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ANTI HEPATOTOXICITY STUDIES OF CRUDE EXTRACT OF
FERONIA LIMONIA IN CCL4 INDUCED TOXICITY
Jayashree V Hanchinalmath and Ramesh Londonkar*
Dept of Biotechnology, Gulbarga University, Gulbarga - 585106, Karnataka, India.
ABSTRACT
The antioxidant activities of Feronia limonia fruit pulp methanolic
extract (MEFL) was investigated by and in vivo assay. The effect of
the extract in reducing carbon tetrachloride induced (CCl4) oxidative
stress in rats was evaluated. Hepatoprotective activity of MEFL was
studied by estimating serum enzyme activities of aspartate
aminotransferase (AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP), Serum Lactate Dehydrogenase (LDH), total
protein (TP) and total bilirubin (TB). The treatment with MEFL
showed a dose-dependent reduction in CCl4 induced elevated serum
levels of enzyme activities with parallel increase in total protein and bilirubin content
indicating the extract could preserve the normal functional status of the liver. MEFL at the
doses of 200 and 400 mg/kg body weight significantly increased the glutathione (GSH), super
oxide dismutase (SOD) and catalase (CAT) activities in a dose dependant manner. Therefore,
the results of this study illustrate that the Feronia limonia methanol extract can shield the
liver against CCl4-induced oxidative damage in rats.
Keywords: Feronia limonia, antioxidant activity, phenolic content, CCl4.
INTRODUCTION
Feronia limonia commonly known as wood apple is an important plant in Ayurveda, the
traditional system of Indian medicine. This fruit is recommended for the treatment of tumors,
asthma, wounds, diarrhea, dysentery, cardiac dysfunction, hepatitis, sore throat and is
considered as tonic, astringent (when unripe), antiscorbutic, and alexiformic agent [1,2] .
Feronia limonia fruit has also been reported to possess hypoglycemic and hypolipidaemic
and wound healing properties[3,4].
WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS SSJJIIFF IImmppaacctt FFaaccttoorr 22..778866
VVoolluummee 33,, IIssssuuee 77,, 11553366--11554466.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357
Article Received on 8 May 2014, Revised on 04 June 2014, Accepted on 24 June 2014
*Correspondence for Author
Prof. Ramesh Londonkar
Dept of Biotechnology,
Gulbarga University, Gulbarga
- 585106, Karnataka, India.
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Londonkar et al. World Journal of Pharmacy and Pharmaceutical Sciences
Antioxidant activity is a fundamental property important for life. Many of the biological
functions, such as antimutagenicity, anticarcinogenicity, and antiaging, among others,
originate from this property[5] Antioxidants are compounds that can delay or inhibit the
oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidative
chain reactions [6]. It has been suggested that natural antioxidants like L-ascorbic acid are
more safe and healthy than synthetic antioxidants such as butylated hydroxytoluene (BHT)
[7]. Hence, there is need for safe, cost effective and less toxic antioxidants with high activity
from natural sources to replace the synthetic chemicals. The importance of the antioxidant
constituents of plant materials in maintaining health and protection from coronary heart
disease. Cancer is another deadly disease raising interest among scientists and food
manufacturers towards the search of functional food with specific health effects [8]. Potential
sources of antioxidant compounds have been searched in several types of plant materials such
as fruits, leaves, seeds, barks, roots and crude plant drugs [9]. Crude extracts of plant
materials rich in phenolics are increasingly of interest in the food industry because they retard
oxidative degradation of lipids and thereby improve the quality and nutritional value of food.
The antioxidative effect is mainly due to flavonoids and phenolic acids present in the plants.
Many of the natural antioxidants, especially phenolics, exhibit a wide range of biological
effects including antibacterial, antiviral, anti-inflammatory, antiallergic, antithrombotic, and
vasodilatory actions [10].
Liver diseases, especially viral hepatitis occurs principally with an enormous impact on
public health. Carbon tetrachloride (CCl4) is widely used in animal models to induce acute
liver injury [11]. It is commonly believed that the toxicity of CCl4 results from its reductive
dehalogenation by the cytochrome P450 enzyme system into the highly reactive free radical
trichloromethyl radical [12-14]. Antioxidant action has been reported to play a crucial role in
the hepatoprotection [15]. In the present study, an attempt has been made to explore the effect
of MEFL in CCl4 induced hepatic damage pertaining to biochemical marker enzymes &
histopathology. The result of this In Vivo study will support the plant as a good herbal
antioxidant agent.
MATERIALS AND METHODS
Chemicals
CCl4 were purchased from Sigma-Aldrich (Mumbai). Liv 52 from Himalaya drugs Ltd. All
other chemicals and reagents were of analytical grade, and they were used as received.
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Londonkar et al. World Journal of Pharmacy and Pharmaceutical Sciences
Plant material and extraction
Fruits of Feronia limonia L were collected from local market Bellary, Karnataka, India
during the month of March, 2012. It was authenticated by the Department of P.G studies and
Research in Botany, Gulbarga University, Gulbarga. Crude fruit pulp extract was prepared by
Soxhlet extraction method. About 50gm of powdered plant material was packed in a thimble
and extracted successively with 350ml of petroleum ether, chloroform, and methanol. The
process of extraction is carried out until the solvent in siphon tube of an extractor become
colorless. The extract was taken in a petriplates and kept in hot air oven and heated at 30-
40ºC till the solvent got evaporated. Solvent free methanolic extract dissolved in 1%
Dimethyl sulphoxide (DMSO) was used for the In Vivo studies.
Animals
Wistar strain Albino rats of inbred colony weighing about 150 – 175 g were used. The
protocol was approved by the Institute’s Animal Ethical Committee (IAEC Reg No. 34800/
CPCSEA Dated: 19.08.2001). Animals were kept in animal house at an ambient temperature
of 250C and 45 – 55% relative humidity, with 12 h each of dark and light cycles. They were
fed with a balanced diet as described by Central Food and Technological Research Institute
(CFTRI, Mysore) and water ad libitum [16].
Acute toxicity experiment
Albino rats were divided into control and test groups (6 animals each). Control group
received the vehicle (3% Tween 80) while the test groups got graded doses (100–4000
mg/kg) of MEFL orally and were observed for mortality till 48 h and the LD50 was
calculated.
Induction of experimental hepatotoxicity
30% CCl4 was prepared in olive oil. Animals of group 2, 3, 4 and 5 were given single dose of
CCl4 at 1ml/kg body weight (bw) intraperitoneally (i.p). Methanolic extract of Feronia
limonia at the dose level of 200 mg/kg bw and 400 mg/kg bw as low dose and high dose were
administered to animals of group 4 and 5 orally with the aid of an intragastric catheter for 7
days. Liv 52 (50 mg/kg bw) was used orally as a standard drug to group 3 at a single dose.
Rats were divided into five groups as following protocol.
GROUP 1: Normal control (n=6, the animals were given saline at 1ml/kg body weight)
GROUP 2: Hepatotoxic control (n=6, the animals were given CCl4 at 1ml/kg body weight)
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GROUP 3: Positive control (n=6, the animals were given CCl4 + Liv 52 for 7 days)
GROUP 4: Treatment group (n=6, the animals were given CCl4 + MEFL low dose for 7 days)
GROUP 5: Treatment group (n=6, the animals were given CCl4 + MEFL high dose for 7
days)
At the end of the experimental period, blood sample from each rat (2 ml) was withdrawn by
cardiac puncture and collected in previously labeled centrifuge tubes and allowed to clot for
30 min at room temperature. Serum was separated by centrifugation at 10,000 rpm for 5 min.
Assessment of hepatotoxicity
Liver functions were evaluated by measuring the serum activity of ALT and AST following
the method of [17] while the activities of ALP and LDH were estimated. The serum
concentrations of TB estimated according to [20] and TP as described by [21].
Assessment of oxidative stress
Liver tissue was homogenized in 10 volume of 100 mM KH2PO4 buffer containing 1 mM
EDTA (pH 7.4) and centrifuged at 12,000 rpm for 30 min at 40C. The activities of the
antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were assayed
in the hepatic tissue homogenate of the control and experimental rats according to the
methods of [22,23]. GSH tissue content was also measured [24].
Histopathological study
Liver was dissected out and divided into two parts. One part was kept in liquid nitrogen for
determination of antioxidant status and the other part was immediately fixed in buffered
formalin 10% and was used for histopathological examination using the standard micro
technique [25].
Statistical analysis of data
Data were presented as means ± SD of three experiments. Analysis of variance was
performed on the data obtained. Significance of differences between means was determined
by least significant differences (LSD) at P ≤ 0.05.
RESULTS
Acute toxicity study
The dose selection for MEFL was based on the acute toxicity study. The study did not show
any adverse effect of doses up to 4000 mg/kg. Accordingly, experimental oral doses of 200
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Londonkar et al. World Journal of Pharmacy and Pharmaceutical Sciences
and 400 mg/ kg equal to one-twentieth and one-tenth of the feasible dose of the extract that
did not cause mortality in rats were selected.
Assessment of hepatotoxicity
The effects of methanol extract of Feronia limonia at dose levels of (200 and 400 mg/kg bw)
on serum marker enzymes are shown in Table 2. Hepatic injury induced by CCl4 has caused
significant rise in marker enzymes such as ALT, AST, ALP activities and decrease in serum
TP, TB levels (P < 0.01). Administration of methanol extract of Feronia limonia at two
different dose levels attenuated the increased levels of the serum enzymes, produced by CCl4,
and caused a subsequent recovery towards normalization almost like that of Liv 52 treatment.
Table 1 Effect of methanol extract of Feronia limonia on serum marker enzymes, total
protein and total bilirubin
Group ALT(IU/L) AST (IU/L) ALP (IU/L) TP (µmol/L) TB (µmol/L) NC 64.18 ± 0.23 81.56 ± 5.54 107.69 ± 1.33 8.14 ± 0.37 0.64 ± 0.04
HC 170.22 ± 0.14 b
134.91 ± 7.33 b
152.56 ± 1.07 b 4.11 ± 0.3 b 1.81 ± 0.09 b
CCl4+ Liv 52 (50mg/kg bw) 91.31 ± 0.42 d 96.07 ± 4.38
d 120.01 ± 1.25
d 7.00 ± 0.24 c 0.72 ± 0.05 c
CCl4+ MEFL (200mg/kg bw)
142.10 ± 0.25 d
126.72 ± 7.16 d
144.30 ± 1.66 d 5.01 ± 0.35 d 1.43 ± 0.06 d
CCl4+ MEFL (400mg/kg bw
117.35 ± 0.31 d
115.80 ± 7.43 d
132.78 ± 2.01 d 6.25 ± 0.65 d 1.09 ± 0.05 d
Results are expressed as mean ± S.E.M; b P < 0.01, compared with NC group; c P < 0.05, d P
< 0.01, compared with CCl4-treated group.
Note: NC (normal control), HC (hepatotoxic control).
Assessment of oxidative stress
CCl4 treatment also resulted in the depletion (P<0.01) of the hepatic antioxidant enzymes.
The activities of GSH, SOD, CAT were depleted to 1.01 ± 0.12, 17 ± 0.17 and 23.16 ± 1.27
respectively of the hepatotoxic control (Table 3). The decline in the activities were noticeably
attenuated (P<0.01) by administration of 200 mg/kg bw and 400 mg/kg bw in MEFL treated
rats. Treatment with MEFL enhanced liver antioxidant enzymes yet after CCl4 treatment.
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Table 2 Effect of methanol extract of Feronia limonia on GSH levels, CAT and SOD
activities
Group GSH (mg/g pro)
CAT (U/g prot) SOD (U/g prot)
NC 1.47 ± 0.43 45.02 ± 1.33 26.54 ± 0.33 HC 1.01 ± 0.12 b 23.16 ± 1.27 b 17.00 ± 0.17 b CCl4+ Liv 52 (50mg/kg bw) 1.32 ± 0.27 d 39.58 ± 1.07 d 22.03 ± 0.25 c
CCl4+ MEFL (200mg/kg bw)
1.16 ± 0.22 d 27.20 ± 1.74 c 18.21 ± 0.66 d
CCl4+ MEFL (400mg/kg bw
1.24 ± 0.26 d 34.10 ± 1.45 d 20.95 ± 0.21 d
Results are expressed as mean ± S.E.M; b P < 0.01, compared with NC group; c P < 0.05, d P
< 0.01, compared with CCl4-treated group.
Note: NC (normal control), HC (hepatotoxic control)
Histopathology
The histopathological examination displayed significant recovery of hepatocytes in the
standard drug and MEFL treated animals, which is again correlated with the biochemical
parameters. The results of the liver histopathological studies (Figs 5a-5e) showed hepatocytes
swelling and necrosis in CCl4-treated rats (Fig 5b) in comparison with normal control rats
(Fig 5a). Treatment with MEFL exhibited a significant protection against hepatocytes injury
and showed complete normalization of the tissues where no fatty accumulation or necrosis
was seen (Fig 5d, 5e). The central vein appeared clearly indicating a potent anti-hepatotoxic
activity. MEFL was found to exhibit a potent anti-hepatotoxicity compared with standard
drug Liv 52 (Fig 5c). The liver section showed the structure of the portal triad and a normal
liver parenchyma, and the central vein appeared clearly. There was no lymphocytic
infiltration and fatty deposition representing a potent anti-hepatotoxicity for the MEFL under
study.
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Londonkar et al. World Journal of Pharmacy and Pharmaceutical Sciences
Fig 5a
Fig 5b Fig 5c
Fig 5d Fig 5e
Fig 5: Light microphotographs (20x) of liver cells of rats a. Normal control b. CCl4
induced c. CCl4 + Liv 52 d. CCl4 + MEFL(200 mg/kg bw) e. CCl4 + MEFL (400 mg/kg
bw)
DISCUSSION
Free radicals are known to play a definite role in a wide variety of pathological
manifestations. Antioxidants due to their scavenging activity are useful for the management
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Londonkar et al. World Journal of Pharmacy and Pharmaceutical Sciences
of these diseases. They exert their action either by scavenging the reactive oxygen species or
protecting the antioxidant defense mechanisms [26]. The present study suggests that Feronia
limonia can be used as a source of antioxidants for pharmacological preparations. Non-
phenolic compounds of the plants such as trace elements may decrease the antioxidant
activity of the phenolic compounds [27].
Liver injury caused by CCl4 in rats was first reported [28] and is extensively and effectively
used by many investigators. Carbon tetrachloride is metabolized by cytochrome P-450 in
endoplasmic reticulum and mitochondria with the formation of CCl3O-, a reactive oxidative
free radical, which initiates lipid peroxidation. In the presence of oxidative stress more of
lipid peroxidation products are formed due to cell damage. In this study, carbon tetrachloride
damage to erythrocytes was confirmed by the increase in SOD, GSH and CAT activities, and
decrease in membrane fluidity. SOD is one of the crucial components in the antioxidant
defense system for the reduction of reactive oxygen species (ROS) and peroxides produced in
the living organism and in detoxification of compounds of exogenous origin, thus playing a
primary role in the maintenance of a balanced redox status[29]. The increase of SOD activity
suggests that the MEFL has an efficient protective mechanism in response to ROS. Catalase
is a very important component of the antioxidant defense system. The increased SOD activity
resulted in the accumulation of hydrogen peroxide, which stimulated increases in CAT
activity. The MEFL increased the activities of Catalase in CCl4 induced liver damage rats to
prevent the accumulation of excessive free radicals and thus protects the liver from
intoxication. GSH is a naturally occurring substance abundant in many living creatures; GSH
depletion increases the sensitivity of cells to various aggressions leading to tissue disorder
and injury[30]. In the present study we demonstrated the efficiency of the extract by using
CCl4 induced rats and found that exogenous MEFL supplementation elevated GSH levels in
rats with CCl4 treatment and thus might provide a mean of recovering reduced GSH levels to
prevent tissue injury. Treatment of experimental animals with the MEFL exhibited an
improved free radical scavenging ensuring decrease in activities of enzymes towards normal.
The field of dietary modification and chemoprevention show considerable effective approach
against oxidative stress and are the focus of research these days. In the assessment of liver
damage by CCl4, the determination of enzyme levels such as AST, ALP and ALT is largely
used. Bilirubin concentration has been used to evaluate chemically induced hepatic injury.
The data showed that the control group demonstrated a normal range of AST, ALP, ALT,
protein and bilirubin levels, while the CCl4-treated group showed elevated levels confirming
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Londonkar et al. World Journal of Pharmacy and Pharmaceutical Sciences
that CCl4 caused liver injury, altered membrane integrity and as a result enzymes in
hepatocytes leak out [31]. However, after treatment with MEFL, the increase in ALT, AST
and ALP were significantly restored. Increase in the level of TP by MEFL indicates
hepatoprotective activity, as stimulation of protein synthesis accelerates the regeneration
process and production of liver cells. These results indicate that the extract has the ability to
protect against CCl4-induced hepatocyte injury, which is in agreement with the previous
study of [32] that reported the protective consequence of polyphenolic compounds against
CCl4-induced liver cirrhosis. Therefore, it is valid to consider that MEFL, possess antioxidant
property which is capable of protecting the hepatic tissue from CCl4-induced injury and
inflammatory changes might be due to the presence of phenolics.
CONCLUSION
Our investigations reveals that MEFL hold potent antioxidant activity. The current study
demonstrates that MEFL exerts effective protection against acute chemical induced hepatic
damage suppressing liver enzyme activities and thus preventing cell membrane damage.
Histopathological study revealed morphological evidence and bioassays revealed functional
evidence. MEFL can thus be proposed to protect the liver against CCl4‐induced oxidative
damage in rats. The in vivo assays indicate that this plant extract is a significant source of
natural antioxidant, which might help prevent the progress of oxidative stress. However, the
components responsible for the antioxidant activity are currently unclear. Efforts are in
progress in our laboratory to isolate and purify the active principle of this medicinal plant.
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