molecules

Article
Hepatoprotective Effects of a Functional Formula of
Three Chinese Medicinal Herbs: Experimental
Evidence and Network Pharmacology-Based
Identification of Mechanism of Action and Potential
Bioactive Components
Sha Li 1 , Ning Wang 1,2 , Ming Hong 1 , Hor-Yue Tan 1 , Guofeng Pan 1,3 and Yibin Feng 1,2, *
1 School of Chinese Medicine, The University of Hong Kong, Hong Kong, China;
u3003781@connect.hku.hk (S.L.); ckwang@hku.hk (N.W.); hong1986@connect.hku.hk (M.H.);
hyhtan@hku.hk (H.-Y.T.); pan.gf1218@163.com (G.P.)
2 Shenzhen Institute of Research and Innovation, The University of Hong Kong, Shenzhen 518057, China
3 Beijing Shijitang Hospital, Capital Medical University, Beijing 100069, China
* Correspondence: yfeng@hku.hk; Tel.: +852-2589-0482; Fax: +852-2872-5476

Received: 20 December 2017; Accepted: 5 February 2018; Published: 7 February 2018

Abstract: Various Chinese herbal medicines (CHMs) have shown beneficial liver protection effects.
Jian-Gan-Bao (JGB), a functional herbal formula, consists of three famous CHMs, including
Coriolus versicolor, Salvia miltiorrhiza and Schisandra chinensis, which has been used as a folk medicine
for several chronic liver diseases. In the present study, we aim systemically to evaluate the effects of
JGB on acute and chronic alcoholic liver diseases (ALD) as well as non-alcoholic fatty liver disease
(NAFLD) in mouse models, and identify its potential bioactive components and mechanism of action.
JGB showed preventive effects for acute and chronic ALD as well as NAFLD, while post-treatment
of JGB showed no significant effect, suggesting the nature of JGB as a health supplement rather
than a drug. Furthermore, a compound-target network was constructed to identify the potential
bioactive compounds and pathways that regulate its hepatoprotective effects. There are 40 bioactive
compounds and 15 related targets that have been identified via this network pharmacology study.
Among them are miltirone, neocryptotanshinone II and deoxyshikonin, with desirable pharmaceutical
properties. Pathways relating to inflammation, fatty acid oxidation, tumor necrosis factor (TNF)
production and cell proliferation were predicted as bioactive compounds and potential underlying
mechanisms, which should be the focus of study in this field in the future.

Keywords: Coriolus versicolor; Salvia miltiorrhiza; Schisandra chinensis; function herbal formula; liver
disease; network pharmacology

1. Introduction
Liver diseases, affecting more than 10% of the world population, remains one of the most serious
public health concerns worldwide [1]. Among the various forms of liver disease, alcoholic liver
disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the two most common types with
high prevalence around the world [2]. ALD, resulting from chronic excessive alcohol consumption,
is one of the most important causes of liver-related death, accounting for an estimated 3.8% of global
mortality [3]. NAFLD, a condition caused by deposited fat in the liver generally due to a person
being obese or overweight, is the most common liver disorder in developed countries. Both ALD
and NAFLD may progress from simple steatosis to hepatitis, fibrosis and cirrhosis [4]. Although
considerable progress has been made in understanding these pathologies, there has been little or no

Molecules 2018, 23, 352; doi:10.3390/molecules23020352 www.mdpi.com/journal/molecules

Molecules 2018, 23, 352 2 of 17

development of new effective therapies for these diseases [3–5]. Therefore, there is an urgent need for
an effective and safe treatment for the prevention and treatment of ALD and NAFLD. In particular,
increasingly evidence indicates that dietary health supplements, such as herbs, fruits and other natural
products, play a beneficial role in preventing hepatic disorder or diseases in a major way through
anti-oxidation and anti-inflammation [1].
Many herbs in Chinese herbal medicines (CHMs) are homologous with food, and have
various bioactivities, such as antioxidant, anti-inflammatory, anticancer and hepatoprotective effects.
Jian-Gan-Bao (JGB), a herbal combination of three famous CHMs, Coriolus versicolor (Yunzhi in Chinese),
Salvia miltiorrhiza (Danshen) and Schisandra chinensis (Wuweizi), has been used as a complementary and
alternative treatment for liver diseases [6]. Yunzhi, Danshen, and Wuweizi are three widely used herbs
in CHMs. Polysaccharides are the main active components identified in Yunzhi, and possess potent
anti-viral, antineoplastic, and immunomodulatory activity in vitro [7–10]. Yunzhi has been used as
adjuvant therapy in cancer patients to boost human immune responses [11,12]. The hepatoprotective
effects of Danshen against various pathogenic factors have been revealed in extensive studies [13–16].
Wuweizi also protects against liver diseases [17–20]. For example, biphenyl dimethyl-dicarboxylate,
a compound isolated from Wuweizi, has been used for the treatment of hepatitis [21]. JGB is a generic
name of the combined formula of Yunzhi, Danshen and Wuweizi. The term Jian-Gan-Bao in Chinese
means liver-function improver. This combination has been commonly used as a herbal supplement to
improve liver function. Therefore, the formulation consisting of these three herbs is considered to be
beneficial for people with liver disorder or disease.
Although JGB has been used in folk medicine for protection against and treatment of several
chronic liver diseases, scientific evidence on its therapeutic effect is still not available. This investigation
aims to systemically evaluate the effects of JGB on acute and chronic ALD as well as NAFLD in mouse
models, and to explore the mechanism underlying the liver-protective effects of this formula. To further
understand the action of mechanism and potential active components of JGB, we applied network
pharmacology approaches in this study. Network pharmacology is an approach based on systems
biology, poly-pharmacology and molecular networks, which has been extensively applied to analyze
relationships between drugs and diseases in recent decade [22,23]. In particular, it has attracted
considerable attention among Chinese medicine researchers for its ability to predict and illustrate
interactive relationships between numerous components and targets of CHMs [24,25]. Network-based
pharmacological analysis is a desirable approach for investigating the mechanisms of action for herbs
and formulae and their potential bioactive components at molecular and systematic levels. In addition,
it is a good tool of in silico prediction of the potential active components and action mechanisms of
herbal medicines, which renders more effective subsequent exploration with experimental approaches.
Network-based pharmacological analysis is a desirable approach for investigating the mechanisms of
action for herbs and formulae and their potential bioactive components at molecular and systematic
levels [25,26].

2. Results

2.1. The Preventive and Curative Effect of Jian-Gan-Bao (JGB) on Acute Alcoholic Liver Disease (ALD)
The preventive and curative potential of JGB on acute ALD was studied. In the preventive
treatment course, JGB exhibited protective effects against alcohol-induced liver damage in mice,
as evidenced by reduced aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
activities as well as histological scores (Figure 1A–C). JGB could significantly reduce the lipid
peroxidation end product malondialdehyde (MDA) (Figure 1D). However, we did not observe any
significant activation of the internal anti-oxidative system, including superoxide dismutase (SOD),
catalases (CAT) and glutathione peroxidase (GSH-Px) (Figure 1E–G). Treatment of JGB could reduce
the recovery time of mice from being drunk, but it was not statistically significant (result not shown).
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In addition,
InIn
addition, in the
inin
addition, the curative
curative
the treatment
treatmentcourse,
curativetreatment course,JGB
course, JGBshowed
JGB showed non-significant
showed non-significant therapeutic
non-significant effect
therapeuticeffect
therapeutic on
effecton liver
onliver
liver
damage
damage
damage(Figure
(Figure
(Figure2A–C).
2A–C).
2A–C).

Figure1. 1. The
Figure The preventive
preventive effect effectofofJian-Gan-Bao
Jian-Gan-Bao(JGB) (JGB) onon acute
acutealcoholic liverliver
alcoholic disease (ALD).
disease (A)
(ALD).
Figure
Serum
1. alanine
The preventive effect of (ALT)
aminotransferase
Jian-Gan-Bao
levels of
(JGB)
mice
on
from
acute alcoholic
different
liver(B)
groups;
disease
Serum
(ALD).
aspartate
(A)
(A) Serum alanine aminotransferase (ALT) levels of mice from different groups; (B) Serum aspartate
Serum alanine aminotransferase
aminotransferase (AST) levels of(ALT)
mice levels different
of mice from different groups; (B)and Serum aspartate
aminotransferase (AST) levels of mice fromfromdifferent groups; groups; (C) Hematoxylin
(C) Hematoxylin eosin
and eosin (H&E) (H&E)
staining
aminotransferase
staining images (AST)
and levels
scoring ofof mice
liver from
histology different
of mice groups;
from (C)
different Hematoxylin
groups; (D) and eosin
Malondialdehyde(H&E)
images and scoring of liver histology of mice from different groups; (D) Malondialdehyde (MDA)
staining
(MDA) images
levels and scoring of liver histology of mice from different groups; (D) Malondialdehyde
levels in liver ofin liverfrom
mice of mice from different
different groups; (E) groups; (E) Superoxide
Superoxide dismutase dismutase (SOD)inlevels
(SOD) levels liver in
of liver
mice
(MDA)
of micelevels
fromindifferent
liver of groups;
mice from (F) different
Catalase groups;
(CAT) (E) Superoxide
levels in liver of dismutase
mice from (SOD) levels
different in liver
groups; (G)
from different groups; (F) Catalase (CAT) levels in liver of mice from different groups; (G) Glutathione
of Glutathione
mice from different
peroxidase groups; (F) Catalase
(GSH-Px) levels in (CAT)
liver of levels
mice in liver
from of mice
different from (JGB-L:
groups. different groups;dose
JGB-low (G)
peroxidase (GSH-Px) levels in liver of mice from different groups. (JGB-L: JGB-low dose group, JGB-M:
Glutathione
group, peroxidase
JGB-M: (GSH-Px)
JGB-middle levelsJGB-H:
group, in liverJGB-high
of mice from different
group). Threegroups. (JGB-L:
biological JGB-lowwere
replicates dose
JGB-middle group, JGB-H: JGB-high group). Three biological replicates were performed for each study.
group, JGB-M: each
JGB-middle * pgroup, JGB-H: 0.01,JGB-high group).with Three biological
group. replicates were
* pperformed
< 0.05, ** pfor study.compared
< 0.01, when < 0.05, with
** p < model when
group.compared model
performed for each study. * p < 0.05, ** p < 0.01, when compared with model group.

Figure 2. Cont.
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Figure
Figure 2. The curative effect of JGBJGB
on acute ALD. (A) Serum ALT ALT
levelslevels
of mice from different groups;
Figure 2.2. The
The curative
curative effect
effect of on acute
of JGB on acute ALD.
ALD. (A) Serum
(A) Serum ALT levels ofofmice
micefrom
fromdifferent
different
(B) Serum(B)
groups; AST levels oflevels
mice offrom different groups;groups;
(C) H&E staining images and scoring of liver
groups; (B) Serum AST levels of mice from different groups; (C) H&E staining images and scoring ofof
Serum AST mice from different (C) H&E staining images and scoring
histology
liver of mice from different groups.groups.
(JGB-L:(JGB-L:
JGB-low dose group, JGB-M:JGB-M:
JGB-middle group,
liver histology
histology ofof mice
mice from different
from different groups. (JGB-L: JGB-low
JGB-low dose group,
dose group, JGB-middle
JGB-M: JGB-middle
JGB-H:
group, JGB-high
group, JGB-H: group).
JGB-H: JGB-high Three
JGB-high group). biological replicates
Three biological
group). Three were
biological replicatesperformed
replicateswere for
wereperformed each
performedfor study.
foreach
eachstudy.
study.

2.2. Effect
2.2.
2.2. Effectofof
Effect ofJGB
JGBon
JGB onChronic
on ChronicPlus
Chronic PlusBinge
Plus Binge ALD
Binge ALD
ALD
After
After successfully
Aftersuccessfully establishing
successfullyestablishing the chronic
establishing the
the chronic plus
chronic binge
plus binge
bingeALD ALD model,
ALDmodel,
model,thethe therapeutic
thetherapeutic
therapeutic potential
potential
potential ofofof
JGB
JGBJGBonon
on chronic
chronic
chronicALD ALD
ALDwaswasstudied.
studied. We
We observed
observed that
observed JGB
that JGB consistently
JGBconsistently relieves
consistentlyrelieves chronic
relieveschronic
chronic plus
plus
plus binge
binge
binge
ALD,
ALD, with
ALD,with reduced
withreduced
reducedALTALT
ALTandand AST
AST activities
activities as
activities as well
well as
well as lower
as histological
lower histological scores
histologicalscoresscoresininin treated
treated
treated mice
micemice
(Figure 3A–C). The microsteatosis and inflammatory cells’ infiltration was significantly relievedbyby
(Figure
(Figure 3A–C).
3A–C). The
The microsteatosis
microsteatosis and inflammatory
inflammatory cells’
cells’ infiltration
infiltration was
was significantly
significantly relieved
relieved by
JGB
JGBJGB treatment
treatment
treatment (Figure
(Figure
(Figure 3C).
3C). Additionally,
Additionally, JGB
JGBJGB could
could
could reduce
reduce
reduce MDA MDA
MDA level
levellevel ininliver
in the theliver
the liver(Figure
(Figure (Figure 3D).
3D).
3D). Similar
toSimilar
Similar to
to the
the observation the observation
in the acuteinmodel,
observation the acute model,
model,
we found weJGB
we
that found
foundhadthatthat JGB had
JGB
no potent had noon
no
effect potent
potent effecton
effect
the activities on the
ofthe
SOD
activities
activities of
of SOD
SOD and
and CAT (Figure 3E–F).
3E–F). However,
However, the
the activity
activity of
of
and CAT (Figure 3E–F). However, the activity of GSH-Px was significantly increased by high-dose GSH-Px
GSH-Px was
was significantly
significantly
increased
increased
treatment ofby
byJGBhigh-dose
high-dose treatment
(Figure 3G). of JGB
Overall, (Figure
(Figure
similar 3G).
3G).
to the Overall,similar
Overall,
observation similar
in thetotothe
theobservation
acute observation
model, JGB in inthe
had the acute
noacute
potent
model,
model,
effect JGB
on JGB had no potent
had no potent
the internal effect on the
anti-oxidative system internal
internal anti-oxidative
of theanti-oxidative
liver. system of the liver.
system of the liver.

Figure
Figure The
3. 3. Theeffect
effectof of
JGB onon
JGB chronic ALD.
chronic (A)(A)
ALD. ALTALTlevels of mice
levels of micefrom different
from groups;
different (B) (B)
groups; ASTAST levels
ofFigure
mice 3. The
from effect of
different JGB on
groups; (C) chronic
H & E ALD. (A)images
staining ALT levels
and of miceoffrom
scoring liver different
histology;
levels of mice from different groups; (C) H & E staining images and scoring of liver histology; (D)
groups;
(D) MDA(B) AST
levels
inlevels
liver of mice
of mice from
from different
different groups;
groups; (C)
(E) H
SOD& Elevels
staining
in images
liver of and scoring
mice from
MDA levels in liver of mice from different groups; (E) SOD levels in liver of mice from different
of livergroups;
different histology;
(F) (D)
CAT
MDA
levels in levels
liver in
of liver
mice of
frommice from
different different
groups; groups;
(G) GSH-Px(E) SOD
levels levels
in liverin liver
of miceof mice
from from different
different groups.
groups; (F) CAT levels in liver of mice from different groups; (G) GSH-Px levels in liver of mice from
groups;JGB-low
(JGB-L: (F) CATdoselevelsgroup,
in liverJGB-M:
of miceJGB-middle
from different groups;
group, (G) GSH-Px
JGB-H: JGB-high levels in liver
group). of mice
Three from
biological
different groups. (JGB-L: JGB-low dose group, JGB-M: JGB-middle group, JGB-H: JGB-high group).
different
replicates groups. (JGB-L:
were performed JGB-low dose group, JGB-M:
p <each JGB-middle
** p <* 0.01, group, JGB-H:
*** p**<p 0.001 JGB-high group).
Three biological replicates for
were each study. *for
performed 0.05,study. p < 0.05, < 0.01,when
*** p compared
< 0.001 when with
Threegroup.
model biological replicates were performed for each study. * p < 0.05, ** p < 0.01, *** p < 0.001 when
compared with model group.
compared with model group.
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2.3.The
2.3. TheHepato-Protective
Hepato-ProtectiveEffect
EffectofofJGB
JGBon
onNon-Alcoholic
Non-AlcoholicFatty
FattyLiver
LiverDisease
Disease(NAFLD)
(NAFLD)Combined
Combinedwith
with
CCl Fibrosis
CCl4 Fibrosis
4

Thetherapeutic
The therapeuticpotential
potential of JGB
of JGB on NAFLD
on NAFLD combined
combined with4 fibrosis
with CCl CCl4 fibrosis was studied.
was studied. The
The results
results showed that the level of ALT was significantly reduced by treatment
showed that the level of ALT was significantly reduced by treatment of JGB, although the dose manner of JGB, although the
dosenot
was manner was not
significant significant
(Figure 4A). (Figure 4A). Furthermore,
Furthermore, JGB could dose-dependently
JGB could dose-dependently reduce AST reduce
levelsASTin
levels in NAFLD mice (Figure 4B). Therefore, JGB possessed preventive
NAFLD mice (Figure 4B). Therefore, JGB possessed preventive action on the progression of fatty action on the progression of
fatty liver disease. The pathological features and histological scores of H&E
liver disease. The pathological features and histological scores of H&E staining indicated that liver staining indicated that
liver inflammation
inflammation was relieved
was relieved by JGBby JGB treatment
treatment (Figure(Figure
4C). To 4C).
studyTohepatic
study hepatic lipid deposition,
lipid deposition, oil-red
oil-red staining was conducted. Scoring revealed results consistent
staining was conducted. Scoring revealed results consistent with the serum test, indicatingwith the serum test,
theindicating
potential
the
of potential
JGB of JGB
in relieving in relieving
fatty fatty4D).
liver (Figure liverIn(Figure 4D).the
particular, In particular, the macrovesicular
macrovesicular steatosis as shown steatosis
in theas
shown in the model group was obviously improved in the JGB group. As
model group was obviously improved in the JGB group. As shown in Figure 4C,D, the lipidosis and shown in Figure 4C,D, the
lipidosishollow
adipose and adipose
space washollow spaceobviously
reduced was reduced obviously
by JGB treatment by (Figure
JGB treatment
4C,D). In (Figure
mice 4C,D).
receivingIn mice
JGB
receiving JGB administration,
administration, liver lobules andliver liverlobules
sinusoid and liver
were sinusoid
obvious, andwere
few obvious,
fat vacuoles andwere
few observed
fat vacuoles in
were observed in hepatic cells. In addition, to assess if JGB could inhibit
hepatic cells. In addition, to assess if JGB could inhibit fibrosis, we conducted picrosirius-red (PSR)fibrosis, we conducted
picrosirius-red
staining. The red(PSR) staining. collagen
stain indicated The red deposition,
stain indicated whichcollagen deposition,
is the clinical markerwhich is the
of fibrosis. clinical
Reduced
markerwere
scores of fibrosis.
found in Reduced
JGB-treated scores wereindicating
groups, found inthat JGB-treated groups,treatment
herbal medicine indicating thatreduce
could herbal
medicine treatment could reduce fibrosis (Figure 4E). Hepatic MDA
fibrosis (Figure 4E). Hepatic MDA level was dose-dependently reduced in mice treated with JGB level was dose-dependently
reduced
(Figure in mice
4F). treated
Treatment of with JGB minimal
JGB had (Figure 4F). Treatment
effects on hepaticof JGB
SOD had
andminimal effects on
CAT activities hepatic
(Figure SOD
4G,H).
and CAT activities (Figure 4G,H). However, it could particularly induce
However, it could particularly induce GSH-Px (Figure 4I), indicating the involvement of anti-oxidative GSH-Px (Figure 4I),
indicating the involvement
GSH-Px in its protective effect. of anti-oxidative GSH-Px in its protective effect.

Figure 4. Cont.
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Figure
Figure 4. TheTheeffect
effectofofJGBJGB onon non-alcoholic
non-alcoholic fattyfatty
liverliver disease
disease combined
combined with
with CCl CCl4 fibrosis.
4 fibrosis. (A)
(A) Serum
ALT levels
Serum ALToflevels
mice of
from
mice different groups; groups;
from different (B) Serum (B)AST
Serum levels
ASToflevels
mice from different
of mice groups; (C)
from different H&E
groups;
staining
(C) H&E images
stainingand scoring
images andof liver histology
scoring of mice from
of liver histology different
of mice groups; (D)
from different Oil-red
groups; (D)OOil-red
stainingO
images and
staining imagesscoring
and of liver histology
scoring of mice from
of liver histology of micedifferent groups; (E)
from different Picrosirius-red
groups; staining
(E) Picrosirius-red
images and
staining imagesscoring
and of liver histology
scoring of mice from
of liver histology of mice different groups; groups;
from different (F) MDA(F)levels
MDAinlevels
liver of
in mice
liver
from
of micedifferent groups;groups;
from different (G) SOD (G)levels
SOD of miceoffrom
levels micedifferent groups;
from different (H) CAT
groups; (H) levels of mice
CAT levels from
of mice
different
from groups;
different (I) GSH-Px
groups; levels levels
(I) GSH-Px of miceoffrom
micedifferent groups.groups.
from different (JGB-L:(JGB-L:
JGB-low dose group,
JGB-low dose JGB-M:
group,
JGB-middle
JGB-M: group, JGB-H:
JGB-middle group,JGB-high group). Three
JGB-H: JGB-high group).biological replicates replicates
Three biological were performed for each study.
were performed for
* p < study.
each 0.05, ***pp<<0.01, ** pp << 0.01,
0.05,*** 0.001***when compared
p < 0.001 whenwith modelwith
compared group.model group.

2.4. A
A Network
Network Pharmacology
Pharmacology Approach
Approach to Predict Potential Active Compounds and Action Mechanisms
To furtherexplore
To further explorewhich which components
components of JGB
of JGB maymay be responsible
be responsible for thefor the hepatoprotective
hepatoprotective effects,
effects,
a networka network pharmacology
pharmacology study was study was performed.
performed. The workflow
The workflow of the network
of the network pharmacology
pharmacology analysis
analysis
of JGB isofshown
JGB is in shown
Figure in 5.
Figure 5. Firstly,
Firstly, knownknown compound compound
targetstargets of Danshen
of Danshen (769), (769),
Wuweizi Wuweizi
(930),
(930), and Yunzhi
and Yunzhi (319) obtained
(319) were were obtained from BATMAN-TCM
from BATMAN-TCM (Supplemental
(Supplemental Table
Table S1). A S1).
totalAoftotal
124 of 124
genes
genes
relatedrelated
to liverto liver diseases
diseases werewere obtained
obtained from
from PubMed
PubMed database(Supplemental
database (Supplemental Table Table S1). Then,
Then,
19 common targets in Danshen and and liver
liver diseases,
diseases, 26 26 common
common targets
targets inin Wuweizi
Wuweizi and and liver
liver diseases,
diseases,
55 common
common targets
targets in Yunzhi and liver diseases were recognized by VENNY 2.1 (Supplemental
Figure S2). Afterwards,
Afterwards, 56 56 responsible
responsible compounds
compounds in in Danshen
Danshen (19),(19), Wuweizi
Wuweizi (35),(35), and
and Yunzhi
Yunzhi (2) (2) for
for
these 30 30targets
targets related
related to diseases
to liver liver diseases were identified
were identified based on BATMAN-TCM.
based on BATMAN-TCM. Then, pharmaceuticalThen,
pharmaceutical
properties analysis properties analysisaccording
was performed was performed
to their according to their oral
oral bioavailability (OB)bioavailability (OB) and
and Caco-2 permeability.
Caco-2 permeability.
As suggested As suggested
by the traditional by the
Chinese traditional
medicine Chinese
systems medicine systems
pharmacology (TCMSP)pharmacology
database and
(TCMSP)
Hong et al. database
(2017), theandthresholds
Hong et used al. (2017), the compounds
as active thresholds used as active
screening compounds
criteria were OB ≥screening
30% and
criteria
Caco ≥ were
0.4. ForOBseveral
≥ 30%compounds
and Caco ≥that 0.4. are
Fornotseveral
includedcompounds
in TCMSP that are notbut
database included
are found in TCMSP
in TCM
database but are found
Database@Taiwan in TCM Database@Taiwan
(http://tcm.cmu.edu.tw/), (http://tcm.cmu.edu.
Lipinski’s Rule (LR) was used tw/), for
Lipinski’s Rule (LR)
active compound
was used for according
identification active compound identification
to the following criteria:according
moleculartoweight
the following
(MW) ≤criteria: molecular
500, chemical weight
composition
(MW)
with not≤ 500,
morechemical
than ten composition
hydrogen bond with not more
acceptors than≤ten
(Hacc 10),hydrogen
lesser thanbond acceptors bond
five hydrogen (Haccdonors
≤ 10),
lesser than five hydrogen bond donors (Hdon ≤ 5), and octanol-water partition
(Hdon ≤ 5), and octanol-water partition coefficient lesser than five (LogP ≤ 5) [22,27]. Compounds coefficient lesser than
five
that (LogP
did not≤ satisfy
5) [22,27]. Compounds
at least two of the that did requirements
above not satisfy at least
were two of the Then
excluded. above16 requirements
compounds were were
excluded.
excluded and Thenthe 16 remaining
compounds40were excludedincluding
compounds and the remaining 40 compounds
several compounds whose including several
phytochemical
compounds whose phytochemical information was not available in these databases were included
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information was not available in these databases were included (Table 1). Lastly, network of
(Table 1). Lastly, network of compounds-targets was constructed by Cytoscape 3.5.0 (Figure 6).
compounds-targets was constructed by Cytoscape 3.5.0 (Figure 6). Based on this network, 15 potential
Based on this network, 15 potential targets were selected and their roles in liver diseases are
targets were selected and their roles in liver diseases are summarized in Table 2. Furthermore,
summarized in Table 2. Furthermore, as revealed by the network pharmacological study, CYP2E1 is
as revealed by the network pharmacological study, CYP2E1 is a potential target of JGB on liver
a potential target of JGB on liver diseases. By detecting the hepatic protein level of CYP2E1 in the
diseases. By detecting the hepatic protein level of CYP2E1 in the ALD model, we found that the
ALD model, we found that the increased CYP2E1 induced by ethanol was significantly decreased by
increased CYP2E1 induced by ethanol was significantly decreased by JGB treatment, consistent with
JGB treatment, consistent with our network pharmacological analysis (Figure 7).
our network pharmacological analysis (Figure 7).

Figure 5. The workflow of the network pharmacological study of JGB.

Figure 5. The workflow of the network pharmacological study of JGB.

Table 1. The 56 compounds from Danshen, Wuweizi, and Yunzhi and their corresponding molecular
Table 1. The 56 compounds from Danshen, Wuweizi, and Yunzhi and their corresponding molecular
properties, OB and Caco.
properties, OB and Caco.
Phytochemical
Phytochemical MW AlogPMW AlogP
Hdon Hdon
Hacc HaccOB OB
(%)(%) Caco-2
Caco-2 Herb
Herb No.
Tanshiquinone
Tanshiquinone BB 280.318 3.769
280.318 1
3.769 1 3 3 DS1
DS1
Miltirone
Miltirone 282.41 4.73
282.41 0
4.73 0 2 2 38.76
38.76 1.23
1.23 DS2
DS2
Miltirone
Miltirone II 312.36 3.353
312.36 1
3.353 1 4 4 DS3
DS3
Dehydromiltirone 280.39 4.29 0 2 24.57 1.14 DS5
Dehydromiltirone 280.39 4.29 0 2 24.57 1.14 DS5
Neocryptotanshinone II 270.35 3.61 1 3 39.46 0.76 DS6
Neocryptotanshinone II 270.35 3.61 1 3 39.46 0.76 DS6
Neotanshinone C 252.265 3.061 1 3 DS4
Neotanshinone C 252.265 3.061 1 3 DS4
Ferruginol 286.452 6.004 1 1 DS7
Ferruginol
Isotenulin 306.354 286.452
1.804 6.004
0 1 5 1 DS7
DS8
Isotenulin
Dihydrokaranone * 218.37 306.354
4.17 1.804
0 0 1 5 28.86 1.39 DS8*
DS9
Salvinone
Dihydrokaranone * 218.37 4.17 0 1 28.86 1.39 DS10
DS9 *
Salviol *
Salvinone 302.50 4.74 2 2 24.31 0.95 DS11
DS10*
Danshensu
Salviol * * 198.19 0.71
302.50 4
4.74 2 5 2 36.91
24.31 -0.27
0.95 DS12
DS11 **
1-Hydroxytaxinine A 492.55 1.453 2 9 DS13
Danshensu * 198.19 0.71 4 5 36.91 −0.27 DS12 *
Cryptoxanthin * 552.96 10.76 1 1 25.16 1.84 DS14 *
1-Hydroxytaxinine A 492.55 1.453 2 9 DS13
Heteratisine 391.50 0.454 2 6 DS15
Cryptoxanthin * 552.96 10.76 1 1 25.16 1.84 DS14 *
Gamma-Sitosterol 414.70 8.084 1 1 DS16
Heteratisine
Isocucurbitacin D 516.67 391.50
1.647 0.454
4 2 7 6 DS15
DS17
Ursolicacid * 456.78 6.47 2 3 17.7 0.56 DS18 *
Samaderin A 330.33 0.054 1 6 DS19
Vitamin B2 * 376.41 0.23 5 10 6.79 −1.22 YZ1 *
Carotene YZ2
Deoxyshikonin 272.32 3.50 2 4 73.85 0.74 WWZ1
Molecules 2018, 23, 352 8 of 17

Table 1. Cont.

Phytochemical MW AlogP Hdon Hacc OB (%) Caco-2 Herb No.

Gamma-Sitosterol 414.70 8.084 1 1 DS16
Isocucurbitacin D 516.67 1.647 4 7 DS17
Ursolicacid * 456.78 6.47 2 3 17.7 0.56 DS18 *
Samaderin A 330.33 0.054 1 6 DS19
Vitamin B2 * 376.41 0.23 5 10 6.79 −1.22 YZ1 *
Carotene YZ2
Deoxyshikonin 272.32 3.50 2 4 73.85 0.74 WWZ1
Nonylphenol WWZ2
(E)-9-Isopropyl-6-Methyl-5,9-Decadiene-2-One 208.34 4.065 0 1 WWZ3
Epiguaipyridine 215.37 3.95 0 1 36.98 1.66 WWZ4
Nootkatone 218.37 3.61 0 1 33.04 1.36 WWZ5
1-Methyl-4-Methylethenylcyclohexene WWZ6
Gamma-Selinene * 204.39 4.95 0 0 22.58 1.84 WWZ7 *
Beta-Chamigrene 204.39 4.71 0 0 31.99 1.82 WWZ8
Clovene 204.39 4.09 0 0 46.49 1.80 WWZ9
Thujopsene 204.39 4.08 0 0 53.81 1.85 WWZ10
Beta-Bisabolene * 204.39 5.33 0 0 29.59 1.88 WWZ11 *
Beta-Pinene 136.26 2.93 0 0 44.77 1.85 WWZ12
Beta-Sesquiphellandrene 218.42 5.39 0 0 30.58 1.82 WWZ13
Myrcene * 136.26 3.69 0 0 24.96 1.84 WWZ14 *
Alpha-Limonene 136.26 3.5 0 0 39.84 1.83 WWZ15
Epsilon-Cadinene * 204.39 4.85 0 0 16.41 1.82 WWZ16 *
D-Limonene WWZ17
Camphene 136.26 2.93 0 0 34.98 1.81 WWZ18
Isolongifolene 204.39 4.08 0 0 46.32 1.8 WWZ19
Longifolene 204.39 4.18 0 0 39.49 1.83 WWZ20
Sesquicarene WWZ21
Clupanodonic Acid 276.46 5.50 1 2 44.01 1.22 WWZ22
1-(1,5-Dimethyl-4-Hexenyl)-4-Methyl Benzene 208.38 5.775 0 0 WWZ23
Isolychnose * 664.60 −7.593 14 20 WWZ24 *
Elemicin * 208.28 2.79 0 3 21.94 1.41 WWZ25 *
Terpinen-4-Ol 154.28 2.55 1 1 81.41 1.36 WWZ26
Delta-Terpineol 154.28 2.47 1 1 55.11 1.28 WWZ27
Linalool 170.28 1.43 1 2 49.73 0.86 WWZ28
3-Phenyldecane * 218.42 6.22 0 0 4.73 1.89 WWZ29 *
Phenyl-2-Propanone 134.19 1.46 0 1 39.66 1.32 WWZ30
Phenylpropyl Alcohol 136.21 2 1 1 36.57 1.18 WWZ31
Campherenol * 222.41 3.80 1 1 18.25 1.35 WWZ32 *
1-Phenyl-1,3 Butanedion 162.18 1.48 0 2 WWZ33
Longispinogenin * 472.83 5.31 3 3 16.07 0.35 WWZ34 *
Myricadiol * 442.80 6.21 2 2 13.58 0.78 WWZ35 *
Note: * compound does not satisfy the screening criteria.
 transcription factor 1 production
Activates several intracellular pathways to regulate
TNF Tumor necrosis factor
inflammation, cell death, and proliferation
CD34 CD34 protein Facilitate cell migration, dedifferentiation
Inflammation, wound healing, hepatocyte damage,
TGF-β
Molecules 2018, 23, 352
Transforming growth factor-β 9 of 17
hepatocyte proliferation, fibrogenesis

Figure
Figure6. Compound-target
6. Compound-target network
networkof JGB thatthat
of JGB is associated withwith
is associated ALDALDand NAFLD. The red
and NAFLD. Theellipses
red
represent liver disease target genes (26); the blue rectangles (14) are candidate compounds
ellipses represent liver disease target genes (26); the blue rectangles (14) are candidate compounds from
Danshen; the yellow
from Danshen; therectangles (25) are candidate
yellow rectangles (25) are compounds from Wuweizi;
candidate compounds fromthe green rectangle
Wuweizi; (1) is
the green
therectangle
candidate (1)compound from compound
is the candidate Yunzhi; thefrom
greyYunzhi;
lines represent
the grey the compound-target
lines interaction. This
represent the compound-target
network comprises
interaction. 66 nodescomprises
This network (26 target66
genes
nodesand(2640target
candidate
genescompounds). The compound
and 40 candidate codes
compounds). Theare
defined in Table
compound 1. are defined in Table 1.
codes

Table 2. The information of major potential targets of JGB in liver diseases.

Target Gene Target Protein Biological Effects Associated with Liver Diseases
CYP2E1 Cytochrome P450 2E1 Oxidative stress and fatty acid oxidation
Peroxisome proliferator-activated Integrator of transcription repression and nuclear
PPARD
receptor delta receptor signaling, lipid accumulation
IGF1 Insulin-like growth factor 1 Activators of the AKT signaling pathway, cell death
TPO Thyroid peroxidase Primary regulator of platelet production
ANXA1 Annexin 1 Anti-inflammation
ADIPOQ Adiponectin Modulates glucose regulation and fatty acid oxidation
Molecules 2018, 23, 352 10 of 17

Table 2. Cont.

Target Gene Target Protein Biological Effects Associated with Liver Diseases
Anticoagulant protein, marker in determining the
PROC Protein C
severity of the liver disease
Mediate the interaction between immune cells and
CCL5 Chemokine CCL5
hepatic stellate cells
FOXP3 Forkhead box P3 Regulatory T cells, immune cells regulation
IL10 Interleukin-10 Modulate Kupffer cells, liver inflammation and fibrosis
IL6 Interleukin-6 Pro-inflammation and anti-inflammation
Sterol regulatory element-binding
SREBF1 Glucose metabolism, fatty acid and lipid production
transcription factor 1
Activates several intracellular pathways to regulate
TNF Tumor necrosis factor
inflammation, cell death, and proliferation
CD34 CD34 protein Facilitate cell migration, dedifferentiation
Inflammation, wound healing, hepatocyte damage,
TGF-β Transforming growth factor-β
Molecules 2018, 23, x FOR PEER REVIEW hepatocyte proliferation, fibrogenesis 10 of 16

Figure
Figure 7. The hepatic
7. The hepatic protein
protein level
level of
of CYP2E1
CYP2E1 induced
induced by
by ethanol
ethanol was
was significantly
significantly decreased
decreased by
by JGB
JGB
treatment. * p < 0.05, **** p < 0.0001 when compared with model group.
treatment. * p < 0.05, **** p < 0.0001 when compared with model group.

3. Discussions and
3. Discussions and Future
Future Perspectives
Perspectives
In this study,
In this study, JGB
JGB showed
showed preventive
preventive effects
effects against acute and
against acute and chronic
chronic ALD,
ALD, as as well
well as
as NAFLD.
NAFLD.
However,
However, JGB had no significant effect after onset of acute alcoholic liver injury. This suggests that
JGB had no significant effect after onset of acute alcoholic liver injury. This suggests that
short-term
short-term administration
administrationof ofJGB
JGBmay
maybe beinadequate
inadequatefor fora atherapeutic
therapeutic effect. Repeated
effect. Repeated consumption
consumption as
preventive
as preventivemeasure is suggested.
measure This also
is suggested. further
This alsoreflects
furtherthereflects
nature of theJGB as a health
nature of JGBsupplement but
as a health
not a drug. Inbut
supplement thenot
aspect of mechanism
a drug. In the aspectof action, JGB potently
of mechanism activated
of action, JGBGSH-Px
potentlyinactivated
NAFLD, which
GSH-Px may
in
partially
NAFLD, explain its protective
which may partially effect.
explainAsitsoxidative
protective stress hasAs
effect. been considered
oxidative ashas
stress a conjoint pathological
been considered as
mechanism in the initiation
a conjoint pathological and promotion
mechanism of liver injury,
in the initiation JGB mightofexert
and promotion liverhepato-protective
injury, JGB mighteffects
exert
through
hepato-protective effects through anti-oxidant [28]. However, in the setting to
anti-oxidant [28]. However, in the setting of ALD, JGB did not appear ofactivate
ALD, JGBthe internal
did not
anti-oxidative enzymes
appear to activate system. As
the internal lipid peroxidation
anti-oxidative enzymes was still reduced,
system. As lipidtheperoxidation
pathways relating to
was still
reduced, the pathways relating to anti-oxidative stress such as the NF-κB pathway, the
sirtuin-FOXO pathway, or the Nrf-2/ARE pathway may be activated by phytochemicals of JGB,
which may have cross-talk with lipid metabolism [29].
Based on the study of network pharmacology, a multitude of compounds and underlying
mechanisms has been predicted. Specifically, 40 bioactive compounds and 15 related targets have
Molecules 2018, 23, 352 11 of 17

anti-oxidative stress such as the NF-κB pathway, the sirtuin-FOXO pathway, or the Nrf-2/ARE pathway
may be activated by phytochemicals of JGB, which may have cross-talk with lipid metabolism [29].
Based on the study of network pharmacology, a multitude of compounds and underlying
mechanisms has been predicted. Specifically, 40 bioactive compounds and 15 related targets have
been identified. Among these 40 compounds, miltirone, neocryptotanshinone II and deoxyshikonin
showed very desirable pharmaceutical properties. Miltirone, a central benzodiazepine receptor partial
agonist from Danshen, might interact with PROC, ADIPOQ, ADRB2, FOXP3, ANXA1, and CCL5,
which may deliver hepatoprotective effects through mediating inflammation and fatty acid oxidation,
leading to the reduced lipid peroxidation observed in our experimental data. Another compound,
neocryptotanshinone II, known as 6,12-dihydroxyabieta-5,8,11,13-tetraen-7-one, is a diterpenoid
isolated from Danshen [30]. Structurally, it is very similar to neocryptotanshinone, which possesses
anti-inflammatory properties [31,32]. They might also have the similar physicochemical, biological
and pharmacological properties. In this network pharmacology study, it is shown to interact
with many vital targets including PROC, IL10, IL6, TNF, IL4, Foxp3, CD34, ANXA1, and CCL5.
5,8-Dihydroxy-2-(4-methyl-3-pentenyl)naphthoquinone, commonly known as Deoxyshikonin, was
initially identified from Lithosperraum erythrorhizon Sieb. et Zucc and was also found to present in
Wuweizi. Deoxyshikonin is considered to be a promising drug candidate for treatment of lymphatic
diseases and wound healing [33], but its application in liver diseases is still not available. Interestingly,
it can also interact with PROC. PROC, a type of anticoagulant protein, is usually used as a marker in
determining the severity of the liver disease [34]. It is the only common gene targeted by Danshen,
Wuweizi, and Yunzhi. We have also found that the hepatoprotective effect of JGB was better than
single herb (data not shown), suggested the collective action of these compounds on PROC. It was
reported that PROC was elevated in patients having fatty liver diseases due to increased hepatic
insulin resistance. These phytochemicals, miltirone, neocryptotanshinone ii and deoxyshikonin
might regulate lipid metabolism through mediating PROC [34], which should be validated by
subsequent experimental approaches. Moreover, among these 15 associated proteins, ADIPOQ,
ADRB2, FOXP3, IL10, PPARD, and PROC could negatively regulate inflammatory response; CD34,
ADIPOQ, FOXP3, and IL10 are negative regulators of tumor necrosis factor; ANXA1, IGF1, IL6, PPARD,
and TNF are associated with cell proliferation. Particularly, targets of ADIPOQ, CYP2E1, IL6, SREBF1,
and TNF are related to NAFLD. The interaction of herbal compounds on these targets and predicted
pathways deserves to be studied in detail in further study. Furthermore, as revealed by the network
pharmacological study, CYP2E1 is a potential target of JGB on liver diseases. Particularly, CYP2E1 plays
a vital role in ALD. CYP2E1 could be induced by alcohol, which is an effective generator of reactive
oxygen species such as hydrogen peroxide and superoxide anion radical, and produces powerful
oxidants such as the hydroxyl radical, resulting in hepatic oxidative stress [3]. Thus, we speculated
that CYP2E1 might be the primary targets of JGB in ALD. By detecting the hepatic protein level of
CYP2E1 in the ALD model, we found that the increased CYP2E1 induced by ethanol was significantly
decreased by JGB treatment, consistent with our network pharmacological analysis. In addition, for the
future translational study of JGB, individual factors, such as genetic predisposition and co-morbidities,
should be carefully considered. Generally, inter-individual differences of patients result in different
responses to pharmacological treatment [35,36]. Individual factors, such as genetic predisposition and
co-morbidities, play an important role in acute and chronic liver diseases [35]. Thus, the impact of
individual factors—which include physiological factors such as age, gender and concomitant diseases,
genetic factors, and environmental factors including co-administered medications, diet, and smoking
behavior—on the therapeutic effect of JGB in liver diseases deserves to be noted and highlighted [37].
Molecules 2018, 23, 352 12 of 17

4. Materials and Methods

4.1. Reagents
Ethanol (99.9%) and carbon tetrachloride (CCl4 , 99.9%) were purchased from Thermo Scientific
(Waltham, MA, USA). Liquid ethanol diet and control liquid dextrose diet were bought from Bio-Serv
(Flemington, NJ, USA). Choline-deficiency amino acid-defined diet (CDAA) was obtained from
Research Diets (New Brunswick, NJ, USA). Kits for alanine transaminase (ALT, catalogue #2930),
aspartate transaminase (AST, catalogue #2920) were bought from Medex Supply (Passaic, NJ, USA).
Kits for malondialdehyde (MDA, catalogue #A003-1), superoxide dismutase (SOD, catalogue #A001-1),
glutathione peroxidase (GSH-Px, catalogue #A005), and catalase (CAT, catalogue #A007-1) were bought
from Nanjing Jiancheng (Nanjing, China). Direct red 80, picric acid and oil-red O were purchased
from Sigma-Aldrich (St. Louis, MO, USA). The JGB aqueous extract was obtained from Vitagreen
(Hong Kong, China), which was produced by mixing the three herbs together and extracting by
indicated solvents with standard operation in good manufacturing practice (GMP) manufacturers.

4.2. Animal Treatment

Male C57BL/6J mice 5 weeks weighting 20 ± 2 g were obtained from Laboratory Animal Unit
of The University of Hong Kong. We had 6 mice in each group, and ketamine (100 mg/kg)/xylazine
(10 mg/kg xylazine) were used to anaesthetize the animals before sacrifice. All animal experimental
protocols were approved by the Committee on the Use of Live Animals in Teaching and Research
(CULATR) of The University of Hong Kong, Hong Kong (document number: 3646-15).
To investigate the preventive effect of JGB on acute ALD, mice were given JGB extract at the dose
of 280, 840 and 2520 mg/kg via gavage for 9 days. Control and model groups of mice were treated
with an equal volume of water. Then 6 h after the last treatment, mice received ethanol (6 g/kg via
gavage). The normal group of mice received isocaloric/isovolumetric maltose-dextrin solution at the
same volume. Recovery time from drunkeness was recorded. Then 9 h after ethanol treatment, mice
were sacrificed, and samples including blood and liver were collected immediately.
The curative effect of JGB on acute ALD was studied as follows. Mice were treated with ethanol
(6 g/kg via gavage) while the control group received isocaloric/isovolumetric maltose-dextrin solution
at the same volume. Mice were given JGB extract via gavage 2 h later. Then, after 9 h, mice were
sacrificed, and samples including blood and liver were collected immediately.
To induce chronic-plus-binge alcoholic liver injury, we used the protocol as described by
Bertola et al. [38]. Briefly, mice were initially fed with control liquid dextrose diet for 5 days for
acclimation. Afterwards, the mice received a liquid ethanol diet for 11 days. On the last day, mice were
given a single dose of ethanol (5 g/kg) via gavage. Normal mice received the control liquid dextrose
diet for 11 days. For the herbal medicine treatment, mice were given JGB extract via gavage daily
throughout the experiment. Similarly, 9 h after the final ethanol treatment, mice were sacrificed to
collect blood and liver samples immediately.
For the establishment of the NAFLD model, mice were fed with a control chow or CDAA diet
for 6 weeks. A low dose of CCl4 (0.4 µL/kg b.w., twice/week) was used to promote fibrosis. As the
CDAA diet combined with CCl4 injection mimics the pathological spectrum of chronic liver disease
well, we applied this model thereafter to study the effects of JGB on the progress from liver steatosis to
fibrosis. For the herbal medicine treatment, mice were given JGB extract via gavage daily throughout
the experiment. At the endpoint, mice were sacrificed, and samples including blood and liver were
collected immediately.

4.3. Biochemical Assays

Blood samples were centrifuged at 3000 rpm for 10 min and the serum samples obtained were
stored at −20 ◦ C for further analysis. The contents of ALT and AST in the serum were detected by
commercial kits. The procedures were conducted as described by the manufacturers’ instructions.
Molecules 2018, 23, 352 13 of 17

Briefly, ALT and AST working reagents were prepared according to instructions and then 100 µL
serum samples were added to 1mL working reagent. Units per liter (U/L) of ALT/AST activity
is the amount of enzyme which oxidizes one µmol/L of NADH per minute. Namely, the average
absorbance per minute was determined, multiply by factor −1746 for results in U/L. The activities
of CAT, GSH-Px, and SOD as well as the production of MDA of liver tissues were determined
by kits bought from Nanjing Jiancheng. The decomposing reaction of H2 O2 by CAT could be
terminated by ammonium molybdate, and resting H2 O2 reacts with ammonium molybdate to
produce faint yellow complex compound with maximum absorption at 405 nm. The determination
of GSH-Px is based on the oxidation of glutathione (GSH) to oxidized glutathione (GSSG) catalyzed
by GSH-Px, which is then coupled to the recycling of GSSG back to GSH utilizing glutathione
reductase (GR) and NADPH. Since GSH-Px is the rate-limiting factor of the coupled reactions,
the decrease in NADPH absorbance measured at 340 nm during the oxidation of NADPH to NADP+ is
indicative of GSH-Px activity. The SOD assay Kit-WST conveniently allows SOD assaying by utilizing
WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium that generates a
water-soluble formazan dye upon reduction with a superoxide anion. SOD inhibits the rate of
the reduction with O2 , which is linearly related to the xanthine oxidase activity. Therefore, the 50%
inhibition activity of SOD (IC50) can be determined by a colorimetric method. The measuring principle
of MDA is that the red product generated by the condensation reaction between MDA and TBA has
maximum absorption at 532 nm.

4.4. Liver Histology

For the H&E staining study, the liver tissues were fixed in 10% buffered formaldehyde, and then
cut into 5 µm thick paraffin sections by a Leica RM 2016 rotary microtome (Leica Instruments Ltd.,
Shanghai, China). The tissues were stained with hematoxylin and eosin staining (H&E staining).
The liver damage was assessed by the following criteria: 0, no obvious injury; 1–3, mild injury
with inflammatory infiltration; 4–6, intermediate injury with microvesicular steatosis or intermediate
necrosis; 7–9, severe injury with structural disorder of hepatic lobules, or macrovesicular steatosis;
and 10, hepatic structure destruction. For hepatic lipid accumulation analysis, oil-red O staining was
performed. Cryostat sections of liver tissues were cut into 10 µm thickness and then stained with
oil red O. Hepatic steatosis scoring on the stained sections was made by three individual examiners
with the criteria as described in previous paper [23,39]: 0, no droplet; 1–3, rare to few small droplet;
4–6, moderate small droplet; 7–9, large droplet; and 10, whole stain. Additionally, picrosirius-red
staining in liver tissue was conducted to evaluate liver fibrosis. Briefly, 5 µm thick paraffin sections
were stained with picrosirius red. Scoring on the stained sections was made according to the following
criteria: 0, no obvious fibrosis signs; 1–3, no portal area fibrosis; 4–6, fibrosis appears in the portal area;
7–9, a destroyed lobule structure fibrosis without cirrhosis; 10, fibrosis and cirrhosis [23].

4.5. Western Blot

Total protein was extracted from liver tissue of mice via RIPA lysis buffer supplemented with
phosphatase inhibitor. The supernatant was transferred and equal amounts of protein were separated
on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and blotted onto
polyvinylidene difluoride membranes. The membrane was then incubated with CYP2E1 (ab28146) and
GAPDH primary antibodies at 4 ◦ C overnight followed by incubation with appropriate horseradish
peroxidase (HRP) labelled secondary antibodies. The blots were subjected to chemiluminescence
analysis (Bio-rad, Hercules, CA, USA).

4.6. Network Pharmacology Construction and Analysis

The constituent compounds and related targeted genes of these three herbs (Danshen, Wuweizi,
Yunzhi) were identified by BATMAN-TCM (Bioinformatics analysis tool for molecular mechanism of
traditional Chinese medicine, http://bionet.ncpsb.org/batman-tcm/). Liver disease targets including
Molecules 2018, 23, 352 14 of 17

ALD and NAFLD were collected from PubMed Database (https://www.ncbi.nlm.nih.gov/pubmed/).

The common targets were recognized by VENNY 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/).
Then, the network of compounds-targets was constructed by Cytoscape 3.5.0. The pharmaceutical
properties’ analysis was performed according to Lipinski’s rule (LR) and the absorption, distribution,
metabolism, and excretion (ADME) system.

4.7. Statistical Analysis

All experiments were performed in triplicate. All the data obtained is presented as the mean ± SD,
and analyzed by one-way analysis of variance (ANOVA) with SPSS. Value of p < 0.05 was considered
to be statistically significant.

5. Conclusions
This study found that JGB showed preventive effects against acute and chronic ALD as well as
NAFLD, while post-treatment with JGB had no significant effect, suggesting the nature of JGB as a
health supplement rather than a drug. In our NAFLD model, JGB can specifically activate GSH-Px,
which may be partially involved in the protective effect. However, the internal anti-oxidative system
including SOD and CAT in the context of ALD are not activated, indicating that JGB does not protect
the liver from alcohol-induced injury by alleviating oxidative stress. It is expected that JGB may
regulate the lipid metabolism of an injured liver to prevent hepatic damage, fatty liver and fibrogenesis,
which needs to be further studied. Based on the construction of network pharmacology, 40 bioactive
compounds and 15 related targets have been identified as possible effective compounds and potential
targets. Among them, miltirone, neocryptotanshinone II and deoxyshikonin are compounds with
good pharmaceutical properties and that act with vital targets of pathways relating to inflammation,
fatty acid oxidation, TNF production and cell proliferation, suggesting that they should be the focus
of future study on this formula. The results provide scientific support for developing this formula,
Jian-Gan-Bao, as a functional supplement for the prevention of alcoholic liver injury and NAFLD.

Supplementary Materials: The following are available online. www.mdpi.com/1420-3049/23/2/352/s1

Acknowledgments: The study was partially financial supported by grants from the research council of the
University of Hong Kong (Project Codes: 104003422, 104003919, 104004092, 104004460), the Research Grants
Committee (RGC) of Hong Kong, HKSAR (Project Codes: 766211, 17152116), Gala Family Trust (Project Code:
200007008), Government-Matching Grant Scheme (Project Code: 207060411), a donation from Vita Green
Health Products Co., Ltd. (Project cord: 200007477) and Shenzhen Basic Research program (project code:
JCYJ20140903112959964). The authors would like to express thanks to Keith Wong, Cindy Lee, and Alex Shek for
their technical support, and express thanks to Brian Yeung for his careful proofreading of this manuscript.
Author Contributions: Yibin Feng conceived and designed the experiments. Sha Li performed the experiments
and drafted the paper. Ning Wang, Ming Hong, Hor-Yue Tan, Guofeng Pan conducted the network
pharmacological study and analyzed the data. All authors revised the paper and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript:

ADIPOQ Adiponectin
ALD Alcoholic liver diseases
ALT Alanine aminotransferase
ANXA1 Annexin 1
AST Aspartate aminotransferase
CHMs Chinese herbal Medicines
CAT Catalases
CCL5 Chemokine CCL5
CD34 Cluster of differentiation 34
CYP2E1 Cytochrome P450 2E1
Molecules 2018, 23, 352 15 of 17

FOXP3 Forkhead box P3

GSH-Px Glutathione peroxidase
IGF1 Insulin-like growth factor 1
IL10 Interleukin-10
IL6 Interleukin-6
JGB Jian-Gan-Bao
MDA Malondialdehyde
MW Molecular weight
NAFLD Non-alcoholic fatty liver disease
OB Oral bioavailability
PPARD Peroxisome proliferator-activated receptor delta
PROC Protein C
SOD Superoxide dismutase
Sterol regulatory element-binding transcription factor
SREBF1
1
TGF-β Transforming growth factor-β
TNF Tumor necrosis factor
TPO Thyroid peroxidase

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Sample Availability: Samples of the compounds are available from the authors.

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