Author’s Accepted Manuscript

Hepatoprotective Potential of Standardized Ficus

Species in Intrahepatic Cholestasis Rat Model:
Involvement of Nuclear Factor-κB, and Farnesoid
X Receptor Signaling Pathways

Seham S. El-hawary, Zeinab Y. Ali, Inas Y.

Younis
www.elsevier.com/locate/jep

PII: S0378-8741(18)33615-8
DOI: https://doi.org/10.1016/j.jep.2018.11.026
Reference: JEP11607
To appear in: Journal of Ethnopharmacology
Received date: 1 October 2018
Revised date: 14 November 2018
Accepted date: 15 November 2018
Cite this article as: Seham S. El-hawary, Zeinab Y. Ali and Inas Y. Younis,
Hepatoprotective Potential of Standardized Ficus Species in Intrahepatic
Cholestasis Rat Model: Involvement of Nuclear Factor-κB, and Farnesoid X
Receptor Signaling Pathways, Journal of Ethnopharmacology,
https://doi.org/10.1016/j.jep.2018.11.026
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 Hepatoprotective Potential of Standardized Ficus Species in Intrahepatic
Cholestasis Rat Model: Involvement of Nuclear Factor-κB, and Farnesoid X
Receptor Signaling Pathways

Seham S. El-hawary a, Zeinab Y. Ali b, and Inas Y. Younis a*

a
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
b
Department of Biochemistry, National Organization for Drug Control and Research (NODCAR),
12553 Giza, Egypt.

* Corresponding author:
Lecturer Dr. / Inas Youssef Younis
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt‎, Egypt.
ORCID ID: https://orcid.org/0000-0002-3084-0161
Phone: 002-01144616340
Fax: +2023628246‎
E. mail address: inas.younis@pharma.cu.edu.eg.

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ABSTRACT
Ethnopharmacological relevance
Ficus is an important commercial crop not only for its nutritive value but also, for its medicinal value.
Several Ficus species have been traditionally used in the Egypt, Indian and Chinese as carminative,
astringent, antibacterial, hepatoprotective, and hypolipidemic agents.
Aim of the study
To standardize and compare the possible hepatoprotective potential of the ethanolic extract of leaves
of five tested Ficus species namely: Ficus mysorensis Roth ex Roem. & Schult, Ficus pyriformis
Hook. & Arn., Ficus auriculata Lour., Ficus trigonata L., and Ficus spragueana Mildbr. & Burret in
the intrahepatic cholestasis rat model induced by 17α-Ethinylestradiol (EE) and to explore the
mechanism of action with respect to their phytochemical constituents.
Materials and methods
Determination of the total phenolic and flavonoid contents, chromatographic examination and acute
oral toxicity test were performed on the tested Ficus extracts. Animals were divided into 8 groups.
Group 1, served as control for 2 weeks. Group 2, untreated cholestatic rats. Groups 3-8, pretreated
with Ficus extracts (100 mg/Kg/day, p.o) or ursodeoxycholic acid (as reference drug) for 2 weeks and
injected by EE in the last 5 days. Serum liver function test, 5′-nucleotidase (5′-N), total bile acids
(TBA), total cholesterol (T.C) and phospholipids were assayed. Also, hepatic Na+/K+-ATPase, nuclear
factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), hepatocyte growth factor (HGF),
hemeoxygenase-1 (HO-1), and markers of oxidative stress were investigated. Furthermore, molecular
docking study was performed to explore the ability of the major constituents of Ficus to interact with
Farnesoid X receptor (FXR).
Results
Four phenolic compounds (gallic, chlorogenic acid, caffeic acids and rutin) were identified.
Chlorogenic acid and rutin represented the major constituents of Ficus extracts. Simultaneous
administration of Ficus extracts with EE effectively. i- preserved liver function, TBA, T.C and
phospholipids, ii- suppressed the pro-inflammatory cytokines (NF-κB and TNF-α), iii- enhanced
hepatic regeneration (HGF) and antioxidant defense system. Furthermore, molecular docking reveals
that rutin and chlorogenic acid effectively act as FXR agonists.
Conclusion

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Among the tested extracts, Ficus spragueana Mildbr. & Burret enriched with phenolics exhibited a
pronounced hepatoprotective activity and may provide a new therapeutic approach for estrogen-
induced cholestasis.

Graphical Abstract

Abbreviations: ALT, Serum alanine aminotransferase; ALP, alkaline phosphatase ; ANOVA, one-
way analysis of variance AST, aspartate aminotransferase; CDCA, Chenodeoxycholic acid; EE,17α-
Ethinylestradiol; ELISA, Enzyme-linked Immune Sorbent Assay; GGT, ɤ -glutamyl transpeptidase;
GST , glutathione S-transferase ; HGF, hepatocyte growth factor; HO-1, Hemeoxygenase-1 ; HPLC,
High performance liquid chromatography; LOD, limits of detection; LOQ, limit of quantifications;
MDA , malondialdehyde ; NF-??B , Nuclear factor-kappa B ; NIH, National Institutes of Health;
NODCAR, National Organization for Drug Control and Research; NO , nitric oxide ; 5′-NT, Serum
levels of 5′-Nucleotidase; OECD, Organization for Economic Co-operation and Development; T.BIL,
total bilirubin; TBA, total bile acid ; T.C , total cholesterol ; TNF­α , tumor necrosis factor alpha ;T.P ,
total protein R2, correlation coefficient; S.C, subcutaneous injection UDCA, ursodeoxycholic acid.

Keywords: Ficus; Cholestasis; Anti-inflammatory; Antioxidant; Nuclear factor-κB; Farnesoid X

receptor.

INTRODUCTION
Cholestasis is a common symptom of liver injury. Either cholestasis is a result of the
interruption of bile formation and/or bile flow at the level of the hepatocytes or the cholangiocytes,

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leads to intrahepatic retention of bile. If no timely treatment, the accumulation of toxic bile acid
initiates oxidative stress and inflammatory signal reactions, resulting in the development and
progression of hepatocellular and bile duct injure, liver fibrosis and ultimately to cirrhosis (Yang et
al., 2016; De Vries and Beuers, 2017).
17α-Ethinylestradiol (EE) is a semisynthetic 17β-estradiol with highly potent estrogen activity.
EE is a main constituent of women oral contraceptives and hormonal replacement therapy. However,
EE- therapy is associated with increased risk of the development of hepatotoxicity such as reversible
intrahepatic cholestasis (Fernández-Martínez et al., 2014). Furthermore, it has been demonstrated that
estrogen induces cholestasis during pregnancy in susceptible woman that possibly associated with an
increased risk of severe fetal adverse events (Chacko and Wolkoff, 2017). Several studies have been
conducted to explore the mechanism of the development of cholestasis and the possibility of the
management (Woolbright and Jaeschke, 2016; Cai et al., 2017). However, the effective medical
therapies for cholestasis are still quite limited. Currently, the first line treatment of cholestatic liver
disease is ursodeoxycholic acid (UDCA). It is well documented that UDCA improved the cholestatic
liver injury in both human and different animal models of intrahepatic cholestasis (Trauner et al.,
2017). Although, UDCA able to slow the progression of cholestasis, up to 40% of patients have
inadequate response to UDCA therapy (Woolbright and Jaeschke, 2016; Zhao et al., 2017). Therefore,
it is extremely important to develop new therapeutic medicines for this disease.
Natural products rich in bioactive compounds are of great interest for protection and treatment
of various diseases (Sokkar et al., 2013; El Hawary et al., 2016; Abd El-Fattah et al., 2017). Ficus is a
unique large genus of family Moraceae. It comprises more than 800 species of evergreen trees, shrubs
and vines present worldwide. Most of these species are native to tropical and subtropical regions, and
commonly known as Fig trees (Abdel-Hameed, 2009). The ethno- pharmacological use of these plants
reveals that they are a promising source of phytomedicine (Mousa et al., 1994). In folk medicine,
many Ficus species have long been used for cardiovascular and liver diseases, respiratory disorders,
menopausal symptoms and cancer-cure (Zingue et al., 2016; Zhang et al., 2018).
Therefore, the present study was designed to investigate the total phenolic and flavonoid
contents of the hydro-ethanolic extracts of leaves of the five Ficus species namely: Ficus mysorensis
Roth ex Roem. & Schult, Ficus pyriformis Hook. & Arn., Ficus auriculata Lour., Ficus trigonata L.‎,
Ficus spragueana Mildb‎r. & Burret, to determine whether these extracts supplementation could have
a hepatoprotective activity against intrahepatic cholestasis induced by EE in adult female rats
comparing with that of UDCA as a reference drug, and explore their mechanism of action.

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1. MATERIALS AND METHODS
1.1 Plant Material

The leaves of the five different species of Ficus (F.) namely: F. mysorensis Roth ex Roem. &
Schult, F. pyriformis Hook. & Arn., F. auriculata Lour., F. trigonata L., and F. spragueana Mildbr.
& Burret were collected from El Orman Botanical Garden, Giza, Egypt on March 2016. They were
kindly identified by Dr. Mohamed El-Gebaly, National Research Institute, Dokki, Giza, Egypt and
vouched specimens were deposited in the herbarium of Pharmacognosy Department, Faculty of
Pharmacy, Cairo University, Cairo, Egypt with numbers: (21316 III‎,‎ 21316 V,‎‎‎21316 VIII‎, ‎21316 IX‎,
and ‎21316 X)‎, respectively.

1.2 Preparation of Crude Alcoholic Extract

Leaves of the five investigated species (500g) were air dried at room temperature and crushed
into fine powder. A hundred grams of each powder were extracted by ethanol: water (3:1) till
exhaustion. The solvent was evaporated under reduced pressure at 45°C using rotary evaporator
(Buchi®R- 300, USA) and stored at 20°C until use.

1.3 Preliminary Phytochemical Screening

The crude Ficus extracts were subjected to preliminary phytochemical screening for detection
of various types of plant secondary metabolites as alkaloids,‎ saponins, flavonoids, anthraquinones,
tannin and cardiac glycosides (Harborne, 1973).

1.4 Preparations of Standard Stock Solutions

Serial dilutions (0.05, 0.10, 0.20, 0.40 and 0.50 µg/ml) of each gallic acid, caffeic acids,
chlorogenic acid and rutin standard (Sigma-Aldrich, St. Louis, MO, USA) were prepared from the
stock solution (1 mg/ml in 50% methanol).

1.5 Determination of Total Phenolic and Flavonoid Contents

Total phenolic contents of the five extracts were determined by Folin–Ciocalteu's method
(Abdel-Hameed, 2009), and expressed as milligrams gallic acid equivalents (GAE) per gram of dry
plant extract. Gallic acid calibration curve was constructed at different concentrations range (80-280
µg/ml) with a correlation coefficient R2 = 0.995. Total flavonoid contents were assayed by aluminum
trichloride method (Shi et al., 2011), using quercetin as standard with concentrations range (10-100
µg/ml) and correlation coefficient of R2 =0.999.

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 1.6 Chromatographic Analysis of Some Phenolic Compounds
High performance liquid chromatography (HPLC) analysis of specific phenolic compounds
was performed as previously reported (Nour et al., 2013) with slight modifications. In brief, the
analysis was carried by HPLC (Shimadzu, model LC20AT series, Japan) equipped with LC-20AD
pump and DGU-20A degasser. The mobile phase consisted of 2% (v/v) acetic acid: acetonitrile with a
ratio of 95:5 in isocratic mode at a flow rate of 1 ml/min and an injection volume of 5 µl. The
chromatographic separation was achieved on by a reversed-phase Inertsil® ODS-3 (5µm, 15cm x 4.6
mm, Sigma-Aldrich, USA). While, the spectrophotometric detector was set at a wavelength of 300 nm
with run time of 23 min. Identification of each compound was performed by comparison of its
retention time with that of standard solutions (gallic acid, caffeic acids, chlorogenic acid and rutin).
Whereas; quantifications of each phenolic compound were achieved from the least square regression
equation of the calibration curve of each standard. Data was acquired by means ± standard deviations
of three independent analyses.
Method validation was performed to confirm linearity, precision and the limits of detection
(LODs) and quantifications (LOQs). The accepted criteria for linearity based on the correlation
coefficient (R2). While, LODs and LOQs were estimated depending upon the signal to noise ratio.
Method selectivity was performed by comparing the retention time of standards with that of the
sample. Specificity was measured by the absence of interference peaks at the retention time (Rt) of the
four standards; methanol was used as a blank solution. In order to ensure linearity, injection of six
concentrations of each standard was prepared in the range of LOQs and injected in HPLC in triplicate
order.

1.7 Animals
Adult female Sprague-Dawley rats, aged 14 weeks and weighing 150-170 g were provided
from the farm of National Organization for Drug Control and Research (NODCAR), Giza, Egypt.
Animals were kept in cages under standard laboratory conditions of artificial lighting 12/12 h
light/dark and 25±2oC. Animals were kept in their cages for a week prior to dosing for adaptation to
the new laboratory conditions. They were free access to conventional laboratory diet and drinking
water. All experiments were performed following the recommendations of the National Institutes of
Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication N85-23, 1985,
revised 1996). The experimental protocols were approved by the Animal Care Committee of National
Organization for Drug Control and Research (NODCAR), Giza, Egypt.

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 1.8 Acute Oral Toxicity Test
To determine the acute oral toxicity testing, a limit test procedure was conducted according to
the Organization for Economic Co-operation and Development (OECD) guideline number 423
(OECD, 2001). Overnight fasted animals were weighted, divided into six groups. Group one served as
control. The other groups received the five tested Ficus extracts once daily at a sole concentration of
2000 mg/kg body weight by gavage using a stomach tube for 14 days. Animals were observed
individually for any signs of morbidity and mortality after dosing during the first 30 minutes with
special attention given during the first 4 hours periodically during the first 24 hours, and daily for 14
days.

1.9 Induction of Intrahepatic Cholestasis

Intrahepatic cholestasis induced in female rats by subcutaneous injection (s.c) of EE supplied
from Sigma Chemical Co. (St. Louis, MO) at a daily dose of 5 mg/kg body weight /day for 5
consecutive days (Zhou et al., 2011).

1.10 Experimental Design

Animals were randomly divided into 8 equally groups, each of 6 rats, as follows:
Group 1: Rats received the vehicle and served as control group for 2 weeks.
Group 2: Untreated EE-induced cholestatic group served as positive control group.
Group 3-7: Rats pretreated once daily with F. mysorensis, F. pyriformis, F. auriculata, F. trigonata,
F. spragueana, extracts, respectively at a dose of 100 mg/Kg body weight, for 2 weeks, and
simultaneous administrated 30 minute before induction of intrahepatic cholestasis in the last
5 days of the experimental period.
Group 8: Rats received UDCA (50 mg/Kg body weight/day, p.o) 30 minute before induction of
intrahepatic cholestasis in the last 5 days of the experimental period (Sanchez Pozzi, 2003).

1.10.1 Blood and tissue sampling

At the end of the experimental period, blood samples were collected from the retro-orbital
sinus of overnight fasted animals under light ether anesthesia. Sera were separated by centrifugation at
800 ×g for 10 minutes. After that all rats were sacrificed by decapitation. Livers were quickly
dissected out, rinsed in ice-cold physiological saline, dried and weighted.
Liver index was calculated (= liver weight/body weight × 100). A 10% of liver homogenates
were prepared in ice-cold saline and centrifuged at 800 ×g for 15 minutes at 4oC. The samples were
kept at −20 °C until further biochemical analysis.

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1.10.2 Biochemical Analysis

1.10.2.1 In serum
Serum alanine aminotransferase, (ALT) and aspartate aminotransferase (AST), alkaline
phosphatase (ALP), ɤ -glutamyl transpeptidase (GGT), total bilirubin (T.BIL), total protein (T.P),
total bile acid (TBA), and total cholesterol (T.C) were assayed by following the manufacturer's
instructions of Randox kits (Antrim, United Kingdom). Serum levels of 5′-Nucleotidase (5′-NT) and
phospholipids were investigated according to Sigma protocol and (Takayama et al., 1977)

1.10.2.2 In liver homogenate

Hepatic membrane-bound Na+/K+-ATPase enzyme activity was assayed (Bonting, 1970).
Enzyme-linked Immune Sorbent Assay (ELISA) technique was used to investigate the hepatic levels
of tumor necrosis factor alpha (TNF­α) and hepatocyte growth factor (HGF) using Quantikine kit
(R&D Systems, USA). Hemeoxygenase-1 (HO-1) was assayed by using a commercial ELISA kit
provided from Enzo Life Sciences International Inc., (New York, USA) according to the
manufacturer’s instruction. Nuclear factor-kappa B (NF-𝜅B) was determined according to Biosource
kit (California, USA). Furthermore, superoxide dismutase, SOD (Marklund and Marklund, 1974),
catalase, CAT (Aebi, 1984), glutathione S-transferase, GST (Buczyński et al., 2006), reduced
glutathione, GSH (Beutler et al., 1963), nitric oxide, NO (Miranda et al., 2001) and malondialdehyde,
MDA (Buege and Aust, 1978) were also determined.

1.11 Molecular Docking Study

In order to explore the molecular mechanism of the hepatoprotective activity of the tested
Ficus extracts, their major constituents (rutin and chlorogenic acid) were subjected to molecular
docking study into FXR which is a ligand-activated transcription factor. FXR is highly expressed in
liver to down-regulate the expression of genes encoding enzymes (cholesterol 7α-hydroxylase and
sterol 12α-hydroxylase) that synthesize bile acids from cholesterol (Li and Guo, 2015).
Therefore, FXR is one of the most important nuclear receptors involved in the gerulation of
bile acid homeostasis in liver (Baptissart et al., 2017). Molecular modeling study was achieved by
Molecular Operating Environment (MOE, 10.2008) software provided by chemical computing group,
Canada. All minimizations were performed with MOE until an RMSD gradient of 0.05 kcal. mol −1Å−1
with MMFF94x force field and the partial charges were automatically calculated.
The X-ray crystallographic structure of FXR co-crystallized with 3-deoxy-chenodeoxycholic
acid (3-deoxy CDCA) as an FXR agonist (PDB ID: 1OT7) was downloaded from the protein data
bank http://www.rcsb.org/pdb/home/home.do; Mi et al., 2003). Chain B and water molecules were

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deleted, and the remaining A chain was prepared for docking study using the Protonate 3D protocol in
MOE with default options. The co-crystallized ligand was used to define the binding site for docking.
Triangle Matcher placement method and London dG scoring function were used for docking.
Docking setup was first validated by re-docking of the co-crystallized ligand (3-deoxy CDCA)
in chain A in the vicinity of the binding site of the receptor with an energy score (S) = −15.36
kcal/mol and RMSD of 0.467Å. This validated setup was then used in predicting the legends receptor
interactions on the binding site for chlorogenic acid and rutin to identify their preferred, energetically
most favorable and binding pose (i.e. the orientation of a ligand relative to its receptor and the ligand’s
conformation).

1.12 Statistical Analysis

The biochemical results were expressed as mean ± SE of six separated determinations. The
biochemical results were analyzed by one-way analysis of variance (ANOVA), followed by a Post hoc
Tukey's multiple comparison test using Statistical Package for the Social Sciences software (SPSS)
version 22 (SPSS Inc., Chicago, IL, USA). The level of significance was set at P < 0.05.

2. RESULTS
2.1 Phytochemical Screening and Total Phenolic and Flavonoid contents

The phytochemical screening of Ficus extracts revealed that the phenolic compounds such as
flavonoids and phenolic acids present in all extracts. Whereas, cardiac glycosides, alkaloids,
saponins and anthraquinones were absent. Whereas, the results of total phenolic and flavonoid
contents depicted in Table 1 show that F. spragueana contained the highest concentration in both
phenolic and flavonoid compounds by values of 136.8 ± 0.29 and 100.7 ± 0.01 mg/g, respectively
followed by F. auriculata, F. trigonata, F. mysorensis and F. pyriformis, respectively.

2.2 Chromatographic Analysis of Some Phenolic Compounds

Table 1
Total phenolic, flavonoids, and the major detected compounds in the five tested Ficus species

Total Total Chlorogenic Caffeic

Gallic acid Rutin
Name phenolic flavonoids acid acid
(µg/g) (µg/g)
(GAE/g) (QE/g) (µg/g) (µg/g)

F. mysorensis 35.56 ± 0.11 28.93 ± 0.04 ----- 0.1465 ± 0.038 ---- 0.2233 ± 0.201

F. pyriformis 24.45 ± 0.01 21.60 ± 0.01 ---- 0.3369 ± 0.013 ---- 0.2463 ± 0.026

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 F. auriculata 60.26 ± 0.29 21.52 ± 0.04 ------ 0.4578 ± 0.014 ---- 0.3600 ± 0.094

0.5095 ±
F. trigonata 48.66 ± 0.11 15.98 ± 0.01 1.3699 ± 0.017 ---- 0.3072 ± 0.712
0.048
100.74 ± 0.1750 ± 0.1137 ±
F. spragueana 136.80 ± 0.29 4.0107 ± 0.217 0.8338 ± 0.036
0.01 0.037 0.004
The data represent the mean of three replicates ± S.D; Abbreviations :‎GAE: Gallic acid equivalent; QE: Quercetin
equivalent.

In the present investigation, HPLC method was efficient in the separation of the phenolic
compounds with high resolution and minimum interference of the matrix (Table 2). The relative
standard deviation (RSD) of all standard varied from 0.69-1.22 %. In order to optimize the
chromatographic conditions, the effect of organic modifier (methanol and acetonitrile) was examined.
Acetonitrile showed the best separation between the peaks with good resolution, so that it was used in
the method development.

A
3 4

1 2

B 2

3 4
1

1 4

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D
Fig.1 HPLC profiles of (A): standard compounds;1-gallic acid; 2- chlorogenic acid; 3-caffeic acid; 4-rutin; (B) F.
spragueana;(C) F. trigonata; (D) F. pyriformis; (E) F. auriculata;(F): F. mysorensis.

The data depicted in Table 2 showed the criteria for validation of HPLC method. The limit of
detection (LODs) was defined as the minimum level of standard that can be detected. The estimation
was based on the signal to noise ratio (S/N). While the limit of quantification (LOQs), was defined as
the lowest quantified concentration for each standard. Acceptance criteria for both LODs and LOQs
were 3 and 10, respectively.
The characteristic HPLC chromatograms of each Ficus extracts allowed the identification and
quantification of the four compounds namely: gallic acid, chlorogenic acid, caffeic acid and rutin.
Chlorogenic acid and rutin were the most abundant phenolic compounds (Fig. 1). The obtained

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results revealed that F. spragueana contained the highest concentration of chlorogenic acid followed
by F. trigonate, F. auriculata, F. pyriformis, and F. mysorensis, respectively. Rutin was more
abundant in F. spragueana as compared to other Ficus species. In addition, caffeic acid was only
detected in F. spragueana.

Table 2 .
Criteria for validation of HPLC method

LODs LOQs
Standards Rt (min) Calibration equation R2 RSD%
(μg/mL) (μg/mL)

Gallic acid 4.90 Y=709795X-2456.3 0.9779 0.69% 0.049 0.161

Chlorogenic acid 11.88 Y=1184398.000X-14084.90 0.9989 0.96% 0.0163 0.053

Caffeic acid 13.39 Y=2659608.67X-8816.37 1.0000 1.22% 0.0183 0.061

Rutin 16.23 Y=647460X-643.28 0.9996 1.06% 0.0168 0.056

Abbreviations ‎Rt: Retention time; R2: Correlation coefficient; LODs: Limits of detection; Limits of quantifications
(LOQs). ‎LODs: is defined as the minimum level of standard that can be detected. LOQs: is defined as the lowest
quantified concentration for each standard.‎

2.3 Biochemical Results

2.3.1 Acute oral toxicity test
The acute oral toxicity test of the ethanolic extract of leaves of the five tested Ficus species
‎showed no mortalities or signs of toxicity. Therefore, acute oral toxicity test implied the safety of the
tested Ficus extracts up to 2000 mg/Kg. Therefore, 1/20 of such dose was chosen to in-vivo study.

2.3.2 Effect on liver index

The data recorded in Table 3 showed the effect of different treatments on liver index after 2
weeks. A marked increase in liver index by 1.37- fold versus normal control level was observed in
EE- treated group. On contrast, restored liver index was recorded in Ficus pretreated animals. This
beneficial effect was more pronounced in F. spragueana- treated group.
To assess the severity of hepatocellular injury, liver function was monitored by measuring ‎the
activities of liver enzymes (ALT, AST, ALP and GGT), T.BIL and total protein in ‎serum. As shown
in Fig. 2, treatment of rats with EE (5 mg/Kg/day, s.c) for 5 ‎consecutive days resulted in a significant
elevation in diagnostic liver enzyme activities of ‎ALT, AST, ALP and GGT, and level of T.BIL in
serum up to 3.5, 3.2, 2.2, 4.3 and 3.26- ‎fold, respectively above normal, and significant depletion in
serum total protein by 28.7% ‎as compared with the control group. ‎

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 However, pretreatment with the five tested Ficus extracts: F. mysorensis, F. pyriformis, ‎F.
auriculata, F. trigonate and F. spragueana (100 mg/Kg body weight/day) for 2 weeks ‎were
significantly counteracted these biochemical changes, confirming their ‎hepatoprotective effect. This
effect was more pronounced in F. spragueana pretreated ‎group as confirmed by significant decrease
in serum ALT, AST, ALP, GGT and T.BIL ‎by 59.3%, 52.3%, 42.1%, 62.2% and 57.7%, respectively,
and significantly increased of ‎total protein by 33.0% as compared with the untreated EE- group. ‎

Table 3
Effect of different treatments on liver index after 2 weeks.

Liver weight
Groups Final body weight (g) Liver index
(g)

Control 171.7 ± 1.84c,d 5.96 ± 0.13a 3.48 ± 0.10a

EE
(5 mg/kg b.wt/day, s.c) 143.3 ± 4.37a 8.16 ± 0.19e 5.70 ± 0.11e
in the last 5 days
F. mysorensis
(100 mg/Kg b.wt/day) 155.9 ± 4.64a,b 7.73 ± 0.27d,e 4.98 ± 0.17d
+ EE
F. pyriformis
(100 mg/Kg b.wt/day) 164.1 ± 4.89c,d 6.96 ± 0.15b,c 4.27 ± 0.18b,c
+ EE
F. auriculata
(100 mg/Kg b. wt/day) 159.3 ± 3.39a,b 7.32 ± 0.53c,d 4.60 ± 0.05c,d
+ EE
F. trigonata
(100 mg/Kg b. wt/day) 164.8 ± 4.05c,d 6.57 ± 0.24a,b 4.01 ± 0.16a,b,c
+ EE
F. spragueana
(100 mg/Kg b.wt/day) 172.3 ± 5.13c,d 6.32 ± 0.18a,b 3.69 ± 0.13a,b
+ EE
UDCA
(50 mg/Kg b.wt/day) 179.7 ± 3.76c 6.19 ± 0.32a 3.46 ± 0.10a
+ EE
The data represent the mean of 6 rats ± S.E .The presence of different ‎superscripts in the same column indicate significant
difference between means ‎tested ‎by ANOVA followed by Tukey HSD multiple comparisons test at 𝑃 < ‎0.05.
Abbreviations: ‎ EE: 17α-Ethinylestradiol F: Ficus, UDCA: ‎Ursodeoxycholic acid; b.wt: body weight. (Liver index = liver
weight/body ‎weight × 100).‎

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Fig. 2 Effect of different treatments on serum diagnostic markers of liver function after 2 weeks. The data represent the
mean of 6 rats ± S.E. The presence of different superscripts over the column indicates significant difference between
means tested by One-way ANOVA followed by Tukey HSD multiple comparisons test at 𝑃 < 0.05. Abbreviations: EE:
17α-Ethinylestradiol F: Ficus, UDCA: Ursodeoxycholic acid; ALT: alanine aminotransferase; AST: aspartate
aminotransferase; ALP: alkaline phosphatase; GGT: ɤ -glutamyl transpeptidase.

Meanwhile, the lowest protective effect was observed in F. mysorensis pretreated group as
manifested by significantly decrease serum ALT, AST, ALP, GGT and total bilirubin only by 19.7%,
11.7%, 12.2%, 10.8% and 24.7%, respectively, and significantly elevated total protein by 9.68% as
compared with the untreated EE- group. Meanwhile, F. trigonate, F. pyriformis and F. Auriculata
exhibited moderate effects.

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 2.3.3 Effect on serum 5′-NT, TBA, total cholesterol and phospholipids
The data in Fig. 3 illustrates the effect of different treatments on serum 5′-NT, TBA, T.C and
phospholipids after 2 weeks. EE induced significant elevation in serum 5′-NT, TBA, T.C and
phospholipids by 2.75, 4.31, 3.34 and 2.85-fold, respectively above the control level.

Fig. 3 Effect of different treatments on serum 5′-nucleotidase, total bile acids, total cholesterol and phospholipids after 2
weeks. The data represent the mean of 6 rats ± S.E. The presence of different superscripts over the column indicates
significant difference between means tested by ANOVA followed by ‎Tukey HSD multiple comparisons test at 𝑃 < 0.05.
Abbreviations: EE: 17α-Ethinylestradiol F: Ficus, UDCA: Ursodeoxycholic acid;‎5′-NT: 5′-nucleotidase; TBA: total bile
acids; T.C: total cholesterol.

The capacity of Ficus extracts to inhibit these biochemical changes varied between the five tested
species. The protective activity was in the order of F. spragueana >F. trigonate > F. pyriformis >F.
auriculata ≥ F. mysorensis pretreated groups, by percentage values of 53.4%, 40.8%, 33.3%, 24.3%
and 14.2%, respectively, for 5′-NT; 64.3%, 49.1%, 42.2%, 33.5% and 25.9%, respectively for TBA;

- 15 -
 50.5%, 48.2%, 42.5%, 30.3% and 12.1%, respectively for T.C, and 45.9%, 41.3%, 35.9%, 23.3% and
9.79%, respectively for phospholipids versus EE- group.

2.3.4 Effect on hepatic levels of Na+/K+ -ATPase, NF-κB, TNF­α, HGF and HO-1
The results in Table 4 illustrated the effect of different treatments on hepatic levels of NF-κB,
TNF­α, HGF, HO-1 after 2 weeks. Administration of EE for 5 days caused a significant inhibition in
Na+/K+ -ATPase by 42.2% and significant elevation in NF-κB, TNF-α, HGF and HO-1 by 261.3%,
358.3% and 113.8% as compared with normal control. Whereas, Ficus extract- treated groups
exhibited a significant increase in the Na+/K+-ATPase enzyme activity by 29.7%, 38.2%, 52.9%,
58.8% and 65.7%, respectively in F. mysorensis, F. auriculata L., F. pyriformis, F. trigonate and F.
spragueana - treated groups, respectively, versus EE-treated group. Also, Ficus treated groups
showed a significant beneficial decrease in hepatic concentration of TNF-α, NF-κB, HGF and HO-1
as compared with EE-treated group.

Table 4
Effect of different treatments on hepatic levels of Na+/K+-ATPase, NF-𝜅B, TNF­α, HGF and HO-1
after 2 weeks

Na+/K+-ATPase NF-κB TNF-α HGF OH-1

Groups
(U/mg protein) (ng/mg protein) (pg/mg protein) (pg/mg protein) (ng/mg protein)

Control 0.90 ± 0.03d 2.84 ± 0.12a 85.5 ± 2.48a 154.2 ± 7.46a 5.12 ± 0.22a

EE
(5 mg/kg b. wt/day, s.c) 0.52 ± 0.04a 10.27 ± 0.44e 391.7 ± 11.4f 329.7 ± 16.0f 30.2 ± 1.31e
in the last 5 days

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 F. mysorensis
(100 mg/Kg b. wt/day) 0.67 ± 0.02b 9.24 ± 0.40de 281.5 ± 8.17e 305.8 ± 14.8ef 23.3 ± 1.32d
+ EE
F. pyriformis
(100 mg/Kg b. wt/day) 0.79 ± 0.03cd 6.02 ± 0.26c 189.4 ± 6.55c 236.4 ± 12.8cd 19.8 ± 0.85d
+ EE
F. auriculata
(100 mg/Kg b. wt/day) 0.71 ± 0.03bc 7.93 ± 0.34d 246.3 ± 10.7d 271.2 ± 13.9de 21.1 ± 1.02d
+ EE
F. trigonata
(100 mg/Kg b. wt/day) 0.82 ± 0.04cd 5.38 ± 0.23bc 168.8 ± 4.90c 218.8 ± 10.9bcd 15.1 ± 0.92c
+ EE
F. spragueana
(100 mg/Kg b. wt/day) 0.86 ± 0.03d 4.79 ± 0.20bc 135.7 ± 5.38b 199.7 ± 9.83abc 12.2 ± 0.83bc
+ EE
UDCA
(50 mg/Kg b. wt/day) 0.87 ± 0.03d 4.25 ± 0.18b 103.9 ± 3.03ab 176.8 ± 8.55ab 10.1 ± 0.69b
+ EE
The data represent the mean of 6 rats ± S.E‎.The presence of different superscripts in the same column indicate significant
difference between means tested by ANOVA followed by Tukey HSD multiple comparisons test at 𝑃 < 0.05.
Abbreviations: EE: 17α-Ethinylestradiol F: Ficus, UDCA: Ursodeoxycholic acid; Na+/K+-ATPase: sodium-potassium
adenosine triphosphatase; one unit of Na+/K+-ATPase is the amount of inorganic phosphorous in µmol liberated per hour.
NF-𝜅B: nuclear factor kappa B; TNF­α: tumor necrosis factor-alpha; HGF: hepatocyte growth factor; HO-1:
hemeoxygenase-1.

2.3.5 Effect on hepatic oxidative stress markers

The data depicted in Fig. 4 showed that EE- induced significant decrease in the hepatic
antioxidant enzyme activities of SOD, CAT and GST by 41.3%, 38.5% and 42.8% and depletion of
GSH level by 37.5% versus control group. Also, EE- induced overproduction of hepatic NO and
MDA by 94.3% and 197.2% versus control animals.
On the other hand, pretreatment with tested Ficus extracts significantly increased the hepatic
antioxidant enzyme activities (SOD, CAT and GST), associated with markedly decreased in MDA
and NO were as compared with the EE-group. Among the five tested Ficus extracts, F. spragueana
exhibited the strongest antioxidant potential followed by F. trigonate, F. pyriformis, F. auriculata and
F. mysorensis- treated groups, respectively.

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Fig. 4 Effect of different treatments on hepatic oxidative stress markers. The data represent the mean of 6 rats ± S.E. The
presence of different superscripts over the column indicates significant difference between means tested by One-way
ANOVA followed by Tukey HSD multiple comparisons test at 𝑃 < 0.05. Abbreviations: EE: 17α-Ethinylestradiol F:
Ficus, UDCA: Ursodeoxycholic acid; SOD: superoxide dismutase; CAT: catalase; GST: glutathione S-transferase; GSH:
reduced glutathione; NO: nitric oxide; MDA malondialdehyde.
2.4 Molecular Docking Study

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 Fig. 5 illustrated the docking results of the major constituents of Ficus, chlorogenic acid and
rutin into FXR binding site in comparable with the well-known FXR agonist (3-Deoxy CDCA). The
ability of the chlorogenic acid and rutin to interact with the key amino acids in the binding site of FXR
indicated by their docking pattern and docking score in comparable with 3-Deoxy CDCA. The
possible key hydrogen bonding interactions accomplished by 3-deoxy CDCA with the key amino
acids are His291, Arg328, Ser329 and Tyr366 in the binding site of FXR.
The binding energy (Table 5) revealed a moderate to high affinity of the chlorogenic acid and
rutin towards FXR binding site. The obtained results revealed that rutin scored a higher binding
energy (-22.38 kcal/mol) than chlorogenic acid (-14.56 kcal/mol) or 3-deoxy CDCA (-15.36
kcal/mol). Furthermore, Table 5 recorded the possible hydrogen bonds between ligand crystal
structure and the amino acid residues at the binding site of the receptor.

A B

C D

E F

Fig. 5 Docking of chlorogenic acid and rutin into FXR binding site in comparable with 3-Deoxy CDCA. (a): Two-
dimensional interaction diagrams of the binding mode of 3-Deoxy CDCA into the binding site residues of FXR protein,
(b): Superimposition of the co-crystallized (red) and the docking pose (blue) of 3-Deoxy CDCA in the FXR binding site,
(c-f): Two and three-dimensional interaction diagrams of chlorogenic acid (c-d), and rutin (e-f) into the binding site of
FXR. Hydrogen bond interactions between the agonist and FXR proteins are indicated by arrows. Abbreviations: FXR:
Farnesoid X receptor; CDCA: chenodeoxycholic acid.

Table 5

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Docking energy scores of chlorogenic acid and rutin compared with 3-Deoxy-CDCA in the binding site of
the Farnesoid X Receptor.

Compounds
Chlorogenic acid Rutin 3-Deoxy-CDCA
(ligands)

Docking Score
-14.56 -22.38 -15.36
(kcal/mol)
CDCA: Chenodeoxycholic acid

3. DISCUSSION

3.1. Quantitative estimation of the major phenolic compounds

Phenolic and flavonoid compounds are naturally occurring biologically active compounds with
various health promoting activities (El Hawary et al., 2016; Abd El-Fattah et al., 2017). In the present
study, the hydro-ethanolic extract of leaves of the five Ficus species namely: F. mysorensis, F.
pyriformis, F. auriculata, F. trigonate and F. spragueana were investigated for their total phenolic
and flavonoids as well as their hepatoprotective activity against intrahepatic cholestasis induced by
synthetic estrogen, EE in adult female rats.
The phytochemical analysis reveals that the hydroethanolic extract of the tested Ficus species
contain a considerable amount of phenolic compounds. Our results are in a good agreement with
previous study (Abdel-Hameed, 2009) which reported that polyphenolic compounds were the main
ingredients of many other Ficus species. Further confirmation of the main phenolic compounds was
performed in the present study by HPLC fingerprint of the tested Ficus extracts at wave length
300nm. The obtained data showed that both chlorogenic acid and rutin represented the major
components.
These finding are broadly consistent with (Veberic et al., 2008) who demonstrated the
presence of chlorogenic and gallic acids as the major phenolic acids in Slovenia’s fruits of F. carica
L. Also, caffeic acid and chlorogenic acid have been detected in the leaves of Ficus exasperata Vahl.
Several clinical studies have been documented the antihypertensive, anti hypercholesterolemic and
hepatoprotective potentials (Oboh et al., 2014; Ali et al., 2017) of Ficus leaves.
Recent advances have revealed dual benefits of these bioactive compounds as nutraceuticals
and food additive to improve human health (El-Hawary et al., 2016; El Tanbouly et al., 2017).
Chlorogenic acid plays an important protective activity against liver damage induced by
pentachlorophenol, an environmental pollutant (Maalik et al., 2016). This effect was attributed to its
antioxidant potential, down regulation of hepatic glucose-6-phosphatase expression and attenuation of
hepatic steatosis.

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 In-vivo clinical trials, has pointed its antioxidant activity against oxidative stress induced
metabolic syndrome (Santana-Gálvez et al., 2017).
Rutin is an important flavonoid glycoside that reported to exert hepatoprotective activity
against carbon tetrachloride induced liver toxicity, through enhancement of endogenous hepatic
antioxidant enzymes, and suppression of the inflammatory cytokines at the gene level (Hafez et al.,
2015).
The hepatoprotective activity was manifested from preservation of normal liver function test,
membrane stabilizing effect and enhancement of antioxidant defense system (Reddy et al., 2017).
Among flavonoids, caffeic acid exerts hepato-renal protective potential mediated through antioxidant
activity (Olayinka et al., 2017). Moreover, Gallic acid is another polyhydroxyphenolic compound with
an excellent antioxidant and hepatoprotective potential mediated through down-regulation of the NF-
κB and inflammatory mediator (Latief et al., 2016).
In the present biochemical investigation, the acute oral toxicity test implies the safety of the
tested Ficus extracts up to 2000 mg/Kg. Therefore, 1/20 of such dose was chosen to in-vivo study. The
intrahepatic cholestasis experimentally induced by EE, which is an accepted animal model closed
mimic to human cholestasis. This model is a widely used model to examine the mechanisms that
involved in estrogen- induced cholestasis to provide a new perspective for treatment or preventing
such cytotoxicity (Wu et al., 2016; Li et al., 2017).
3.2. Evaluation of liver function
In agreement with the previous study, the observed elevation of serum liver enzymes in EE-
induced cholestatic group above their normal levels reflect their cellular leakage into circulation and
loss of functional hepatocyte membrane integrity. Therefore, they used as the main diagnostic markers
for intrahepatic cholestasis due to their high sensitivity (Wu et al., 2016). It's well known that ALP
and GGT are membrane bounded enzymes located on the membrane of the epithelia cells of hepatic
bile duct. Also, 5’-NT is normally found in the cell membrane of many tissues but is mainly located in
the cholangiole and sinusoid in the liver. Consequently, the significant elevation in serum 5’-NT in
EE- group could be clinically serve as an indicator in the diagnosis of the hepato-biliary diseases
(Hyder, 2016).
On the other hand, the significant decrease in these diagnostic enzymes that observed after
administration of Ficus extracts is a clear manifestation of their membrane stabilizing effect and
hepatoprotective efficacy (Singh et al., 2016). In addition, the decrease in serum total protein in EE-
group reflects the decrease in protein synthesis and functioning mass of the liver. As consequence, the
significant increase in total protein Ficus treated groups promoting the regeneration of the
hepatocytes, improving the liver function, and confirmed the hepatoprotective effect of the tested

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Ficus extracts. This beneficial effect was highly observed in both UDCA and F. spragueana- treated
groups, followed by F. trigonate, F. pyriformis, and F. auriculata. Meanwhile the lowest effect was
detected in F. mysorensis- treated group.
3.3. Evaluation of serum TBA, total cholesterol and phospholipids
Serum level of TBA alone or in combination with aminotransferases are the most commonly
used as biomarkers for intrahepatic cholestasis (Manzotti et al., 2017). It is well documented that bile
acids are cholesterol derivatives that synthesized only in the hepatocytes. The synthesis of bile acid
represents the major route for cholesterol elimination from the body (Li and Li, 2017). The major
constituents of the bile, bile acids, cholesterol and phospholipids forming mixed micelles to increase
cholesterol solubility and decrease bile acid toxicity in the bile (Boyer et al., 2013). Therefore, the
decreased bile flow in the present study due to impaired secretion by hepatocytes through intra-
hepatic bile ducts leading to retention of several substances that normally excreted into bile are
retained (Fickert and Wagner, 2017).
In this context, the marked elevated serum level of TBA, total cholesterol and phospholipids
that were detected in the EE- group is clear manifestation of reduction of the bile flow. These results
are in good agreement with the previous studies who reported that EE-induced intra-hepatic
cholestasis was associated with a significant elevation in serum TBA level, total cholesterol and
phospholipid (Trauner et al., 2017).
These significant elevations were well prevented in the animal’s pretreatment with Ficus
extracts. Therefore, our results suggested that Ficus extracts provide hepatoprotective effect against
EE- induced internal cholestasis attributed to their ability to increase the bile flow rate that promoting
the hepatic excretory capacity for elimination of bile acids and cholesterol from the body. The
maximum effect was detected with F. spragueana and UDCA while F. mysorensis exhibited the
lowest effect against intrahepatic cholestasis induced by EE. Several other defense mechanisms will
be discussed to confirm such hypothesis as hepatic levels of Na+/K+-ATPase.
3.4. Evaluation of hepatic levels of Na+/K+-ATPase
Recently, it well demonstrated that canalicular membrane fluidity affected the permeability,
signal transduction transporter function of the cell membrane as well as activity of membrane-
associated enzymes (Ezzat et al., 2016; Thakkar et al., 2017). In the present study, Na+/K+-ATPase
activity was significantly inhibited in the EE-induced intrahepatic cholestatic animals as compared
with the control animal. This inhibition attributed to the alteration of the canalicular membrane
fluidity under the effect of the intracellular accumulation of bile acids, which can bind to
phospholipids of the cell membrane by their detergent properties result in inhibition of Na+/K+-
ATPase activity and the normal bile transporter. Our result was consistent with the previous study that

- 22 -
reported the involvement of Na+/K+-ATPase inhibition in the pathogenesis of cholestasis (Ikeda et al.,
2017).
Meanwhile, administration of Ficus extract to EE-treated animals resulted in significant
enhancement of the Na+/K+-ATPase enzyme activity, confirming the membrane stabilizing effect of
Ficus extract, which maintenance of the normal membrane fluidity and the biliary flow. This
enhancement effect was decrease in the order of UDCA ≥ F. spragueana ≥ F. trigonate ≥ F.
pyriformis ≥ F. auriculata > F. mysorensis. Therefore, membrane-stabilizing effect may consider as a
mechanism by which Ficus provide hepatoprotection.
3.5. Evaluation of hepatic levels of NF-κB and TNF-α
NF-κB is a well-known a key regulator of inflammation and represents a major inducible
protein transcription fact. Upon activation by various stimuli, NF-κB directly triggers inflammation
reactions by enhancing synthesis of other proinflammatory cytokines, such as TNF-α, interleukin-1β,
IL-6, adhesion molecules and growth factors, and indirectly through regulating the cell proliferation
and apoptosis (Liu et al., 2017). Recent studies confirmed that the over expression of proinflammatory
cytokines enhanced by the toxic accumulation of the bile acids within hepatocytes, triggering hepatic
inflammatory response and initiate the development and progression of cholestasis (Cai and Boyer,
2017).
In agreement with previous reports (Yu et al., 2016; Cai and Boyer, 2017), the present study
revealed that both hepatic NF-κB and TNF-α were significantly elevated in response to administration
of EE. As consequence, the observed suppression of the liver NF-κB and TNF-α in Ficus extract
treated groups, suggesting the tested Ficus extracts effectively suppress the release of
proinflammatory cytokines such as TNF-α, through down-regulation of NF-κB signaling pathway.
The maximum effect was detected in‎F. spragueana- treated group, and the lowest effect was
observed in F. mysorensis- treated group. Meanwhile, F. trigonate, F. pyriformis and F. Auriculata
exhibited moderate effects.
Therefore, this suppressive effect affords another mechanism by which Ficus exhibited
hepatoprotection.
3.6. Evaluation of hepatic levels of HGF
HGF is mitogenic toward hepatocytes, exhibited multiple functions involved regulation of cell
growth and enhancement of liver regeneration and repair mechanism mediated by signaling pathway
imitated by binding of HGF to specific MET receptor (Zhang and Zhang, 2017). It was demonstrated
that HGF effectively act as a medical therapy during the progression of cholestasis in rodents
mediated by antioxidant, anti-inflammatory, and antiapoptotic activity and preservation of the immune
homeostasis (Ilangumaran et al., 2016).

- 23 -
 In agreement with recent investigation which confirmed the important role of the signaling
molecules in the process of liver regeneration (Tao et al., 2017), the observed decline in serum level
of HGF in EE- induced intrahepatic cholestasis, was significantly reversed in Ficus extracts treated
groups confirming their cytoprotective mechanism to reduce the toxic accumulation of bile acids
within hepatocytes. This repairing potential was decrease in the order of UDCA ≥ F. spragueana ≥ F.
trigonate ≥ F. pyriformis ≥ F. auriculata > F. mysorensis.
Therefore, up-regulation of the HGF signaling pathway affords another potential mechanism
by which Ficus exhibited hepatoprotection.
3.7. Evaluation of hepatic oxidative stress markers
Regulation of redox state plays an important role in the cell functions (Sokkar et al., 2013); (El
Hawary et al., 2016). Oxidative stress and mitochondrial dysfunction are known to be a common
feature of cholestatic syndrome (Li et al., 2016; Van Thuy et al., 2017). A previous study
demonstrated that the cytotoxicity of 17-EE mediated through the overproduction of its oxidized a 0-
quinone metabolite result in mitochondrial dysfunction, exhaustion of the hepatic antioxidant defense
mechanisms, that involved in the onset and progression of intrahepatic cholestasis induced by EE
(Wan and O’Brien, 2014). As consequence, antioxidant agents or enzymes catalyzing reduction or
conjugation with GSH could provide a perspective for the treating 17-EE-induced liver injury (Singh
et al., 2016). By this concept, the significant decline in hepatic HO-1, SOD and CAT enzyme
activities and GSH content, increases the deleterious effects of EE due to the accumulation of reactive
radicals confirmed the implication of oxidative stress in the intrahepatic cholestasis induced by EE. It
was reported that the initial stages of biliary cirrhosis is characterized by abnormal metabolism of NO
and thiol oxidation (Van Thuy et al., 2017).
In the present study, the significant elevated level of hepatic NO and lipid peroxide product
(MDA) is a clear manifestation of excessive production of oxidized metabolites, and reactive radicals
(Z. Y. Ali et al., 2017). This process can alter hepatocyte membrane fluidity and permeability leading
to loss of membrane integrity and cellular leakage as mentioned earlier. Furthermore, ROS has been
recognized as functional signaling molecules, play an important role in triggering the inflammatory
response through activation of redox-sensitive transcription factors such as NF-κB signaling pathway
(Li et al., 2016; Zhang and Zhang, 2017).
The previous studies provided many evidences that supported the ameliorating of oxidative
stress, enhanced HO-1 and inactivation of NF-κB signaling pathway represent a strategy to control the
expression of pro-inflammatory cytokines which contribute to the development of cholestasis (Chen et
al., 2015); (Yu et al., 2016); (Y. Zhao et al., 2017). Therefore, the present study suggested that
protection from the development of hepatic oxidative stress via antioxidant constituents of Ficus

- 24 -
extracts confirming their hepatoprotective potential against intrahepatic cholestasis induced by EE.
Also, UDCA alleviates all the biochemical changes during induction of intrahepatic cholestasis by EE
(Woolbright and Jaeschke, 2016; Li et al., 2017).
In order to explore the mechanism by which Ficus extracts exert its hepatoprotective activity,
a molecular modelling was applied using Molecular Operating Environment software. FXR is selected
as one of the most important nuclear receptors involved in the regulation of bile acid homeostasis and
highly involved in liver regeneration.
3.8. Evaluation of the Molecular Docking Study
Binding of FXR with its ligand (mainly bile acids) activates signaling pathway that regulates
the expression of target genes responsible for the enterohepatic circulation of bile acids (synthesis and
transport), cholesterol homeostasis and inflammation (Yang et al., 2016; Gonzalez et al., 2017). There
is a general agreement that EE-mediated down regulation of FXR as a result the bile transporters are
impaired leading to the development of cholestasis (Cariello et al., 2018). Discovering new FXR
agonist has received attention as a promising therapeutic strategy for metabolic, inflammatory and
hepatobiliary diseases (Yu et al., 2016; Thakkar et al., 2017).
In the present, molecular docking results indicated that both rutin and chlorogenic acid had
high affinity to interact with the key amino acids in the binding site of FXR compared to that of a
potent and selective FXR agonist 3-Deoxy CDCA, suggesting their action as FXR agonist. Also, our
data reveals that rutin displayed the most promising effect over 3-Deoxy CDCA. Based on these
observations, the present study proved that rutin and chlorogenic acting as novel FXR agonists to
regulate the enterohepatic bile acids homeostasis.
Furthermore, this study provides direct evidences that the immunosuppressive, anti-oxidant,
and membrane stabilizing effects also responsible for the hepatoprotective activity of the Ficus
extracts against intrahepatic cholestasis induced by EE. Taken together, the present study describes for
the first time, the potential hepatoprotective activity of F. spragueana against EE-induced liver
toxicity over the tested Ficus species.

4. Conclusion
Among the tested Ficus extracts, Ficus spragueana would be a promising hepatoprotective
agent and a new strategy approach for cholestatic liver diseases by modulating the NF-κB, and FXR
signaling pathways. However, further clinical investigations are required to isolate these biological
markers (chlorogenic acid & rutin) from Ficus spragueana Mildb‎r. & Burret, to establish deeply the
molecular mechanisms of action, and to provide the evidentiary basis for approvals of safe and
effective prescriptions as complementary and alternative remedy. In addition, to investigation of their

- 25 -
mechanism of action on fresh hepatic tissues and perform gene expression at mRNA levels especially
on FXR.

Funding
This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.

Declarations of interest: none

Author's contributions
All authors listed in the manuscript had contributed equally. The final manuscript was read and
approved by all authors.

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Highlights:
 HPLC analysis of phenolics in the leaf extracts of five Ficus species
 Quantification of chlorogenic acid and rutin by HPLC
 Five Ficus species were tested for their hepatoprotective potential
 Modulation of the NF-κB, and FXR signaling pathways.
 A new strategy approach for estrogen-induced cholestasis.

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