Hepatoprotective activity of aqueous methanolic extract of Viola odorata against paracetamol-induced liver injury in mice

Abstract

Traditionally Viola odorata is used for liver protection. To provide scientific
support to its traditional use, aqueous methanolic extract of V. odorata (250
mg/kg and 500 mg/kg) was given to mice intoxicated with paracetamol.
Obtained results demonstrated that the extract significantly (p<0.01-0.001)
reduced paracetamol induced increase levels of serum hepatic enzymes and
total bilirubin. Histopathological studies showed that the plant attenuated the
hepatocellular necrosis and inflammation. HPLC results showed the presence
of hepatoprotective flavonoids (isorhamnetin and luteolin) in the extract. It
was concluded from the present study that V. odorata has hepatoprotective
activity against paracetamol-induced liver injury in mice.

Full text

Introduction
Now-a-days, liver diseases are one of the major issues
of all the medical community due to the higher rate of
mortality and morbidity. There are lot of medicines and
natural plants available in commercial market for liver
diseases. These products do not cure all the liver
symptoms. Therefore, there is a need to find out the
perfect remedy. Many plants have been identified as
hepatoprotective like Trianthema decandra (Balamurugan
and Muthusamy, 2008), Cocculus hirsutus (Thakare et
al., 2009), Carica papaya (Sadeque and Begum, 2010),
Carissa spinarum (Hegde and Joshi, 2010), Convolvulus
arvensis (Ali et al., 2013), Dodonaea viscosa (Khan et al.,
2013), Trichodesma sedgwickianum (Saboo et al., 2013),
OfIpomoea staphylina (Bag and Mumtaz, 2013), Khamira
Gaozaban Ambri Jadwar Ood Saleeb Wala (Akhtar et
al., 2013) and Morus nigra (Mallhi et al., 2014).
Viola odorata L. (family Violaceae), commonly known as
sweet violet or banafsheh, is cultivated in all over the
world as an ornamental plant. Pharmacologically, it has
been reported that V. odorata has anti-inflammatory
(Koochek et al., 2003), antipyretic (Khattak et al., 1985)
and antibacterial activity (Arora and Kaur, 2007).
Flavonoids have been identified in V. odorata (Siddiqi et
al., 2012) and they may have hepatoprotective activity
(Ali et al., 2013). Traditionally V. odorata is worthwhile
to cure Jaundice (Amiri et al., 2014). Phytochemical pro-
file and traditional use of V. odorata in liver injury
appealed us to scientifically evaluate its hepatoprotec-
tive potential.
Materials and Methods
Plant material: Aerial parts of V. odorata were collected
from Faisalabad (Pakistan) and identified from Depart-
ment of Botany, University of Agriculture, Faisalabad,
Pakistan. The plants were kept in herbarium for future
reference. The plants were shade-dried and powdered
by a mechanical grinder. The powdered material was
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ISSN: 1991-0088; DOI: 10.3329/bjp.v9i2.18049
Abstract
Traditionally Viola odorata is used for liver protection. To provide scientific
support to its traditional use, aqueous methanolic extract of V. odorata (250
mg/kg and 500 mg/kg) was given to mice intoxicated with paracetamol.
Obtained results demonstrated that the extract significantly (p<0.01-0.001)
reduced paracetamol induced increase levels of serum hepatic enzymes and
total bilirubin. Histopathological studies showed that the plant attenuated the
hepatocellular necrosis and inflammation. HPLC results showed the presence
of hepatoprotective flavonoids (isorhamnetin and luteolin) in the extract. It
was concluded from the present study that V. odorata has hepatoprotective
activity against paracetamol-induced liver injury in mice.
Article Info
Received: 19 February 2014
Accepted: 28 February 2014
Available Online: 25 April 2014
Keywords:
Hepatoprotective
Silymarin
Viola odorata
Number of Figures: 2
Number of Table: 1
Number of Refs: 36
Correspondence: MIQ
e-mail: mrimranqadir@hotmail.com
This work is licensed under a Creat ive Commons Attribution 3.0 License. You are free to copy, distribute and perform t he work. You must attribute the work in the
manner specified by the author or licensor.
Hepatoprotective activity of aqueous methanolic extract of
Hepatoprotective activity of aqueous methanolic extract of Hepatoprotective activity of aqueous methanolic extract of
Hepatoprotective activity of aqueous methanolic extract of
Viola
Viola Viola
Viola
odorata
odorata odorata
odorata
against paracetamol
against paracetamolagainst paracetamol
against paracetamol-
--
-induced liver injury in mice
induced liver injury in miceinduced liver injury in mice
induced liver injury in mice
M. Imran Qadir1, Muhammad Ali1, Muhammad Ali2, Mohammad Saleem1
and Muhammad Hanif3
1College of Pharmacy, GC University, Faisalabad, Pakistan; 2Institute of Molecular Biology & Biotechnology,
Bahauddin Zakariya University, Multan, Pakistan; 3Faculty of Pharmacy, Bahauddin Zakariya University, Multan,
Pakistan.

extracted by the method of cold maceration using
aqueous methanol (70:30) as solvents. After soaking the
powdered extract for 48 hours, it was passed though
muslin cloth and then through filter paper for filtration.
Rotary evaporator was used for concentrating the
extracts.
Experimental animal: Swiss Albino mice weigh about 20-
35 g were used. Mice obtained from the NIH Islamabad.
The animals were kept in animal house of College of
Pharmacy, housed in standard condition and feed with
standard pellet diet and water ad libitum (Vadivu et al.,
2008).
Experimental protocol: Animals were divided into five
groups containing six animals each. Group I was
control group received distilled water only. Group II
received paracetamol at the dose of 250 mg/kg orally
dissolved in water daily for 7 days. Group III was
treated with silymarin as standard drug at the dose of
50 mg/kg p.o daily for 7 days and received paracetamol
3 hours after silymarin. Group IV was treated with 250
mg/kg p.o of aqueous methanolic extract of V. odorata
for 7 days and received paracetamol 250 mg/kg p.o for
7 days 3 hours after the extract dose. Group V was
treated with 500 mg/kg p.o aqueous methanolic extract
of V. odorata for 7 days and received paracetamol 250
mg/kg p.o for 7 days 3 hours after the extract dose
(Sabir and Rocha, 2008).
After completing the last dose mice sacrificed by
cervical dislocation. By decapitate sedated mice we can
draw the maximum Blood from mice and the blood was
collected in eppendroff tubes and the clot was
dispersed with glass rod and then centrifuged at 3000
rpm for 15 minutes. Then separated out the serum that
was used to estimate SGPT, SGOT and serum alkaline
phospatase to present the parameter of LFT and serum
total bilirubin used as excretory function test. The liver
from animals was separated for histopathological
studies (Ali et al., 2013).
Identification of active constituent by HPLC: Hepatoprotec-
tive activity may be due to flavonoids (Ali et al., 2013).
HPLC may be used to separate and identify the flavo-
noids. HPLC protocol was used as described elsewhere
(Saddiqe et al., 2011). By using the said protocol, reten-
tion time for quercetin is 2.05, myricetin 1.75, isorham-
netin 2.72, kaepferol 2.6, luteolin 1.98.
Statistical analysis: One way ANOVA (analysis of
variance) was used for statistical analysis. Results were
represented by mean ± SE.
Results
Serum biochemical parameters are shown in Table I.
Paracetamol administration caused damage to hepato-
cytes demonstrated by raised level of liver enzymes
(ALT, AST, and ALP) and total bilirubin as compared to
control. Elevated levels of these enzymes are indicative
of cellular damage and loss of functional integrity of
hepatocytes. V. odorata pretreatment significantly restor
-ed elevated levels of ALT with 250 and 500 mg/kg
(p<0.01), AST with 250 & 500 mg/kg (p<0.01), ALP
with 250 mg/kg (p<0.01) and with 500 mg/kg (p<0.001)
and total bilirubin with 250 mg/kg (p<0.01) and with
500 mg/kg (p<0.001). Both doses (250 mg/kg and 500
mg/kg) provided significant approximately similar
reduction in biochemical parameters except ALP & total
bilirubin. Results for histopathological examination are
given in Figure 1. The normal (control) group receiving
distilled water only has normal nuclei. While the
administration of paracetamol causes necrosis, infla-
mmation and ballooning of cells. The group treated
with silymarin has mild inflammation and necrosis and
ballooning was also absent. The groups treated with
both doses (D, E) of V. odorata have few inflammatory
cells but most of the cells gained normal structures as
were present in control group (A).
Phytochemical analysis for qualitative determination of
active constituents through HPLC showed presence of
two flavonoids: isorhamnetin and luteolin (Figure 2).
Discussion
The liver is the major organ of the body and
metabolizes many nutrients carbohydrates, proteins,
lipids; and also metabolize the drugs, and excrete them
from the body (Saleem et al., 2010). Many drugs and
chemicals cause the damage to the liver. The existing
hepatoprotective drugs are not full of the efficacy.
Efficacy of a drug may be increased by increasing the
drug delivery to the target site by the use of polymers
(Khalid et al., 2009; Hussain et al., 2011) or through
Table I: Effect of aqueous methanolic extract of Viola odorata on liver enzymes and total bilirubin
Treatment groups ALT (U/L) AST (U/L) ALP (U/L) Total bilirubin
(g/dL)
Normal (control) 44.0 ± 2.7 62.8 ± 3.0 218.0 ± 8.3 0.7 ± 0.0
Paracetamol (250 mg/kg) 134.5 ± 12.1 90.5 ± 5.2 325.0 ± 12.6 1.6 ± 0.1
Silymarin 50 mg/kg + paracetamol (250 mg/kg) 47.3 ± 4.0b 44.8 ± 3.6b 172.3 ± 18.1b 0.7 ± 0.0b
Extract (250 mg/kg) + paracetamol (250 mg/kg) 81.0 ± 6.3a 65.8 ± 3.6a 287.8 ± 11.1a 0.8 ± 0.0a
Extract (500 mg/kg) + paracetamol (250 mg/kg) 57.8 ± 3.6a 58.8 ± 4.2a 205.8 ± 8.9b 0.8 ± 0.0b
Data are mean ± SE; ap<0.01, bp<0.001
Bangladesh J Pharmacol 2014; 9: 198-202 199

nanotechnology (Naz et al., 2012; Ehsan et al., 2012),
synthesis of new drugs, either by the use of proteomics
(Qadir, 2011), or synthesis from lactic acid bacteria
(Masood et al., 2011), or marine microorganisms (Javed
et al., 2011). However, now a days, the trend is also
being changed to the use of herbal products for their
use as their anti-inflammatory (Qadir, 2009), hypoten-
sive (Qadir, 2010), hypoglycaemic (Qadir and Malik,
2009; Qadir and Malik, 2010), amoebicidal (Asif and
Qadir, 2011), anti-fertility, cytotoxic, antibiotic (Amin et
al., 2012), spasmolytic, bronchodilator (Janbaz et al.,
2013), antioxidant (Janbaz et al., 2012) and hepatoprotec-
tive (Ahmad et al., 2012).
V. odorata was considered to be a hepatoprotective
traditionally (Amiri et al., 2014), which was proven by
this study. Results showed that paracetamol damages
the liver but after taking standard hepatoprotective
drug (silymarin), liver cells cured. Mice which treated
with V. odorata were also looking healthy but the results
were less significant than the silymarin. Significant
reduction (p<0.01) of biochemical markers was obser-
ved with aqueous methanolic extract of V. odorata in
paracetamol-treated mice. Two doses (250 mg/kg and
500 mg/kg) were screened to evaluated hepatoprotec-
tive potential of this plant. There were no significant
difference (p>0.05) between two doses in terms of liver
enzymes e.g. ALT, AST and ALP. This is indicative that
V. odorata doesn’t cause dose dependent reduction of
liver enzymes. On the other hand the two doses have
significant difference (p<0.01) when compared each
other for reduction of total bilirubin. Direct proportion
relationship of dose-response can be confirmed by more
focused studies.
The biochemical results were further supported by
Figure 1: Histopathological pictures of (A) normal hepatocytes, (B)
paracetamol-treated, (C) silymarin-treated, (D) 250 mg/kg extract
treated, (E) 500 mg/kg extract treated
Figure 2: HPLC chromatogram of aqueous methanolic extract of Viola odorata
200 Bangladesh J Pharmacol 2014; 9: 198-202

histopathological findings. Paracetamol administration
lead to damages normal hepatocyte`s structure that
were restored with silymarin. Similar effects were obser
-ved with plant extract showing improvement in infla-
mmation-necrosis score and reversal of normal hepato-
cytes.
Results were further supported by HPLC, in which two
flavonoids were found isorhamnetin and luteolin.
These flavonoids belong to flavonols class which is well
reported to have hepatoprotective activity (Singab et al.,
2005; Chattopadhyay et al., 2003).
It was concluded from the present study that V. odorata
has hepatoprotective activity against paracetamol-
induced liver injury in mice.
Acknowledgement
Authors are thankful to Mr. Asif Masud and Mr. Bilal Ahmed
for their contribution to accomplish this study.
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