Evaluation of hepatoprotective activity of Allium sativum ethanolic extract in thioacetamide induced hepato-toxicity in albino Wistar rats.

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AMERICAN JOURNAL OF RESEARCH IN MEDICAL SCIENCES, 2018
VOL 3, NO. 2, PAGE 48–53
10.5455/ajrms.20180107060815
ORIGINAL ARTICLE Open Access
Evaluaon of hepatoprotecve acvity of Allium savum ethanolic extract in
thioacetamide-induced hepatotoxicity in albino Wistar rats
Krishna Mohan Chinnala, Praisy Pinky Jayagar, Gayathri Moa, Raghuma Chowdary Adusumilli, Madhan Mohan
Elsani
Department of Pharmacology, School of Pharmacy, Nalla Narasimha Reddy Educaon Society’s Group of Instuons, Hyderabad, India
ABSTRACT
Aim: The present study was designed to evaluate the hepatoprotecve eect
of ethanolic extract of Allium savum (EEAS) on thioacetamide (TAA)-induced
hepatotoxicity in rats.
Methods: Male Wistar rats were administered 200 and 400 mg/kg/p.o. of EEAS for 21
days and simultaneously administered TAA 50 mg/kg/s.c. 1 hour aer the respecve
assigned treatments for every 72 hours. At the end of experimental methods, all the
animals were sacriced by cervical decapitaon. Blood samples were collected. Serum
was separated and analyzed for various parameters like aspartate transaminase (AST),
alanine transaminase (ALT), alkaline phosphatase (ALP), and total bilirubin using com-
mercially available test kits.
Results: Results indicate that EEAS administraon blunt TAA-induced increase in acvi-
es of dierent marker enzymes of hepatocellular injury, viz., AST, ALT, ALP, and total bil-
irubin signicantly (p < 0.001), suggesng that EEAS possibly has a protecve inuence
against TAA-induced hepatocellular injury and degenerave changes.
ARTICLE HISTORY
Received January 07, 2018
Accepted July 15, 2018
Published July 24, 2018
KEYWORDS
Allium savum;
thioacetamide;
hepatoprotecve acvity
Introducon
The liver is the largest glandular organ in the body
and performs multiple critical functions to keep the
body free from toxins and harmful substances. The
liver synthesizes, concentrates, and secretes bile
acids and excretes other toxicants, such as biliru-
bin [1]. In addition to producing bile, the liver also
plays a number of important roles, viz
the blood to get rid of harmful substances such as
alcohol and drugs, stores some vitamins and iron,
stores the sugar glucose, converts stored sugar to
functional sugar when the body’s sugar (glucose)
levels fall below normal, breaks down hemoglobin
as well as insulin and other hormones, converts
ammonia to urea, which is vital in metabolism,
and destroys old red blood cells [2]. Hepatotoxicity
implies chemical-driven liver damage that causes
acute and chronic liver diseases. More than 900
drugs have been implicated in causing liver injury
and it is the most common reason for a drug to be
withdrawn from the market [3]. Hepatotoxicity and
drug-induced liver injury also account for a sub-
stantial number of compound failures, highlighting
the need for drug screening assays, such as stem
cell-derived hepatocyte-like cells, that are capable
of detecting toxicity early in the drug development
process [4,5].
Garlic (Allium sativum) is used worldwide as
a food additive, spice, and medicine [6]. Allium
sativum, a member of the Liliaceae family, is a com-
          
herbs most commonly used in modern folkloric
medicine. Garlic was an important medicine to the
ancient Egyptians as listed in the medical text Codex
Ebers (ca. 1550 BC) especially for the working class
involved in heavy labor because it was an effective
Contact Madhan Mohan Elsani elsanimadhan@gmail.com Associate Professor, Department of Pharmacology, School of Phar-
macy, Nalla Narasimha Reddy Educaon Society’s Group of Instuons, Hyderabad, India.
© EJManager. This is an open access arcle licensed under the terms of the Creave Commons Aribuon Non-Commercial License (hp://
creavecommons.org/licenses/by-nc/3.0/) which permits unrestricted, noncommercial use, distribuon and reproducon in any medium, provided
the work is properly cited.

www.ajrms.com 49
Hepatoprotecve acvity of Allium savum
remedy for many ailments such as heart problems,
headache, bites, worms, and tumors. Allium sativum
also reported for its several pharmacological
activities such as anti-diabetic and hypolipidemic
activity [7], anticancer activity [8], antioxidant
activity [9], antihypertensive activity [10], in vitro
anti-fungal activity [11], and antiplatelet activity
[12]. The present study was designed to evaluate
the hepatoprotective effect of aqueous extract of A.
sativum on thioacetamide (TAA)-induced hepato-
toxicity in rats.
Methods
Collecon and authencaon of plant material
The A. sativum plant material (raw bulblets) was
collected from the local market of Hyderabad, Tel-
angana and was authenticated by Ms. D. Kavitha,
Asst. Professor, Department of Botany, Osmania
University, Hyderabad.
Preparaon of plant extract
The raw bulblets of the A. sativum were cleaned,
sliced, crushed into small pieces, and dried and then,
the dried pieces were pulverized into coarse powder.
The coarse powder was prepared by mechanical
grinding. The powdered material (100 g) was sub-
jected to continuous hot extraction in a Soxhlet
apparatus at a temperature of 50°C–60°C by using
ethanol as a solvent. The solvent was evaporated at
room temperature to obtain a viscous mass. It was
       
molten mass was brown in color and was stored in
a desiccator until use.
Preliminary phytochemical analysis
The ethanolic extract of A. sativum (EEAS) was
subjected to preliminary phytochemical analysis
       -
noids, terpenes, steroids, glycosides tannins and
the absence of proteins, sterols, phenols, glycosides,
saponins, and terpenes. The extract was suspended
in distilled water using 1% sodium carboxymethyl
cellulose (SCMC) as a suspending agent for oral
administration to animals.
Experimental animal
A total of 30 male Wistar strain rats weighing
150–220 g, were procured from the Sainath Agen-
cies [Reg.no. 282/99/ Committee for the Purpose of
Control And Supervision of Experiments on Animals
(CPCSEA)] Hyderabad, Telangana and were housed
at CPCSEA approved animal house of School of Phar-
macy, Nalla Narasimha Reddy Education Society’s
Group of Institutions, Hyderabad. The animals were
kept in polypropylene cages (six in each cage) under
standard laboratory condition (12 hours light and
12 hours dark cycle) and had free access to com-
mercial pellet diet (Hindustan Lever Ltd., Bombay,
India) with water ad libitum. The animal house tem-
perature was maintained at 25°C ± 2°C with relative
humidity at (50% ± 15%). The study was approved
by the Institutional Animal Ethical Committee of
Nalla Narasimha Reddy School of Pharmacy (002/
Institutional Animal Ethics Committee (IAEC)/
NNRG/2017). Ethical norms were strictly followed
during all experiments.
Hepatoprotecve acvity
       
group consisting of six animals. Group I served as
control group received the 1% SCMC (2 ml/kg).
Group II served as disease control received TAA 50
mg/kg/s.c., for every 72 hours for 21 days. Group III
received standard drug (silymarin 50 mg/kg p.o.)
for 21 days and simultaneously administered TAA
50 mg/kg/s.c. 1 hour after the respective assigned
treatments for every 72 hours. Groups IV and V
treated with EEAS 200 mg/kg/p.o., 400 mg/kg/p.o.
for 21 days and simultaneously administered TAA
50 mg/kg/s.c. for every 72 hours (Table 1). At the
end of the experimental period, all the animals
     
samples were collected and allowed to clot. Serum
was separated by centrifuging at 2,500 rpm for
15 minutes and analyzed for various biochemical
parameters [13].
Assessment of liver funcon
Biochemical parameters such as aspartate trans-
aminase (AST), alanine transaminase (ALT), alka-
line phosphatase (ALP), and total bilirubin were
analyzed using commercially available test kits
(Robonik India Pvt. Ltd.). The liver was removed,
weighed, and morphological changes were
        
formalin for histopathological studies.
Table 1. Grouping of animals.
S. no Group Treatment Dose and route
1 I Control (vehicle) Feed and water
ad libitum
2 II Disease control (TAA) 50 mg/kg s.c.
3 III TAA followed by silymarin 50 mg/kg p.o.
4 IV TAA followed by EEAS 200 mg/kg p.o.
5 V TAA followed by EEAS 400 mg/kg p.o.

50 Am J Res Med Sci • 2018 • Vol 3 • Issue 2
Krishna Mohan Chinnala, Praisy Pinky Jayagar, Gayathri Moa, Raghuma Chowdary Adusumilli, Madhan Mohan Elsani
Aspartate transaminase
AST levels in serum were estimated using gluta-
mate oxaloacetate transaminase (GOT)/AST test kit
using the International Federation of Clinical Chem-
istry (IFCC) method without pyridoxal phosphate.
GOT also known as Aspartate aminotransferase is
a transaminase, GOT catalyzes the transfer of the
      
give L-glutamate. GOT is widely distributed in the
body, but the highest levels are found in heart, liver,
skeletal muscles, and kidneys. Damages to cells of
these tissues induce GOT activity increase in serum.
The activity was measured at 340 nm [14–16].
 GOT Oxaloacetate + L-Glutamate
Oxaloacetate + NAD + H+ MDH L-Malate + NAD+
Alanine transaminase
ALT levels in serum were estimated using an ALT
test kit using the IFCC method. Glutamate-pyruvate
transaminase (GPT) also known as ALT is a transami-
nase. GPT catalyzes the transfer of the amino group of

highest levels were found in the liver and the kidneys,
and in smaller amounts in heart and skeletal muscle.
GPT activity is increased when hepatic cells are dam-
aged (liver cells necroses or injury of any cause). The
levels were measured at 340 nm [16–18].
 GPT Pyruvate + L-glutame
Pyruvate + NADH + H+ LDH L-Lactate + NAD+
Alkaline phosphatase
ALP levels in serum were estimated using the ALT
test kit using the IFCC method. ALP is an enzyme of
the hydrolase class of enzymes and acts in an alka-
line medium. It is found in high activity on the liver,
biliary tract epithelium, and in the bones. Normal
levels are age-dependent and increase during bone
development. Increased levels are associated mainly
with liver and bone diseases, hepatotoxicity caused
by drugs are osteomalacia, as well as obstruction.
p-nitro phenyl phosphate is converted to nitro phe-
nol and phosphate by ALP. The rate of formation of
p-nitro phenol is measured as an increase in absor-
bance which is proportional to ALP activity in the
sample and measured at 405 nm [15,16].
p-nitro phenyl phosphate ALP p-nitro phenol + Phosphate
Total bilirubin
Total bilirubin levels were estimated using total

method. Total bilirubin is the sum of unconjugated
bilirubin and conjugated fractions. Bilirubin is ele-
vated in the hemolysis or lysis of increased break-
down of red blood cells, hepatitis, and cirrhosis
azo abstraction of the biliary tract e.g., gallstone.
In the determination of total bilirubin, bilirubin
is coupled with diazotized sulphanilic acid in the
presence of caffeine benzoate solution to produce
azobilirubin which has a maximum absorbance at
546 nm. Direct bilirubin presence of diazotized
sulphanilic acid forms a red colored azo compound
in an acidic medium which has a maximum absor-
bance at 546 nm [15,16,19].
Histopathology of liver
      
carefully dissected out for histopathological studies.
After rinsing in normal saline, sections were taken
        
10% formal saline, dehydrated with 100% etha-

processed into 4–5 m thick sections stained with
hematoxylin-eosin and observed under a photomi-

Stascal analysis
Graph pad prism software, version 6.0 was used in
the statistical analysis of experimental data. The
statistical analysis was carried out using analysis
of variance (ANOVA) followed by Dunnet’s multiple
comparison test. p values p < 0.0001, p < 0.01, and
p
Results
Eect of EEAS on body weights and liver weights
against thioacetamide-induced animals
p < 0.001) decrease in body weight
was observed in TAA-induced Group II animals
(disease control group) when compared with
the control group, silymarin (50 mg/kg) treated
and EEAS (200 and 400 mg/kg) treated animals
   p < 0.01) dose-dependent
maintenance in bodyweight when compared
      
(p < 0.001) increase in the liver weights was
observed in disease control group animals when
compared with the control group animals whereas
animals treated with silymarin and EEAS (200
  p < 0.001)
protection as compared with the disease control
group (Tables 2 and 3).

www.ajrms.com 51
Hepatoprotecve acvity of Allium savum
Eect of EEAS on biochemical parameters against
thioacetamide-induced animals
The liver function test was carried out by measuring
the serum biochemical parameters and was
      
of AST, ALP, ALT, and total bilirubin levels in the
disease control group (p < 0.001). This is clearly
indicating that the incidence of liver damage is
due to TAA administration. Administration of EEAS
at the doses 200 and 400 mg/kg and standard
drug silymarin (50 mg/kg) remarkably prevented
TAA-induced hepatic damage in a dose-dependent
     
group animals (Table 4).
Histopathological studies
Figure 1 discusses the histological examina-
tion of the liver. The control group shows the
maintenance of normal lobular architecture
and normal histology. In the group treated with
TAA, the portal tract showed small aggregates of
      
    
    
were seen. In the group treated with EEAS 200
and 400 mg/kg, the portal tract showed dose-de-
pendent observations such as a small aggregate of
     
    
damage in the group treated with the TAA and pro-
tective effect of the ethanolic extract.
Discussion
Liver is one of the vital organs of the animal body
and plays a central role in transforming and clear-
ing the chemicals, but it is susceptible to the toxic-
ity from these agents. Certain medicinal agents, like
paracetamol, when taken in overdoses or some-
times even within therapeutic ranges, may damage
the liver [13]. Liver diseases which are still a global

    
-
     -
tunately, treatments of choice for liver diseases are
controversial because the conventional or synthetic
drugs for the treatment of these diseases are insuf-
      
Table 2. Eect of EEAS on body weights against TAA-induced animals.
S. no Groups Treatment Dose Before treatment (g) Aer treatment (g)
1 I Normal -215.20 ± 1.23 218.7 ± 2.171
2 II TAA + vehicle 50 mg/kg/s.c. 215.16 ± 1.28 201.7 ± 7.032***
3 III Silymarin 50 mg/kg/p.o. 219.12 ± 2.86 218.3 ± 9.18**
4 IV EEAS 200 mg/kg p.o. 215.26 ± 2.56 211.84 ± 6.24**
5 V EEAS 400 mg/kg p.o. 219.62± 3.12 217.12 ± 5.57**
Values are expressed as mean ± standard error of the mean of six animals.
Stascal signicance test for comparisons was done by one-way ANOVA, followed by “Dunnet’s
mulple comparison test.”
Comparisons were done between (a) Goup I vs Group II and (b) Group II vs Groups III–V. **p < 0.01,
***p < 0.001, ns = non-signicant.
Table 3. Eect of EEAS on liver weights against
TAA-induced animals.
S. no Groups Treatment Dose Liver weights (g)
1 I Normal -6.825 ± 0.066
2 II TAA + vehicle 50 mg/kg /s.c. 9.633 ± 0.121***
3 III Silymarin 50 mg/kg/p.o. 6.90 ± 0.061***
4 IV EEAS 200 mg/kg p.o. 8.99 ± 1.085***
5 V EEAS 400 mg/kg p.o. 7.55 ± 0.086***
Values are expressed as mean ± standard error of the mean of six
animals.
Stascal signicance test for comparisons was done by one-way
ANOVA, followed by “Dunnet’s mulple comparison test.”
Comparisons were done between (a) Goup I vs Group II and (b)
Group II vs Groups III–V. **p < 0.01, ***p < 0.001, ns = non-
signicant.
Figure 1. Histopathological studies of Liver .

52 Am J Res Med Sci • 2018 • Vol 3 • Issue 2
Krishna Mohan Chinnala, Praisy Pinky Jayagar, Gayathri Moa, Raghuma Chowdary Adusumilli, Madhan Mohan Elsani
[20,21]. Since ancient times, mankind has made
use of plants in the treatment of various ailments
because their toxicity factors appear to have lower
side effects [22]. Many of the currently available
drugs were derived either directly or indirectly from
medicinal plants. Recent interest in natural thera-
pies and alternative medicines has made research-
ers pay attention to the traditional herbal medicine.
In the past decade, attention has been centered on
      
plant origin for the treatment of various diseases
[23]. In this regard, the present study was carried
A.
sativum ethanolic bulb extract against TAA-induced
hepatotoxicity in rats. The preliminary phytochem-
ical analysis of EEAS showed that the plant has
    
and the absence of proteins, sterols, phenols, gly-
cosides, saponins, and terpenes which are used for
many disorders including tuberculosis [24]. Injury
to hepatocyte and bile duct cells leads to accumu-
lation of bile acid inside the liver, which promotes
further liver damage [25]. Administration of TAA
at doses of 50 mg/kg/i.p. alternatively for every 72
hours for 21 days results in the hepatic damage in
animals [26]. The mechanism behind its toxicity is
thought to be associated with its toxic metabolite
(s-oxide). It interferes with the movement of RNA
from the nucleus to the cytoplasm which may cause
membrane injury [13,27]. It reduces the number
of viable hepatocytes as well as the rate of oxygen
consumption and also decreases the volume of bile
and its content, i.e., bile salts, cholic acid, and deoxy-
cholic acid.
-
tion of serum biochemical parameters like AST, ALP,
ALT, and total bilirubin which are primary markers
for a liver function test. The elevation of these bio-
markers because of systemic damage of the hepatic
cells by reactive oxygen species released by the

serum biomarkers dose dependently and the above
observations also explain that the EEAS has shown
clear protection at the cellular level by preventing
      
   
the disease control animals, which indicates that
the severity of the hepatic damage and treatment

From the above observation, it is concluded that
the herbal drug possesses hepatoprotective activ-
ity and it has been proven by inhibiting the reactive
oxygen species produced by the administration
of TAA. Further studies to characterize the active
principles and to elucidate the mechanism are in
progress.
References
         -
-
icol Sci 2002; 65:166–76.
[2] Abdel-Misih SR, Bloomston M. Liver anatomy. Surg
Clin North Am 2010; 90(4):643–53.
        -
sis & treatment in gastroenterology. Lang Medical
Books/McGraw-Hill, New York, NY, 2003.
        
1983; 15:3–14.
[5] Greenhough S, Hay DC. Stem cell-based toxicity
screening: recent advances in hepatocyte genera-
tion. Pharm Med 2012; 26(2):85–9.
[6] Singh UP, Prithviraj B, Sarma BK, Madhavi Singh,
Ray AB. Role of garlic (Allium sativum) in human

22.
[7] Thomson M, Al-Amin ZM, Al-Qattan KK, Shaban LH,
Ali M. Anti-diabetic and hypolipidaemic properties
of garlic (Allium sativum) in streptozotocin-induced
       
15.
[8] Ranjani R, Ayya Raju M. Anticancer properties of
Allium sativum    
Res 2012; 3(2):191–6.
[9] Liu C, Yang X, Yao Y, Huang W, Sun W, Ma Y. Deter-
mination of antioxidant activity in garlic (Allium
sativum) extracts subjected to boiling process in

Table 4. Eect of EEAS on ALT levels against TAA-induced animals.
S. no Groups Treatment Dose ALT (U/l) AST (U/l) ALP (U/l) Total bilirubin (mg%)
1 I Normal - 76.050 ± 0.090 147.54 ± 0.32 207.54 ± 1.32 0.28± 0.24
2 II TAA + vehicle 50 mg/kg/s.c. 245.388 ± 0.2642** 284.963 ± 2.65** 484.963 ± 2.65** 4.76 ± 0.45**
3 III Silymarin 50 mg/kg /p.o. 114.798 ± 0.131*** 172.80 ± 0.32** 199.80 ± 1.32*** 0.56 ± 0.36**
4 IV EEAS 200 mg/kg p.o. 194.512 ± 0.19* 245.267 ± 1.89** 315.267 ± 2.19ns 0.72 ± 0.92**
5 V EEAS 400 mg/kg p.o. 122.475 ± 0.147** 186.360 ± 0.53*** 246.360 ± 2.23*** 0.66 ± 0.23**
Values are expressed as mean ± standard error of the mean of six animals.
Stascal signicance test for comparisons was done by one-way ANOVA, followed by “Dunnet’s mulple comparison test.”
Comparisons were done between (a) Goup I vs Group II and (b) Group II vs Group III–V. **p < 0.01, ***p < 0.001, ns = non-signicant.

www.ajrms.com 53
Hepatoprotecve acvity of Allium savum
[10] Nwokocha CR, Ozolua RI, Owu DU, Nwokocha MI,
Ugwu AC. Antihypertensive properties of Allium
sativum (garlic) on normotensive and two kidney

26:213–8.
[11] Saman S, Mohammad Hosein SZ, Saber Y, Amin
H, Nazereh H. In vitro Anti fungal effect of Allium
sativum and Thymus vulgaris on two pathogenic

[12] Pablo FC, Alejandra C, Claudio RG, Philipp WS.
Effect of cooking on garlic (Allium sativum L.) anti-
 

      
Satish K. Protective effect of Pisonia aculeata on
thioacetamide induced hepatotoxicity in rats. Asian

[14] Henderson AR, Moss DW. Creatinine, uric acid, and
urea. In: Saunders WB (ed.). Tietz fundamentals of
clinical chemistry. 5th edition, Elsevier, Philadel-
phia, PA, p 352, 2001.
[15] Tietz NW. Molecular diagnostics. In: Saunders WB
(ed.). Clinical guide to laboratory tests. 3rd edition,
WB. Saunders, Philadelphia, PA, p 76, 1995.
[16] Young DS. Effects of drugs on Clinical Lab. Tests. 4th
edition, AACC Press, Washington, DC, 1995.
       
In: Burtis CA, Ashwood ER (eds.). Tietz textbook of
clinical chemistry. 3rd edition, W.B. Saunders Com-
pany, Philadelphia, PA, pp 477–540, 1999.
[18] Thomas L. Clinical laboratory diagnostics. 1st edi-
tion, TH Books, Frankfurt, Germany, 1998.
[19] Vassault A, Grafmeyer D, Naudin C. et les membres
de la commission Validation de techniques de la
SFBC. Protocole de validation de techniques (Doc-
ument B). Ann Biol Clin 1986; 44:686–745.
   
-
lium of Antrodia cinnamomea protects hepatic cells
from ethanol-induced oxidative stress through

77.
  
activity of the extract of Homalium letestui stem
against paracetamol-induced liver injury. Avicenna

        
      
and antiproliferative activities of date palm pollen
(Phoenix dactylifera) on experimentally-induced

8(40):1–13.
[23] Majid S, Foo S, Luyt B, Xue Z, Yin-Leng T, Chang Y,
et al. Adopting evidence-based practice in clinical
decision making: nurses’ perceptions, knowledge,

[24] Nair SS, Gaikwad SS, Kulkarni SP, Mukne AP. Allium
sativum constituents exhibit anti-tubercular activ-
ity in vitro and in RAW 264.7 mouse macrophage
cells infected with Mycobacterium tuberculosis
H37Rv. Pharmacogn Mag 2017; 13(50):209–15.
[25] Shapiro H, Ashkenazi M, Weizman N, Shahmurov M,
Aeed H, Bruck R. Curcumin ameliorates acute thio-
   
Hepatol 2006; 21:358–66.
 
S. Hepatoprotective effect of three herbal extracts
      

[27] Isabel F. Dysregulation of apoptosis in hepatocel-
  
15:513–20.