated protein kinase 3 (MAPK3), Th17,
and other pathways; inhibit inflammation; modulate
immune function; and reduce lung injury [8].
Yin-Chai-Xiao-Du decoction is a recommended
recipe for patients with severe COVID-19 by the
Guidelines for the Prevention and Treatment of
Pulmonary Dampness Plague of Taiyin (Coronavirus
Pneumonia) [11] and is derived from the ancient
prescription Gan-Lu-Xiao-Du decoction.
Gan-Lu-Xiao-Du decoction was first mentioned in Wei
Zhixiu’s Xu Mingyi Lei'an (Supplement to Classified
Case Records of Celebrated Physicians) in the Qing
Dynasty that was released in approximately 1770 C.E.
The prescription was composed of Huangqin
(Scutellariae radix), Yinchen (Herba Artemisiae
Scopariae), Guanghuoxiang (Pogostemonis herba),
Lianqiao (Forsythia suspensa), Shichangpu (Acorus
tatarinowi), Roudoukou (Semen myristicae), Bohe
(mint), Mutong (Akebia quinata), Shegan (blackberry
lily), and Chuanbeimu (Fritillaria cirrhosa). In
addition, it is commonly used for treating acute
infectious diseases [9]. Yin-Chai-Xiao-Du decoction
has evolved based on the formula of Gan-Lu-Xiao-Du
decoction. It is composed of Chaihu (Bupleuri radix),
Huangqin (Scutellariae radix), Jinyinhua (Lonicerae
japonicae flos), Lianqiao (Forsythia suspensa),
Shichangpu (Acorus tatarinowi), Yujin (Radix
curcumae), Guanghuoxiang (Pogostemonis herba),
Peilan (Herba Eupatorii), Yinchen (Herba Artemisiae
Scopariae), Roudoukou (Semen myristicae), Danggui
(Radix angelicae sinensis), Mudanpi (Cortex moutan
radicis), Yiyiren (Coicis semen), Shishangbai
(Selaginella doederleinii hieron), and Gancao
(Glycyrrhizae radix et rhizoma). In clinical application,
patients with COVID-19 mainly show a low fever after
the fever is reduced. Therefore, Chaihu (Bupleuri radix)
in the original prescription is replaced by Qinghao
(Artemisiae annuae herba) to subside the low fever
after the fever is reduced. This study reveals the
possible molecular mode of action of
Yin-Chai-Xiao-Du decoction for treating COVID-19
through network pharmacology (Figure 1).
Figure 1 The composition chart of Yin-Chai-Xiao-Du decoction

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Materials and methods
Virtual screening of active ingredients of
Yin-Chai-Xiao-Du decoction
We searched the Traditional Chinese Medicine
Systems Pharmacology Database and Analysis
Platform (http://ibts.hkbu.edu.hk/lsp/tcmsp.php) using
the keywords of Bupleuri radix, Scutellariae radix,
Lonicerae japonicae flos, Forsythia suspensa, Acorus
tatarinowi, Radix curcumae, Pogostemonis herba,
Herba Eupatorii, Herba Artemisiae Scopariae, Semen
myristicae, Radix angelicae sinensis, Cortex moutan
radicis, Coicis semen, Selaginella doederleinii hieron,
Glycyrrhizae radix et rhizoma, and Artemisiae annuae
herba to get the candidate ingredients of
Yin-Chai-Xiao-Du decoction. To obtain the active
ingredients of Yin-Chai-Xiao-Du decoction, these
ingredients were further screened by the criteria of oral
bioavailability (OB) ≥ 30% and drug-like (DL) ≥ 0.18
[10].
Potential target prediction and gene name
annotation
The chemical ingredients of Yin-Chai-Xiao-Du
decoction were entered into the PubChem database
(https://pubchem.ncbi.nlm.nih.gov/) by name, and the
3D structures were identified and stored in .sdf format.
These data were then uploaded to the PharmMapper
platform server
(http://www.lilab-ecust.cn/pharmmapper/) [11] to
complete the target prediction of the chemical
composition and obtain the relevant targets of the
active ingredients. The UniProt database
(https://www.uniprot.org/) was used to standardize the
target proteins as per the active ingredients and obtain
the gene names as per the targets.
Screening of disease targets
The online Mendelian inheritance in man database [12]
(http://omim.org/) and GeneCards database [13]
(https://www.genecards.org) were searched for
relevant targets using the keyword “COVID-19”.
Concurrently, the coexpressed gene of severe acute
respiratory syndrome coronavirus 2 for the human
receptor angiotensin I converting enzyme 2 (ACE2)
was identified from GeneMANIA [14]
(http://genemania.org/) to obtain the disease-related
targets. The Venny platform (version 2.1,
http://bioinfogp.cnb.csic.es/tools/venny/) was used to
intersect the targets of Yin-Chai-Xiao-Du decoction
and the disease and further obtain the targets for the
treatment of COVID-19.
Protein-protein interaction (PPI) network
Via the STRING database, the PPI was formed by the
intersecting targets from drug and disease, and the PPI
network was constructed with Cytoscape 3.7.2
(http://www.cytoscape.org/). Then we used the
MCODE plugin to perform cluster analysis of the PPI
network. Proteins can hardly achieve their biological
and physiological functions alone, so the similarly
related groups, called protein complexes or functional
modules, accomplish specific cellular tasks. MCODE
[16] is a clustering algorithm that can quickly detect
densely connected areas in a large-scale target network
and score the correlation of targets in the module. To
select highly related clusters, the parameter K-Core
was set at 2, i.e., the identified module contains at least
three edges.
Gene Oncology (GO) genetic biological process
analysis
Cytoscape’s ClueGO plugin was used to perform gene
function GO enrichment analysis on the clusters
obtained from common targets. The ClueGO parameter:
“Homo Sapiens” in the control panel and “GO
biological process” in “Ontologies/Pathways” in
“ClueGO Settings” were selected, and only GO entries
with P ≤ 0.01 were shown.
Kyoto Encyclopedia of Genes and Genomes (KEGG)
gene pathway enrichment analysis
The intersecting targets of the herb and disease were
imported into the KOBAS 3.0 database
(http://kobas.cbi.pku.edu.cn) for pathway analysis.
Settings: “Gene Symbol” in “Type” and “Homo
Sapiens” in “Species” were selected, “KEGG” was
checked, P ≤ 0.05, and further pathway enrichment
analysis was performed on the genes identified in the
screen.
Construction of “herb-ingredient-target-pathway”
network
Cytoscape 3.7.2 was used to construct a network
diagram. We first developed the network and property
tables of “herb-active-ingredient-key-target-path”, and
then imported them into the Cytoscape for drawing. In
the output result, the herb, active ingredients, targets,
and pathways were shown as colored and shaped nodes.
The ingredients and potential targets were connected
by edges and so were the target proteins and pathways.
Results
Search results of the chemical constituents of
Yin-Chai-Xiao-Du decoction
A total of 1,992 active ingredients were obtained from
the screening, including Chaihu (Bupleuri radix) 288,
Huangqin (Scutellariae radix) 58, Jinyinhua
(Lonicerae japonicae flos) 236, Lianqiao (Forsythia
suspensa) 150, Shichangpu (Acorus tatarinowi) 105,
Yujin (Radix curcumae) 222, Guanghuoxiang
(Pogostemonis herba) 94, Peilan (Herba Eupatorii) 60,
Yinchen (Herba Artemisiae Scopariae) 53, Roudoukou
(Semen myristicae) 71, Danggui (Radix angelicae

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sinensis) 125, Mudanpi (Cortex moutan radicis) 55,
Yiyiren (Coicis semen) 38, Shishangbai (Selaginella
doederleinii hieron) 30, Gancao (Glycyrrhizae radix et
rhizoma) 280 and Qinghao (Artemisiae annuae herba)
127. Further, all the chemical constituents found were
screened with OB ≥ 30% and DL ≥ 0.18, which
revealed 293 active ingredients that met the screening
criteria, as shown in Figure 2 and Table 1.
Search results of the targets of the active
ingredients
The names of the active ingredients identified by the
screen were entered into the PubChem database, and
the obtained structures of the ingredients were
imported into the PharmMapper to predict the targets.
A total of 3,758 targets were obtained, whereas 168
targets remained after deduplication. Table 1 shows the
ingredients, active ingredients, and targets of herbs
identified in the search and screen.
Searching for disease genes and screening of key
targets
We obtained 1,196 COVID-19 targets from the online
Mendelian inheritance in man database, 251
COVID-19 targets from the GeneCards database, and
5,556 ACE2 coexpressed genes from the GeneMANIA.
Then, by intersecting the targets of the active
ingredients of the herbs with the disease targets, we
finally obtained 138 possible targets as per the
treatment of fever by Yin-Chai-Xiao-Du decoction.
Cytoscape was used to construct the target interaction
network of Yin-Chai-Xiao-Du decoction, including
138 nodes and 2,640 edges in the model. The target
network model is characterized as the average degree
of node connection of 23, the clustering coefficient of
0.554, and the network density of the topological
structure of 0.355. As shown in Figure 3 the darker the
color and the larger the size, the denser the targets are.
Accordingly, interleukin (IL)-6, MAPK3, JUN, TNF,
VEGFA, and CC motif ligand 2 (CCL2) had higher
degrees and stronger interactions with other proteins
(Figure 3).
Further cluster analysis of 138 nodes was conducted
using the MCODE plugin, and six clusters were
obtained (Figure 4). An analysis of the biological
functions (GO) of the clusters using ClueGO plugin
showed that the mechanisms were related to smooth
muscle adaptation, positive regulation of cyclase
activity, regulation of protein deacetylation, regulation
of muscle cell apoptotic process, response to reactive
oxygen species, cellular response to chemical stress,
and cellular response to oxidative stress, etc.
Results of KEGG gene pathway enrichment
analysis
Using the KOBAS 3.0 database, 120 enriched
pathways were detected and the top 20 were chosen as
per the P-value. The results showed that the targets
were remarkably enriched pathways in terms of
multiple signaling pathways of ILs, immune system,
cytokine in the immune system, neuroactive
ligand-receptor interaction, IL-17, TNF, and relaxin
(Figure 5).
Figure 2 The number of main chemical constituents of each herb in Yin-Chai-Xiao-Du decoction

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Table 1 The number of targets of chemical constituents in Yin-Chai-Xiao-Du decoction
Herb name Number of active ingredient Number of targets
Chaihu (Bupleuri radix) 17 336
Huangqin (Scutellariae radix) 24 558
Jinyinhua (Lonicerae japonicae flos) 23 207
Lianqiao (Forsythia suspense) 23 209
Shichangpu (Acorus tatarinowi) 4 114
Yujin (Radix curcumae) 15 90
Guanghuoxiang (Pogostemonis herba) 11 220
Peilan (Herba Eupatorii) 11 147
Yinchen (Herba Artemisiae Scopariae) 13 395
Roudoukou (Semen myristicae) 12 343
Danggui (Radix angelicae sinensis) 2 95
Mudanpi (Cortex moutan radicis) 11 185
Yiyiren (Coicis semen) 9 87
Shishangbai (Selaginella doederleinii hieron) 4 27
Gancao (Glycyrrhizae radix et rhizoma) 92 237
Qinghao (Artemisiae annuae herba) 22 508
Figure 3 PPI map of common targets. Each node represents a target. The darker color and larger size indicate a
higher degree of nodes, which are more possible to be key targets. The proteins with higher degrees include
MAPK3, JUN, TP53, IL-6, TNF, VEGFA, CCL2, and EGF, which all have stronger interactions with other
proteins.

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Figure 4 The six clusters in the target network of Yin-Chai-Xiao-Du decoction. Each node represents a target
and each edge represents an interaction relationship. The highly correlated nodes are enriched into a subcluster. The
darker the color and the larger the size of the node, the higher its degree value is. Each cluster contains at least three
edges.
Figure 5 KEGG enrichment analysis of the targets of Yin-Chai-Xiao-Du decoction. The abscissa is the
negative logarithm of the P-value, and a longer band means a higher correlation; the ordinate is the name of the
KEGG pathway.
Construction of the herb-ingredient-target-pathway
network
A table of “herb-ingredient-target-pathway” network
was developed and imported into Cytoscape 3.7.2
software. The red triangle nodes represent drugs, blue
rectangle nodes represent pathways, and green ellipse
nodes represent active ingredients. The
herb-ingredient-target-pathway of Yin-Chai-Xiao-Du
decoction for the treatment of COVID-19 was
constructed by the targets with orange rectangle nodes.
As shown in the figure, each active ingredient acted on

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multiple targets. The more connected nodes and the
higher correlations, the more likely it is a key
ingredient, and it is then at a core position in the
mechanisms of Yin-Chai-Xiao-Du decoction for the
treatment of COVID-19. After the network analysis,
we found that quercetin, kaempferol, isorhamnetin,
stigmasterol, beta-sitosterol, and luteolin were the
top-ranked core ingredients (Figure 6).
We selected the ingredients of the top-ranked 10
targets, and the herb-ingredient network constructed by
Cytoscape showed that the ingredients with the highest
degrees were quercetin, kaempferol, isorhamnetin,
stigmasterol, beta-sitosterol, and luteolin. Among them,
quercetin and kaempferol were shared by Jinyinhua
(Lonicerae japonicae flos), Lianqiao (Forsythia
suspensa), Chaihu (Bupleuri radix), and Qinghao
(Artemisiae annuae herba); stigmasterol and
isorhamnetin were shared by Chaihu (Bupleuri radix),
Huangqin (Scutellariae radix) and Qinghao
(Artemisiae annuae herba); luteolin and beta-sitosterol
were shared by Jinyinhua (Lonicerae japonicae flos)
and Lianqiao (Forsythia suspensa); quercetin and
isorhamnetin were shared by Yinchen (Herba
Artemisiae Scopariae) and Guanghuoxiang
(Pogostemonis herba); luteolin and quercetin were
shared by Roudoukou (Semen myristicae) and
Lianqiao (Forsythia suspensa). These data suggest that
Jinyinhua (Lonicerae japonicae flos), Lianqiao
(Forsythia suspensa), Chaihu (Bupleuri radix),
Huangqin (Scutellariae radix), Guanghuoxiang
(Pogostemonis herba), Yinchen (Herba Artemisiae
Scopariae), Roudoukou (Semen myristicae), and
Qinghao (Artemisiae annuae herba) are the core herbs
of Yin-Chai-Xiao-Du decoction for the treatment of
COVID-19 (Figure 7).
Figure 6 Herb-compound-target-pathway network. The red triangle nodes represent herbs, blue rectangle nodes
represent pathways, green ellipse nodes represent active ingredients, and orange rectangle nodes represent targets.

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Figure 7 Herb-top 10 ingredients network of Yin-Chai-Xiao-Du decoction. The green nodes represent the herbs,
and the blue nodes represent the core ingredients.
Discussion
Yin-Chai-Xiao-Du decoction consists of Chaihu
(Bupleuri radix), Huangqin (Scutellariae radix),
(Forsythia suspensa), Shichangpu (Acorus tatarinowi),
Yujin (Radix curcumae), Guanghuoxiang
(Pogostemonis herba), Peilan (Herba Eupatorii),
Yinchen (Herba Artemisiae Scopariae), Roudoukou
(Semen myristicae), Danggui (Radix angelicae
sinensis), Mudanpi (Cortex moutan radicis), Yiyiren
(Coicis semen), Shishangbai (Selaginella doederleinii
hieron), Gancao (Glycyrrhizae radix et rhizoma) and
Qinghao (Artemisiae annuae herba). The network
pharmacology analysis showed that the key ingredients
of Yin-Chai-Xiao-Du decoction for treating COVID-19
are shared by many herbs. For example, stigmasterol
and isorhamnetin are shared by Chaihu (Bupleuri radix)
and Huangqin (Scutellariae radix). Stigmasterol shows
good anti-inflammatory activity by considerably
reducing the expression of IL-1β, IL-6, MCP-1, and
COX-2 [17], and synergistic isorhamnetin can inhibit
oxidative stress caused by inflammation [18].
Kaempferol is shared by Jinyinhua (Lonicerae
japonicae flos) and Lianqiao (Forsythia suspensa).
Kaempferol can scavenge oxygen-free radicals and
reduce tissue damage caused by hypoxia [19], and
inhibit the expression of the TNF-α and IL-6
inflammatory factors by attenuating the activity of
MAPK and AKT pathways, thereby exerting an
anti-inflammatory effect [20]. Luteolin and
beta-sitosterol are shared by Roudoukou (Semen
myristicae) and Qinghao (Artemisiae annuae herba).
Luteolin can reduce the activity of catalase and
superoxide dismutase in lung tissue, thereby reducing
the level of oxidative damage and lipid peroxidation
and further alleviating acute lung injury in mice [21].
Besides, it can also enhance acute lung injury by
inhibiting the MEK/ERK and PI3K/Akt pathways and
weakening the activation of neutrophils [22].
Beta-sitosterol has a certain protective effect on acute
lung injury caused by lipopolysaccharide. It can
improve the oxidative stress response and inhibit the
accumulation and release of TNF-α inflammatory
factors [23]. In addition, it can also inhibit the
infiltration of neutrophils into tissues, reduce the
production of β-glucuronidase and superoxide, regulate
the complement system, and further regulate immunity
system [24]. Furthermore, quercetin is shared by
Yinchen (Herba Artemisiae Scopariae) and
Guanghuoxiang (Pogostemonis herba). It has the
effects of reducing lung inflammation and having
antioxidant and antiviral effects. Other studies have
shown that it can reduce the expression of TGF-β1,
α-SMA, and TNF-α, inhibit the apoptosis of rat
alveolar cells, and reduce the inflammation and
fibrosis injury of rat lung tissue. Meanwhile, it can
enhance pulmonary fibrosis by inhibiting the signal
pathway of SphK1/S1P [25–27]. These two effects are
synergistic. Jinyinhua (Lonicerae japonicae flos),
Lianqiao (Forsythia suspensa), Chaihu (Bupleuri
radix), Huangqin (Scutellariae radix), Guanghuoxiang
(Pogostemonis herba), Yinchen (Herba Artemisiae
Scopariae), Shishangbai (Selaginella doederleinii
hieron), and Qinghao (Artemisiae annuae herba) are

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TMR | July 2020 | vol. 5 | no. 4 | 197
doi: 10.12032/TMR20200601185
the core herbs for the Yin-Chai-Xiao-Du decoction that
have anti-inflammatory, antioxidant,
immunoregulation, and lung injury improvement
effects.
Using network pharmacology to screen ingredients,
predict targets, and analyze the protein network, we
found that the active ingredients of Yin-Chai-Xiao-Du
decoction could act on MAPK3, IL6, VEGFA, TNF,
and CCL2, which are at the center of the network for
treating COVID-19. The network topology analysis
showed that the treatment of COVID-19 by
Yin-Chai-Xiao-Du decoction involved multiple gene
functions and was very complex.
After the coronavirus invades the lung cells and
infects the human body by binding with the S protein
on the envelope to the ACE2 receptor [28], it is
recognized by the host cell. Subsequently, the body
regulates different molecular signaling pathways such
as the NF-κB pathway and the MAPK/JNK pathway to
trigger inflammation, resulting in the expression of
proinflammatory factors and antiviral genes [29–30].
More so, activated immune cells release a large
number of cytokines and chemokines, including
TNF-α, interleukins (IL-1, IL-6, and IL-12), and
chemokines (CCLs), leading to the initiation of
inflammatory cytokine storms, which promote the
transfer of immune cells to the primary infection site
[31]. Then, leukocytes and lymphocytes are recruited
and attracted by the boosted cytokines and chemokines
to the lesion sites. They are continuously activated and
proliferated in attempting to eliminate the virus by
releasing inflammatory mediators [32]. Several
immune cells and tissue fluids gather in the lungs to
regulate the key genes, such as vascular endothelial
growth factor, which changes the permeability of the
blood vessel and leads to airway obstruction, lung
inflammation, and edema. The patient will experience
critical conditions such as extreme difficulty in
breathing, blood oxygen desaturation, unconsciousness
that further leads to multiple organ failure and even
death [33]. Therefore, it is speculated that COVID-19
may activate the IL receptor and the MAPK pathway,
increase the expression of inflammatory cytokines
such as TNF-α and IL-6, and incur a local
inflammatory response, therefore resulting in cytokine
storms and immune disorders. Yin-Chai-Xiao-Du
decoction may play an anti-inflammatory role by
inhibiting these proinflammatory cytokines. It can
modulate immune functions by influencing the number
of immune cells and prevent multiple organ failures
caused by endogenous inflammatory mediators to
reduce lung damage.
Meanwhile, COVID-19 patients have congestion
and edema of the respiratory tract mucosa caused by a
large number of inflammatory cell infiltration,
angiectasis, and increased exudation. The large
quantity of mucus secretion can be retained in the
bronchus and block the normal function of tracheal
smooth muscle, producing symptoms of cough,
expectoration, and asthma [34]. GO enrichment
analysis showed that the targets of Yin-Chai-Xiao-Du
decoction were remarkably enriched in the biological
processes of smooth muscle adaptation, cell response
to reactive oxygen species, and cellular response to
chemical stress.
KEGG enrichment showed that the treatment of
COVID-19 by Yin-Chai-Xiao-Du decoction involved
multiple pathways such as the anti-inflammatory and
immune regulation pathways of IL-17 and TNF. Via
corresponding receptors, the IL-17 family transmits
signals to activate downstream pathways such as
NF-κB and MAPKs. The IL-17 family can induce the
expression of antibacterial peptides, cytokines, and
chemokines, which plays a vital role in acute and
chronic inflammatory reactions [35]. The TNF
signaling pathway mainly acts on inhibiting the
cytokines in the immune response and inflammatory
storm activated by COVID-19, alleviating the immune
response, and eliminating the inflammation. TNF can
bind to the recombinant human type I TNFR1 and
TNFR2, which further mediate inflammation and
immune regulation via the cascade reaction of the
NF-κB pathway and MAPK [36].
Furthermore, the KEGG results showed that the core
targets were enriched in the relaxin signaling pathway,
which acts on COVID-19 mainly through the
following four aspects. First, severe COVID-19 cases
in the recovery stage can have pulmonary fibrosis for a
long time. Specifically, cases with acute respiratory
distress syndrome have a poorer prognosis [37]. Mou
et al. [38] examined the lung CT of 24 patients with
COVID-19. They found that the lesions began to be
absorbed and showed fibrosis on the lung CT of the
severe and critical patients. Relaxin has anti-fibrotic
effects, which have been validated in non-germinal
tissue, such as the lung, heart, kidney, and liver tissue
[39]. Secondly, relaxin can promote the regeneration of
muscle fibers by inhibiting inflammation and fibrosis
and participate in the healing of injured ligaments and
skeletal muscles [40]. Most patients with COVID-19
are complicated with a concurrent condition of
myalgia, which can be relieved by relaxin. More so,
clinical studies have shown that the levels of serum
myocardial necrosis markers increased regardless of
the patient’s condition [41], indicating that COVID-19
may damage cardiac muscle cells. Experiments
showed that relaxin could effectively prevent and
reverse many adverse reactions in cardiovascular
animal models (ischemic/reperfusion injury,
myocardial infarction, hypertensive heart disease, and
cardiomyopathy), and serelaxin, a recombinant form of
relaxin, is in the extended phase III clinical trial for
acute heart failure [42]. Finally, Jordan et al. [43] have
confirmed that relaxin can prevent the death of brain
cells under hypoxia conditions and may have a direct
protective effect on nerves, which help to protect the

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TMR | July 2020 | vol. 5 | no. 4 | 198
doi: 10.12032/TMR20200601185
brain tissue of patients with COVID-19 under hypoxia.
Interestingly, in this network pharmacology study,
chlorogenic acid and saikoside were not selected for
subsequent analysis because of the low OB value
(chlorogenic acid: 11.93% vs. screen criteria: OB ≥ 30)
and low DL value (saikoside: 0.09 vs. screen criteria:
DL ≥ 0.18). Besides, baicalein and artemisinin were
not selected for subsequent analysis because their
numbers of targets were 33 and 28, respectively, and
ranked 17 and 44, respectively, among all ingredients.
Nevertheless, more pharmacological evidence has
shown that these ingredients also have
anti-inflammatory, antiviral, and immunity regulation
effects [44–51]; however, their values for COVID-19
treatment need to be further studied.
Conclusion
In summary, the core herbs of Yin-Chai-Xiao-Du
decoction for the treatment of COVID-19 are
(Forsythia suspensa), Chaihu (Bupleuri radix),
Huangqin (Scutellariae radix), Yinchen (Herba
Artemisiae Scopariae), Guanghuoxiang (Pogostemonis
herba), Roudoukou (Semen myristicae), and Qinghao
(Artemisiae annuae herba). The key ingredients
include quercetin, kaempferol, isorhamnetin,
stigmasterol, beta-sitosterol, and luteolin. The core
targets are IL-6, MAPK3, TNF, CCL2, and the core
signaling pathways are those of IL-17, TNF, and
relaxin.
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