1
Date: 11/03/2014
Professor Ahmed Alkhazim
The supervisor of National Plan for Science & Technology
King Saud University
Riyadh, Saudi Arabia
Dear Prof. Alkhazim,
I am pleased to submit to you the first year report of the research project entitled “Evaluation of the
efficacies of natural products for their antiviral activities against hepatitis B virus (HBV) gene expressions
and replication”, number "11-MED-1585-02". The report contains detailed information about what we
have made in the project for the first year, including the various technical issues like plants collection,
identification, extraction, and assessment of cytotoxicity/anti-hepatitis B activities.
Should you have any concerns or questions, please do not hesitate to contact me. .
Sincerely,
Mohammed S. Al-Dosari, Ph.D.
Principal Investigator
College of Pharmacy
Department of Pharmacognosy
P.O Box 2457, Riyadh 11451
E-mail: msdosari@yahoo.com
2
Submitted for
National Plan for Science and Technology
King Saud University
Project title
Evaluation of the efficacies of natural products for their antiviral activities against hepatitis B virus
(HBV) gene expressions and replication
Project number
11-MED-1585-02
Project Investigators
Mohammed S. Al-Dosari, Ph.D., PI
Mohammad Khalid Parvez, Ph.D., CO-I
Adnan J. Al-Rehaily, Ph.D., CO-I
Year
2013
3
1. Abstract
Hepatitis B virus (HBV) chronic infection remains a serious global health issue, including Kingdom of
Saudi Arabia. Despite availability of many effective anti-HBV agents, the emergence of nucleot(s)idebased drug-resistance strains restricts the therapeutic approaches. As an alternative approach, many nonnucleot(s)ide-based drugs as well as natural products have been reported for their anti-HBV potentials.
We therefore, collected 48 plants based on their known hepatoprotective activities in traditional/folk
practices or in experimental animals as well as those with antiviral activities against other viruses closely
related to HBV. The ethanol-extracts of these plants were first tested for cytotoxicity on cultured HepG2
cells and then evaluated or confirmed for hepatoprotective properties. So far, of the 27 plants tested for
cytotoxicity on cultured HepG2 cells and their IC50(microg/ml) values determined using MTT-assay. Out
of these, 19 plants were evaluated for their hepatoprotective properties experimentally because of their
reported folk medicinal use. Of these, Acacia Mellifera leaf (IC50: 684 microg/ml) showed the most
promising hepatoprotection in 2,7-dichlorofluorescein (DCFH)-toxicated HepG2 cells, in vitro as well as
CCl4-injured liver of rats, in vivo evaluated by biochemical and histological parameters. Further, to screen
the anti-HBV efficacies of the selected plant extracts, HepG.2.15 cell culture supporting HBV DNA
replication and gene expressions was optimized with standard antiviral drug Lamivudine (3TC). As an
initiative, A. Mellifera leaf extract was evaluated for anti-HBV screening by measuring the HBsAg
secretion in the culture supernatant, using ELISA-Kit. Our preliminary dose- and time-dependent
experiments showed a promising effect of A. Mellifera on suppressing HBsAg. In conclusion, (1) we have
collected and authenticated 48 plants of interest, (2) of the 27 plants extracted and tested for toxicity, 19
were evaluated for hepatoprtection, in vitro, (3) A. Mellifera extract showed potential hepatoprotective
properties in vivo that warranted its therapeutic use, and (4) the anti-HBV efficacy of A. Mellifera needs
further experimental validation using HBeAg-ELISA. Collection of more plants and evaluations of
hepatoprotective and anti-viral efficaies of the remaining plants are currently under progress.
4
Acknowledgments
The financial support from the National Plan for Science and Technology (Grant No. 11-MED-1585-02),
KACST, Saudi Arabia is gratefully acknowledged.
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Table of Contents
1.0. Abstract
2.0. Introduction
3.0. Objectives
4.0. Materials and methods
4.1. Human liver cell and HBV-cell cultures
4.2. Statistical analysis
4.3. Analysis effects of phytoproducts on HBsAg expressions
4.4. Biochemical and histological profiling
4.5. Animals and treatment with plant extracts
4.6. In vitro hepatoprotective activity assay
4.7. Preparation of plant extracts
4.8. Cytotoxicity test
4.9. Selection of plants or phytoproducts, including those of ‘heaptoprotective’ properties
5.0. Results
5.1. Establishment of human liver cell and HBV cultures
4.2. Analysis of collected plants (n= 48)
5.3. Hepatoprtective effect of A. Mellifera (AM) on cultured liver cells
5.4. Hepatoprotective effect of AM on biochemical markers, in vivo
5.5. Histological improvement by AM
5.6. Optimization of HBV antigen expressions in culture
5.7. Optimization of Lamivudine (3-TC) as standard anti-HBV drug
6.0. Discussion
7.0. Future work
6
List of Figures and List of Tables
Table-1: List of plants collected, authenticated, extracted and screened
Table-2: Effect of A. mellifera exract on CCl4-induced hepatotoxicity-related parameters
Table-3: Effect of A. mellifera exract on CCl4-induced lipid profile changes
Table-4: Biochemical parameters (liver tissue) treated with A. mellifera exract
Figure-1: MTT-cell proliferation assay showing hepatoprotective effect of A. mellifera extract against
DCF-induced hepatotoxicity in HepG2 cells
Figure-2: Histogram showing rat liver with normal hepatocytes and central vein
Figure-3: Histogram showing CCl4-injured rat liver with necrosis and fatty degenerative changes
Figure-4: Histogram showing A. mellifera (250 mg)+CCl4 treated rat liver with congested central vein
with necrosis and fatty changes
Figure-5: Histogram showing A. mellifera (500 mg)+CCl4 treated rat liver with normal hepatocytes and
central vein with full recovery
Figure-6: Histogram showing Silymarin+CCl4 treated rat liver with normal hepatocytes and central vein
with full recovery
Figure-7: HBsAg ELISA showing antiviral activity of A. mellifera
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2.
Introduction
Hepatitis B virus (HBV) infection continues to be one of the most widespread viral infections in human
worldwide, including the Arabian Peninsula (Evans et al., 1998; Qirbi and hall, 2001; Toukan, 1997). The
prevalence of chronic HBV infection is estimated as >400 million with an annual death rate of 1.2 million
people. Chronic hepatitis B (CHB) is a serious public health issue because it causes broad spectrum of
chronic liver diseases (CLDs) like, fulminant liver failure, chronic active hepatitis, liver cirrhosis and
hepatocellular carcinoma (HCC) (Evans et al., 1998). Especially in Saudi Arabia, prevalence of Hepatitis
B surface antigen (HBsAg) ranges from 7.4 to 17% showing high endemicity (Al Faleh, 1998; Atiyah and
Ali, 1980). In areas of low prevalence, the disease is found primarily amongst adolescents and adults as a
consequence of parental or sexual exposure. Persistent HBV infection, particularly if associated with
cirrhosis, is the most important risk factor for the development of HCC, also reported in the Kingdom
(Abdo et al., 1980; Fasir et al., 1996). Vaccination against HBV is the cornerstone in the global strategy
in the prevention against further spread of the disease, in particular, perinatal and early horizontal
transmission. Nevertheless, upon the implementation of HBV vaccination of Saudi children, the
prevalence has dramatically reduced (Alfaleh et al, 2008).
Moreover, no satisfactory anti-HBV therapeutic breakthrough has been achieved so far due to lack of a
suitable animal model or cell culture system to mimic clinical HBV infection. For those individuals with
chronic infection, control of active disease to avoid its progression to cirrhosis and HCC lies in the
efficacy of anti-HBV agents and their appropriate use in the clinical setting. For many years, interferonalpha (IFN-alpha) has been the only registered antiviral cytokine available. However, significant side
effects and non-response to IFN-alpha in a proportion of HBV patients, has limited its use. In recent
years, nucleos(t)ide analogues therapy has emerged as promising antiviral therapy. Although the use of
nucleos(t)ide analogues could potentially change the course of HBV infection due to the emergence of
drug-specific resistant (HBV-pol/RT) mutants, has opened
up new frontiers and challenges in the
treatment of CHB (Durantel et al, 2005). Currently, combination chemotherapies with more than one
8
nucleos(t)ide analogues are being evaluated as the new approach towards management of CHB infection.
Further, there are numerous natural products from plants of diverse geographic origin and based on local
cultural practices have described to have therapeutic benefits for viral hepatitis. Although, a number of
plant products are shown ‘hepatoprotective’ (Alqasoumi. 2010; Abdel-Kader et al., 2009; Al-Howiriny et
al., 2004; Al-Ghamdi, 2003;
Ali et al, 2001), their ‘anti-HBV’ activities have not been studied.
Therefore, to overcome the antiviral drug-resistance and adverse-side effects, the evaluation and
discovery of novel natural products of anti-HBV activities are mandatory.
The HepG2.2.5 line is a derivative of human hepatoma cells, HepG2 that has been stably transfected with
HBV infectious genome (Simon et al., 1985). Hep2.2.15 efficiently supports HBV DNA replication and
secretes viral antigens in the culture supernatant, and is used to screen anti-HBV candidates universally.
In the present study we therefore, used this established system to screen the antiviral activities of plant
products.
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3.
Objectives
The aims of the present study were to screen and identify novel anti-HBV activities of the
‘hepatoprotective’ natural products in a cell culture-based model.
1. Establishment of liver cell culture-system to model HBV infection in vitro
2. Cytotoxicity test of the natural products (extracts) on cultured liver cells
3. Screening of natural products for their anti-HBV activities
4. Evaluation of the efficacy of potential antiviral product(s) on HBV DNA replication
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4. Materials and methods
4.1.Human liver cell and HBV-cell cultures
Human hepatoma cell lines: HepG2 and HuH7, including HepG2.2.15 (HBV stable cells) was obtained
from International Center for Genetic Engineering & Biotechnology (CGEB), New Delhi, India. Cell
were maintained in complete Dulbecco’s modified Eagle’s media (DMEM) supplemented with 100 mL/L
FCS, 5 mg/L insulin and with or without 10g/L DMSO at 37 ℃ in a humidified incubator supplied with
5% CO2. Cells were passaged and frozen time to time to maintain the stock in liquid nitrogen or 1500C
and were revived when required.
4.2. Selection of plants or phytoproducts, including those of ‘heaptoprotective’ properties
The potential candidates for anti-HBV screening was selected, based upon their known hepatoprotection
activities experimentally or in traditional/folk practices as well as those with antiviral activities against
other viruses closely related to HBV. However, those extracts showing very high cytotoxicity would be
excluded from the antiviral analysis. The botanical authentications for all study plants were done in the
college by an expert taxonomist and voucher specimen submitted. The other novel plant products of
taxonomically related family or genus may also be included
4.3. Cytotoxicity test
A test for cytotoxic effect(s), if any, of the extracts and their solvents used was carried out on HepG2 cells
prior to further analysis using MTT-cell proliferation assay.
4.4. Preparation of plant extracts
The plant materials were shade-dried and powdered. For each plant, 100g of powdered materials was
soaked in a suitable volume of 80% aqueous ethanol at 25-30°C with frequent agitation until the soluble
matter dissolved completely. The mixture was then strained and the damp solid material was extracted
two times with fresh solvent. The combined liquids were clarified by decantation after standing and
filtration. The extract was evaporated using a rotary evaporator under reduced pressure at 40 °C. The
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obtained greenish brown semi-solid extract (yield 10.83 -7.6%) was stored at -20°C and then used for
evaluation of biological activities.
4.5. In vitro hepatoprotective activity assay
HepG2 cells were seeded (104 cells/well, in triplicate) in a 96-well flat-bottom plate and grown over
night. Cytotoxicty of 2,7-dichlorofluorescein (DCF) was determined by the MTT. DCF (IC50: 100
microM) was used as a cytotoxic dose, prepared in dimethylsulfoxide (DMSO). Plant extracts were
dissolved in DMSO (100 mg/ml), followed by dilution with RPMI media to 4 doses (25, 50, 100, and 200
mg/ml). The final concentration of DMSO used on in vitro never exceeded >0.1%, and therefore tolerated
by cultured cells. The culture monolayer were replenished with RPMI containing 100 𝜇M DCF plus a
dose of the extract, including untreated as well as DCF-treated controls. Cells were incubated for 48 h at
37 0C with 5% CO2. Cells were then treated with 20 μl MTT reagent/well and further incubated for 4 h,
and then 100 μl detergent was added to each well to dissolve the formazan crystals. The optical density
(OD) was recorded at 490 nm in a microplate reader and the data analyzed as per the standard protocol.
4.6. Animals and treatment with plant extracts
Male Wister rats were obtained from the Experimental Animal Care Center (EACC) of the College.
Animals were housed in polycarbonate cages in a room free from any source of chemical contamination,
artificially illuminated (12 h dark/light cycle) and thermally controlled (25 ±2 0C). After acclimatization,
animals were randomized and divided into five groups (I–V) of six animals each. Group I served as
untreated control and fed orally with normal saline 1mL. Group II was received CCl₄ in liquid paraffin
(1:1) 1.25 ml /kg⋅bw intraperitoneally (IP). Groups (III, IV and V) received CCl₄ in liquid paraffin (1:1)
1.25 ml/kg⋅bw intraperitoneally (IP). Groups II and III treated with A. Mellifera extract at a dose of 250
mg/kg.bw and 500 mg/kg⋅bw respectively for three weeks. Group V was treated with the standard drug
silymarin at a dose of 10 mg/kg⋅bw for three weeks. All animals received human care in compliance with
the guidelines of the Ethics Committee of the Experimental Animal Care Society, College of Pharmacy,
King Saud University, Riyadh.
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4.7. Biochemical and histological profiling
Animal’s blood samples were collected for estimating serum aspartate transaminase (AST), alanine
transaminase (ALT), gamma-glutamyl transferase (GGT),
alkaline phosphatase (ALP), bilirubin
cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), very low-density
lipoproteins (VLDL),
triglycerides (TG) and malondialdehyde (MDA) levels using commercially
available kits. Animals were sacrificed and liver tissues were dissected for further analysis of non-protein
sulfhydryl (NP-SH) and total proteins(TP) using commercial kits.
For the histological study, the liver tissues were fixed in 10% buffered formalin and processed using a
VIP tissue processor. The processed tissues were then embedded in paraffin blocks and sections of about
5 µm thickness were cut by employing an American optical rotary microtome. These sections were
stained with haematoxylin and eosin using routine procedures. The slides were examined microscopically
for pathomorphological changes.
4.8. Analysis effects of phytoproducts on HBsAg expressions
The levels of secreted HBsAg and HBeAg in the culture supernatants was analyzed by commercial
enzyme immunoassays (BioRad HBsAg Moalisa Microelisa).
4.9. Statistical analysis
All values were represented using ANOVA, followed by Dunnett’s multiple comparison test.
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5. Results
5.1. Establishment of human liver cell and HBV cultures
HepG2 and HepG2.2.15 cultures were established, and enough stocks were frozen for further use.
5.2. Analysis of collected plants (n= 48)
Of the 48 plants collected, 27 plants extracts were prepared and tested for cytotoxicity on cultured HepG2
cells and their IC50(microg/ml) values determined using MTT-assay (Tables-1 and 2). P. tomentosa (code10; family: Asclepiadaceae) showed a very high cytotoxicity (IC50 = 38.33 microg/ml) and therefore
excluded, from the study. Nineteen plant extracts were further evaluated for hepatoprotective properties in
vitro. Of these, A. Mellifera (Code-9; IC50: 684 microg/ml) showed the most promising hepatoprotection
in DCF-toxicated HepG2 cells, in vitro as well as CCl4-injured liver of rats, in vivo evaluated by
biochemical and histological parameters.
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Table 1. List of plants collected, authenticated, extracted and screened
Plant
Family
Capparis decidua (stem)
Euphorbia hirta (aerial parts L,S, F)
Abutilon figarianum (leaves)
Pulicaria crispa ( aerial parts L, S,
F)
Ipomoea cairica (L.) sweet (aerial
parts S,L F)
Guiera senegalensis (leaves)
Psidium guajava ( Leaves )
Balanites aegyptiaca (bark) :
Acacia mellifera (leaves)
Acacia mellifera (bark)
Pergularia tomentosa
Marrubium vulgare
Ficus benghalensis ( leaves)
Ficus benghalensis ( bark)
Combretum molle (bark)
Euphorbiaceae
Crassulaceae
16
Jatropha curcas (seeds)
Cleome droserifolia (aerial parts
L,S,F)
Ficus palmata (L)
17
Cassytha filiformis (S)
Lauraceae
18
19
20
Dodonea angustifolia (L)
Senna obtusifolia (F)
Senna occidentalis (F)
Sapindaceae
Fabaceae
Fabaceae
21
22
23
24
25
26
27
28
29
30
31
Delonix elata
Achyranthes aspera
Citrus maxima, (L)
Datura inoxia, (L)
Clerodendrum inerme
Euphorbia tirucalli (stem)
Ricinus communis ( L )
Flaveria trineriva
Juniperus procera
Juniperus phonicea
Delonix regia
Fabaceae
Amaranthaceae
Rutaceae
Solanaceae
Verbenaceae
Euphorbiaceae
Euphorbiaceae
Asreraceae
Cupressaceae
Cupressaceae
Fabaceae
1
2
3
4
5
6
7
8
9
9a
10
11
12
12a
13
14
15
Capparaceae
Euphorbiaceae
Malvaceae
Asteraceae
Place of
collection
Tabouk, KSA
Sudan
Sudan
Sudan
Cytotoxicity (IC50(
μg/ml)
127
189.2
175
203.1
Anti-HBV
activity
Pending
Pending
Pending
Pending
Convolvulaceae
Riaydh,KSA
225.5
Pending
Combretaceae
Myrtaceae
Zygophyllaceae
Fabaceae
Fabaceae
Asclepiadaceae
Labiatae
Moraceae
Moraceae
Combretaceae
Sudan
Sudan
Sudan
Sudan
Sudan
Riaydh, KSA
Elhadda, KSA
Riaydh, KSA
Riaydh, KSA
Jabel Shada,
KSU
Out of KSA
Way to Duba,
KAS
Jabel Burma,
KSA
Jabel Fabya,
KSA
Shargeia, KSA
Shargeia, KSA
Wady Lajab,
KSA
Shargeia, KSA
Riaydh, KSA
Riaydh, KSA
Riaydh, KSA
Riaydh, KSA
Riaydh, KSA
Riaydh, KSA
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
297.4
263.6
590
684
122.8
38.33
381.42
870
251.73
280
Pending
Pending
Pending
In progress
Pending
Excluded
Pending
Pending
Pending
Pending
401.5
156.5
Pending
Pending
402.58
Pending
948
Pending
186.68
778.33
1085
Pending
Pending
Pending
538
552.66
280
272.1
841.66
187.39
414.57
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Moraceae
15
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Argemome ochroleuca
Bouganvillea spectablis
Albizia procera
Rumex dentatus
Atriplex suberecta
Avera Javanica
Chenopodium glaucum
Chenopodium ambrosioides
Boerhavia diffusa
Haplophylum tuberculum
Fumaria parviflora
Anagallis arvansis
Bacopa muniera
Senna alexandria
Coccinea grandis
Delonis alata
Eruca sativa
Papaveraceae
Nyctaginaceae
Fabaceae
Polygonaceace
Chenopodiaceae
Amaranthaceae
Chenopodiaceae
Chenopodiaceae
Nyctaginaceae
Rutaceae
Fumariaceae
Primulaceae
Scrophulariaceae
Fabiaceae
Cucurbitaceae
Fabaceae
Brassicaceae
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
Eltaif, KSA
South, KSA
South, KSA
South, KSA
South, KSA
South, KSA
South, KSA
South, KSA
South, KSA
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
Pending
16
5.3. Hepatoprtective effect of A. Mellifera (AM) on cultured liver cells
MTT test showed a cytotoxic effect of DCF on HepG2 cell. While DCF-toxicated cells were
recovered to about 100% with 100 mg/ml of AM extract, supplementation with 200 mg/ml of
AM further enhanced the hepatocytes proliferation by about 20% (Figure-1).
1.4000
Sutvival fraction
1.2000
1.0000
0.8000
0.6000
0.4000
0.2000
0.0000
200
mg/ml
100
50
25
DCF only
mg/ml
mg/ml
mg/ml
100 μM
___________________________________
+100 μM DCF
Cells only
Figure-1. MTT-cell proliferation assay showing hepatoprotective effect of A. mellifera extract against DCFinduced hepatotoxicity in HepG2 cells
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5.4. Hepatoprotective effect of AM on biochemical markers, in vivo
Based on in vitro hepatoprotective activity, the effects of AM extract was further examined in laboratory
animals model. As shown in Table-2 the administration of CCl₄ dramatically elevated the serum AST,
ALT, GGT ALP and bilirubin levels compared to the normal control group (P<0.0001), indicating
significant hepatotoxicity of CCl₄ treatment. In contrast, administration of AM extract significantly
decreased the above elevated parameters in CCl₄-treated rats when compared to the CCl₄-treated group.
Moreover CCl₄-induced toxicity caused significant elevation in lipid profile including cholesterol,
triglycerides, LDL-C, and VLDL-C and reduction in the HDL-C levels in serum. The three-week
pretreatment of rats with AM extract in different doses, dose-dependently and significantly, reduced the
cholesterol, triglycerides, LDL-C, and VLDLC levels and significantly improved HDL-C level (Table-3).
Silymarin, on the other hand, significantly prevent the CCl₄-induced elevated levels of marker enzymes
and lipid profile. Furthermore, our results indicated that treatment with CCl₄ resulted in a significant
increase in MDA and a significant decrease in NP-SH and TP concentration (Table-4). Treatment of rats
with AM extract resulted in a significantly diminished level of MDA and significantly enhanced NP-SH
and TP levels.
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Table-2. Effect of AM exract on CCl4-induced hepatotoxicity-related parameters
Treatment
Dose
group
mg/kg
Normal
CCl4
AM+ CCl4
1.25mg/kg
250
AST(U/L)
ALT(U/L)
GGT(U/L)
ALP (U/L)
Bilirubin
(mg/dl)
106.15±4.36
37.91± 1.61
3.26± 0.21
308.83±8.81
0.5±0.02
379.83±11.70***
303.83±12.12***
18.20±
586.16±11.92***
2.74±0.1.0***
a
a
0.89***a
a
a
322.50±12.08**b
264.83±10.74*b
12.41±0.73***b
512.0±11.40**b
1.66±0.10***
b
AM+ CCl4
Silymarn +
CCl4
500
10
283.66±9.82***b
166.16±9.34b
178.16±6.27***
99.75±3.55 ***
6.90±0.38***b
5.78± 0.26***b
398.50±o.486***
1.16±0.10***
b
b
325±33±12.10***
0.94±0.04***
b
b
All values represent mean ± SEM. ∗ 𝑃 < 0.05; ∗∗ 𝑃 < 0.01; ∗∗∗𝑃 < 0.001; ANOVA, followed by Dunnett’s multiple comparison
test., a As compared with no rmal group. bAs compared with CCl4 only group.
19
Table 3. Effect of AM extract on CCl4-induced lipid profile change
Treatment
Dose
group
mg/kg
Normal
CCl4
1.25ml/kg
TC(mg/dl)
TG (mg/dl)
HDL-C(mg/dl)
LDL-C(mg/dl)
VLDL-C
mg/dl)
88.09±6.5
78.26±4.09
50.82±1.25
72.24±6.34
15.56±0.65
212.69±7.84***a
184.05±8.09***a
22.14±0.56***a
175.80±8.42***a
36.81±1.69**
*a
AM+ CCl4
250
169.04±11.25***
168.59±9.17 b
28.26±1.25**b
135.22±10.18*b
33.71±1.83 b
117.37±7.01***b
36.76±1.02***b
112.89±4.02***b
23.47±1.40**
b
AM+ CCl4
500
136.50±4.70***b
*b
Silymarn +
CCl4
10
124.60±5.94***b
98.55±5.60***b
35.34±1.81***b
104.74±5.93***b
19.71±1.12**
*b
All values represent mean ± SEM. ∗ 𝑃 < 0.05; ∗∗ 𝑃 < 0.01; ∗∗∗𝑃 < 0.001; ANOVA, followed by Dunnett’s multiple comparison
test., a As compared with normal group. bAs compared with CCl4 only group.
20
Table 4. Biochemical parameters (liver tissue) treated with AM
Treatment
Dose mg/kg
TP (mg/dl)
MDA (nmol/g)
NP-SH(mg/dl)
95.84±6.27
0.96±0.11
8.16±0.42
1.25ml/kg
30.78±3.13***a
8.55±1.07***a
4.25±0.42***a
AM+ CCl4
250
36.21±3.22b
5.57±0.77*b
6.20±0.56*b
AM+ CCl4
500
56.63±3.80***b
2.257V0.20***b
6.28±0.48*b
Silymarin +
10
56.64±7.61***b
2.87±0.64***b
7.36±0.54**b
group
Normal
CCl4
CCl4
All values represent mean ± SEM. ∗ 𝑃 < 0.05; ∗∗ 𝑃 < 0.01; ∗∗∗𝑃 < 0.001; ANOVA, followed by Dunnett’s multiple comparison
test., a As compared with normal group. bAs compared with CCl4 only group.
21
5.5. Histological improvement by AM
The histological examination of rat liver tissues, revealed evidence of hepatic necrosis and fatty
degenerative changes in CCl4-injured animals. Compared to this, the AM extract-treated (250 mg/kg/day)
animals exhibited congested central vein with mild necrosis and fatty changes. On the other hand, the
higher dose (500 mg/kg/day) of AM and Silymarin administration showed normal hepatocytes and central
vein with full recovery (Figure 2-6). This finally confirmed the hepatoprotective efficacy of AM.
Figure-2. Histogram showing rat liver with normal hepatocytes and central vein
Figure-3. Histogram showing CCl4-injured rat liver with necrosis and fatty degenerative changes
22
Figure-4. Histogram showing A. mellifera (250 mg)+CCl4 treated rat liver with congested central vein
with necrosis and fatty changes
Figure-5. Histogram showing A. mellifera (500 mg)+CCl4 treated rat liver with normal hepatocytes and
central vein with full recovery
23
Figure-6. Histogram showing Silymarin+CCl4 treated rat liver with normal hepatocytes and central vein
with full recovery
5.6. Optimization of HBV antigen expressions in culture
The HepG2.2.15 culture supernatants were tested for HBV surface antigen (HBsAg) expressions, in
culture flasks as well as 96-well plate by ELSA. HepG2 cells were used as negative (uninfected/healthy)
control. Two commercially available HBsAg ELISA-kits from BioRad and DiaScore were used.
5.7. Optimization of Lamivudine (3-TC) as standard anti-HBV drug
Lamivudine (3-TC), the FDA-approved nucleos(t)ide analog used as potential antiviral against chronic
hepatitis B. Pure Lamivudine was procured (Sigma) and the stock was prepared in DMSO (1%). The
HepG2.2.15 cultures were treated with with different concentration of Lamivudine and the supernatants
were tested for HBV surface antigen (HBsAg) expressions in 96-well plate by ELSA. HepG2 cells served
as negative control. Lamivudine was optimized to completely inhibit HBV gene expressions at a
concentration of 2 M.
24
6. Discussion
Natural or phytoproducts are always been explored as an alternative therapeutic approach against human
diseases, including liver abnormalities like viral hepatitis B. In an extraction process of phytoproducts,
aqueous ethanol is generally employed in an attempt to extract as many compounds as possible. This is
based on the ability of alcoholic solvents to increase cell wall permeability, facilitating the efficient
extraction of large amounts of polar and medium to low-polarity constituents.
In the present study, we so far have collected and identified 48 plants; the collection was based on their
known hepatoprotective activities in traditional/folk practices or in experimental animals as well as those
with antiviral activities against other viruses closely related to HBV. To conduct our experiments, plant
ingredients were pulled out of the whole plant using ethanol extraction. So far, plants extracts of 27 plants
were tested for their cytotoxic activities on cultured HepG2 cells using MTT-assay. Out of these 27
plants, 19 plants were evaluated for their hepatoprotective properties experimentally because of their
reported folk medicinal use. Out of these, 19 plants were evaluated for their hepatoprotective properties
experimentally because of their reported folk medicinal use. Of these 19 plants, Acacia Mellifera leafs
(IC50: 684 microg/ml) showed the most promising hepatoprotection in vitro in 2,7-dichlorofluorescein
(DCFH)-toxicated HepG2 cells and in vivo using CCl4-injured rat's liver. AM not only protected the cells
against DCF-induced toxicity, but also promoted cell recovery and proliferation.
CCl4 is a common hepatotoxin used in the experimental study of liver diseases that induces free radical
generation in liver tissues (Ganie et al., 2013). Liver damage caused by acute exposure to CCl4 causes
clinical symptoms such as jaundice, and elevated levels of liver enzymes in the blood (Tirkey et al.,
2005). The liver enzymes such as such as AST, ALT, and ALP found within organs and tissues are
released into the blood stream following cellular necrosis and cell membrane permeability and are used as
a diagnostic indicator of liver damage. In this investigation, treatment with two doses of AM extarct: 250
and 500 mg/kg showed the ability to reduce the ALT, AST, GGT, ALP and bilirubin level significantly in
a dose-dependent manner. Similar trend was observed for the serum cholesterol and triglycerides and
25
HLD level where AM was able to reduce the level of these parameters in CCl4-induced rats. The effect of
AM was comparable to standard drugs Silymarin suggesting a protective effect of the extract. The
significant reduction in levels of LDLP-C, LDLP-C and total cholesterol in the AM-treated rats and an
increase in HDL-C level further indicates the hepatoprotective potential of the AM extract. MDA is a
metabolite that is produced during peroxidation of biological membrane of polyunsaturated fatty acid, and
the amount of MDA is used as an indicator for lipid peroxidation of cell membrane which could cause
cell damage (Suhail et al., 2009 ). The levels of MDA had reduced in both CCl4-induced liver damage
treated with AM and Silymarin which further suggested the hepatoprotective and curative activities of
AM. NPSH are involved in several defense processes against oxidative damage (Babu et al., 2001). In
the current study, the liver NP-SH level in CCl4-treated group was significantly diminished when
compared with the control group. Pretreatment of rats with AM or sliymarin replenished NPSH
concentration as compared with CCl4 only treated animals suggesting free radical scavenging activity of
our extract. The levels of TP in serum were related to the function of hepatocytes. Diminution of TP is a
further indication of liver damage in CCl4-injured animals. In this study, the level of TP has been restored
towards the normal value indicating its hepatoprotective action Furthermore, most of the parameters in
the group which received CCl4 plus AM extract having nearer value of the group received CCl 4 plus
standard drug Silymarin indicating that AM extract is able to inhibit CCl4-induced hepatotoxicity.
Moreover, the histological examination of rat livers also confirmed the potential hepatoprotective efficacy
of SM.
This hepatoprotective effect of AM extract could be attributed to the presence of antioxidant and free
radical scavenging factors for example, phenolic and flavonoid compounds which were reported to have
hepatoprotective activity (Tirkey et al., 2006; Ai, et al., 2013; Saboo et al., 2013). The hepatoprotective
activity of flavonoids is due to their ability to reduce free radical formation and to scavenge free radicals
(Nogata et al., 2006).
As an initiative, A. Mellifera leaf extract was evaluated for anti-HBV screening by measuring the HBsAg
secretion in the culture supernatant, using ELISA-Kit. Our preliminary dose- and time-dependent
26
experiments showed a promising effect of A. Mellifera on suppressing HBsAg and needs further
experimental validation using HBeAg-ELISA.
27
7. Future work
The remaining plant out of the 48 collected or from any future collected plants would be processes and
screened for cytotoxicity and/or hepatoprotection, the extract no. 16, 19, 21, 22, 23, 24, 27 and 29 with
very high IC50 values would be also studied for their hepatoprotective activities in vitro as well as in vivo.
To study the effects of the extracts on HBV replication, HBeAg ELISA-Kit will be optimized and all
selected non-toxic extracts would be screened for antiviral activities by HBsAg and HBeAg ELISA Kits.
Additionally, viral DNA replication will be monitored by quantifying HBV DNA load, for the potential
antiviral extract. Furthermore, extracts showing promising antiviral activity might be further fractionated
with different organic solvents of varying polarity to identify the potential active ingredient.
28
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Publications/presentations
Parvez MK. Emerging and re-emerging viral diseases: risks and controls. In Microbial Pathogens and
Strategies for Combating Them: Science, Technology and Education, Ed. by A. Mendez-Vila (Formatex,
Spain, 2013), Vol. 3: pp. 1619-1626.
Parvez MK, Emerson SU and Al-Dosari MS. Molecular characterization of hepatitis E virus ORF1 gene
supports a papain-like cysteine protease (PCP)-domain activity. European Association of Study of Liver
(EASL)-Interantional Liver Congress 2013(ILC2013), Amsrterdam, 2013.
Arbab AH, Parvez MK, Al-Dosari MS, Al-Said M and Rafatullah S. Attenuation of CCl4-induced
oxidative stress and Hepatotoxicity by Acacia mellifera extract in Rats. European Association of Study of
Liver (EASL)-Interantional Liver Congress 2014(ILC2014), London, 2014.
Arbab AH, Parvez MK, Al-Dosari MS, Al-Said M and Rafatullah S. Attenuation of CCl4-induced
oxidative stress and Hepatotoxicity by Acacia mellifera extract in Rats. 2014 [under preparationn].