Chinese Medicine, 2010, 1, 18-22
doi:10.4236/cm.2010.11003 Published Online June 2010 (
Copyright © 2010 SciRes. CM
Antihepatotoxic Effect of Feronia Limonia Fruit against
Carbon Tetrachloride Induced Hepatic Damage in
Albino Rats
Rucha Upadhyay1, Narendra Deo Pandey1, Shahid Suhail Narvi1, Amita Verma2, Bahar Ahmed2
1Department of Chemistry Motilal Nehru National Institute of Technology, Allahabad, India
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delh, India
Received March 10, 2010; revised April 28, 2010; accepted May 10, 2010
Feronia limonia Swingle, though mentioned as a high utility medicinal plant in Ayurveda for the treatment
of liver disorders, but has no scientific investigations. Therefore, the present study was undertaken to inves-
tigate the hepatoprotective activity of extracts of Feronia limonia fruit on albino rat liver, damage induced by
single dose of carbon-tetrachloride (1.5 ml/kg, p.o.). The oral administration of extracts (500 mg/kg, p.o.) for
7 days has shown significant hepatoprotective action. There is reduction in the elevated levels of serum en-
zymes such SGOT, SGPT, ALP and Serum bilirubin and elevation in level of GSH, GPX,GLY and total pro-
tein in Feronia limonia protected rats.
Keywords: Anti-Hepatotoxic, Carbon Tetrachloride, Feronia Limonia Swingle
1. Introduction
Feronia limonia Swingle (Syn. Feronia elephantum
Correa, Fam Rutaceae) are described in ayurvedic litera-
ture as Kapittha is a moderate-sized tree with straight
sharp strong spines, 1.2-3.7 cm long, which is widely
distributed throughout Bangladesh, Srilanka, Java and
India [1-3]. The fruits of the plant are edible and consid-
ered to be a stomachic, astringent, diuretic, cardiotonic
and tonic to the liver and lungs [2]; the bark is occasion-
ally used for biliousness [4]; the leaves are aromatic and
carminative and are used for the treatment of indigestion
and minor bowel infections of children [2]; the roots are
sometimes given for the treatment of snake-bites [2].
Previous phytochemical work was mainly focused on the
isolation of coumarins [5-7], steroids and volatile com-
ponents [8, 9] of the plant.
Feronia limonia has great reputation in Ayurvedic
medicine for treatment of liver disorders. No scientific
and methodical investigations have so far been reported
in literature regarding its action on liver. Hence the pre-
sent study is focused to evaluate the hepatoprotective
potentials of the Feronia limonia fruits against CCl4 in-
duced liver injury in rats.
2. Experimental
2.1. Plant Collection
Fruits of Feronia limonia were collected during Sep-
tember-October 2006 from wild area in the vicinity of
Allahabad. The fruit of plant was authenticated by Dr.
M.P. Sharma, Reader and taxonomist, Department of
Botany, Hamdard University, New Delhi. A voucher
specimen (No. PD-FP-32) of plant was kept in herbarium
of Hamdard University, New Delhi.
2.2. Preparation of Extract
Coarsely powdered dry fruits of Feronia limonia (500 g)
were extracted to exhaustively with petroleum ether
(60-80˚C), chloroform and methanol using a sohxlet ap-
paratus. The extracts thus obtained were dried under re-
duce pressure yielding 6.9%, 17.4%, 31.8% powder with
reference to dry starting material respectively.
2.3. Experimental Animals
Male albino rats of wistar strain (150-200 g) were em-
ployed for assessing the antihepatotoxic activity. They
were procured from the Central Animal House of Jamia
Hamdard, New Delhi Sanction Letter No. (173/CPCSEA),
after approval under the project proposal number-326.
They were maintained under controlled condition of light
(12/24 h) and temperature (23 ± 1˚C). Food pellets (Hind-
ustan lever Ltd. Mumbai, India) and tap water were pro-
vided ad libitum. For experimental purposes animals
were kept fasting but were allowed free access to water.
2.4. CCl4-Induced Hepatotoxicity
The animals were divided into six groups of six animals
each. Group I served as normal control which received
normal saline only. Group II served as CCl4 control and
received CCl4:liquid paraffin (1:1, 1.5 ml/kg, b.w., p.o.)
on first day. Group III served as standard control and re-
ceived single dose of CCl4:liquid paraffin (1:1, 1.5 ml/kg,
b.w., p.o.) on first day and thereafter received treatment
with standard drug silymarin (Silybon-70) (10 mg/kg,
b.w., p.o.) for 7 days. Groups IV-VI received single dose
of CCl4:liquid paraffin (1:1, 1.5 ml/kg, b.w., p.o.) on first
day and thereafter treated with petroleum, chloroform
and methanolic extract of fruits of Feronia limonia
(500 mg/kg, b.w., p.o.) [10], respectively, for 7 days.
2.5. Assessment of Liver Functions
Rats of all groups were anaesthetized with 1.2 g/kg b.w,
of a 25% w/v aqueous solution of urethane (Loba-Chemie,
Bombay), given on 8th day. The blood collected by
puncturing the orbital plexus was allowed to coagulate at
ambient temperature for 30 min. and the rats were sacri-
ficed by decapitation. Serum was separated by centrifu-
gation at 3500 rpm for 10 min. The livers of all animals
were removed and processed for histological investiga-
In serum, alanine aminotransferase (ALT), aspartate
aminotransferase (AST) [11], alkaline phosphatase (ALP)
[12], total serum protein (TSP) [13] and bilirubin [14]
were measured. Glutathione (GSH) [15], glycogen (GLY)
[16] and glutathione peroxidase (GPX) [17] were esti-
mated in liver homogenate.
2.6. Histological Investigations
Liver slices fixed for 48 h in 10% formosaline were
processed for paraffin embedding following the standard
microtechnique [18]. Sections (5/~m) of livers stained
with haematoxylin and eosin were evaluated for histopa-
thological changes under a light microscope.
2.7. Statistical Analysis
The data were expressed as mean ± S.E.M. (n = 6). Re-
sults were analyzed statistically by one-way ANOVA
followed by Dunnett’s test. The difference was consid-
ered significant if p < 0.05.
3. Results and Discussion
3.1. CCl4 Induced Hepatotoxicity
It is well established from the earlier studies that the
cleavage of carbon-chlorine bond (C-Cl bond) of carbon
tetrachloride leads to the formation of trichloromethyl
peroxy radical (CCl3·O2), which is involved in the
pathogenesis of liver injury [19].The abnormal higher
levels of serum SGPT, SGOT, ALP and bilirubin; and
decrease in total protein observed (Table 1) was the con-
sequence of carbon tetrachloride induced liver disfunc-
tion and denotes the damage to the hepatic cells [20].
Feeding of Feronia limonia extracts to such carbon tet-
rachloride-treated rats reduced the enhanced level of se-
rum glutamate oxaloacetate transaminase (SGOT) by
16.74, 32.18, and 42.20%, serum glutamate pyruvate
transaminase (SGPT) by 14.46, 31.87 and 34.12%, alka-
line phosphatase (ALP) by 20.71, 25.98 and 29.64% and
serum bilirubin by 39.38, 40.92, 42.19% which seems to
offer the protection and maintain the functional integrity
of hepatic cells. Glutathione (GSH) constitutes the first
line of defense against free radicals. Reduction in liver
GSH and decrease in GPX activity in carbon tetrachlo-
ride-treated rats (Table 2) indicated the damage to the
hepatic cells, which was confirmed by the earlier reports
[20]. Administration of Feronia limonia promoted the
conversion of GSSG (oxidised glutathione) into GSH by
the reactivation of hepatic glutathione reductase enzyme
in carbon tetrachloride-treated animals. The availability
of sufficient amount of GSH thus increased the detoxifi-
cation of active metabolites of carbon tetrachloride
through the involvement of GPX. But the restoration of
liver glutathione (GSH) by 13.82, 37.50 and 59.04% and
liver glutathione peroxidase (GPX) by 18.71, 21.37and
39.83% after the enduration of the Feronia limonia ex-
tracts to such carbon tetrachloride-treated rats account
for the protective efficacy of the extract. Decrease in
total serum protein (TSP) (Table 1) and liver glycogen
(GLY) (Table 2) observed in the rats treated with carbon
tetrachloride may be associated with the decrease in the
number of hepatocytes, which in turn may result into the
decreased hepatic capacity to synthesize protein and
GLY and consequently decrease in the liver weight (Ta-
ble 2) but when the Feronia limonia extracts were given
along with the carbon tetrachloride, the significant in-
crease in the glycogen (GLY) by 31.61, 15.92 and
39.94%, total serum albumin (TSA) by 37.76%, 53.64%,
63.51% and total serum protein (TSP) by 40.83, 45.41
and 50.41 % was observed indicating the antihepatotoxic
activity of the extracts and also accounting for the increase
Copyright © 2010 SciRes. CM
Copyright © 2010 SciRes. CM
Table 1. Effect of Feronia limonia extracts on the level of serum SGOT, SGPT, ALP, bilirubin and total protein.
Group SGOT (unit/ml)SGPT (unit/ml) ALP (unit/ml) Total Bilirubin (g/dl)Total albumin (g/dl) Total Protein (g/dl)
Group I (Normal) 59.49 ± 2.40**48.22 ± 2.87** 47.71 ± 1.62** 2.12 ± 0.20** 4.57 ± 0.28** 7.47 ± 0.32**
Group II (CCl4) 175.54 ± 3.80 134.94 ± 5.02 82.06 ± 2.07 3.91 ± 0.07 2.33 ± 0.21 4.80±0.25
Group III
95.14 ± 3.27**
76.00 ± 3.04**
51.45 ± 1.44**
2.23 ± 0.18**
3.58 ± 0.15**
7.44 ± 0.18**
Group IV
(Petroleum ether)
146.15 ± 2.46**
115.42 ± 4.54**
65.06 ± 4.42**
2.39 ± 0.12**
3.21 ± 0.19*
6.76 ± 0.25*
Group V
119.05 ± 2.94**
91.93 ± 2.57**
60.74 ± 2.36**
2.31 ± 0.18**
3.58 ± 0.14**
6.98 ± 0.21**
Group VI
101.45 ± 2.70**
88.89 ± 2.91**
57.73 ± 1.53**
2.24 ± 0.23*
3.81 ± 0.17**
7.23 ± 0.17**
Values are mean ± S.E. (n = 6).
*P < 0.05 as compared with Group II., **P < 0.01 as compared with Group II
Table 2. Effect of Feronia limonia extracts on the level of liver GSH, GPX, GLY, body weight (before and after treatment)
and liver weight.
Body weight
Group GSH
(g/mg protein)
(mg/gm of wet tissue)
(micrograms of glutathione
utilized/min/mg protein)Before treatment
After treatment
Liver weight
Group I (Normal) 6.15 ± 0.25** 28.17 ± 0.33** 7.89 ± 0.02** 160.9 ± 5 175.1± 4 6.1 ± 0.4
Group II (CCl4) 3.76 ± 0.19 18.60 ± 0.20 4.97 ± 0.06 162.4 ± 2 168.3 ± 3 6.08 ± 0.7
Group III
6.07 ± 0.32**
26.99 ± 0.65**
7.13 ± 0.34**
(43.46%) 170.02 ± 6 186 ± 7 5.96 ± 0.4
Group IV
(Petroleum ether)
4.28 ± 0.27*
24.48 ± 0.28**
5.90 ± 0.15*
(18.71%) 176 ± 4 191 ± 6 6.75 ± 0.2
Group V
5.17 ± 0.32*
21.56 ± 0.38**
6.05 ± 0.04**
(21.73%) 165 ± 3 178 ± 5 5.80 ± 0.4
Group VI
5.98 ± 0.39**
26.03 ± 0.37**
6.95 ± 0.06**
(39.83%) 172 ± 4 188 ± 6 6.54 ± 0.6
Values are mean ± S.E. (n = 6).
*P < 0.05 as compared with Group II., **P < 0.01 as compared with Group II
in the liver weight most probably through the hepatic cell
regeneration. It was conclusively observed that the meth-
anolic extract exhibited most significant result among all
the extracts.
3.2. Histopathology
Histomorphology of the livers of the untreated control
(Figure l(a)), the CCl4 treated (Figure l(b)) and the pe-
troleum, chloroform, methanolic extract of Feronia li-
monia plus toxin treated (Figures 1(d, e, f)) respectively.
CCl4 administration caused gross necrosis of the centri-
lobular hepatocytes characterized by nuclear pyknosis,
karyolysis and eosinophilic infiltration (Figure l(b)).
Treatment with the extracts of the Feronia limonia re-
versed, to a large extent, the hepatic lesions produced by
CCl4, as is obvious from the absence of eosinophilia and
presence of fewer necrotic zones (Figures 1(d, e, f)).
Further, the methanolic extract exhibited most significant
recovery of hepatic cells, which is in accordance with the
results obtained in biochemical parameters.
Copyright © 2010 SciRes. CM
(a) (b)
(c) (d)
(e) (f)
Figure 1. (a) Histology of the liver of control rat showing normal hepatic cells architecture, (b) Histology of the liver of car-
bon tetrachloride-treated rats showing necrosis with the obliteration of architecture in hepatic cells, (c) Histology of the liver
treated with silymarin showing recovery of the hepatic cells, (d) Histology of the liver treated with petroleum extract of Fero-
nia limonia showing recovery of the hepatic cells, (e) Histology of the liver treated with chloroform extract of Feronia limonia
showing recovery of the hepatic cells, (f) Histology of the liver treated with methanol extract of Feronia limonia showing re-
overy of the hepatic cells.
Copyright © 2010 SciRes. CM
4. Conclusions
It appears from our results that the mode of action of
Feronia limonia fruit extracts in affording the hepato-
protective activity against carbon tetrachloride may be
due to the cell membrane stabilization, hepatic cell re-
generation and activation of antioxidative enzymes such
as glutathione reductase, glutathione peroxidase, super-
oxide dismutase and catalase.
5. References
[1] J. D. Hooker, “The Flora of British India,” L. Reeve &
Co, London, 1875, p. 178.
[2] K. R. Kirtikar, B. D. Basu and I. C. S. An, “Indian Me-
dicinal Plants,” Orient Enterprises, Dehradun, India, 1993,
Vol. 1, pp. 496-498.
[3] M. Yusuf, J. U. Chowdhury, M. A. Wahab and J. Begum,
“Medicinal Plants of Bangladesh,” BCSIR Laboratories.
Chittagong, 1994, pp. 112-114.
[4] L. M. Perry, “Medicinal plants of East and Southern
Asia,” The MIT Press, Cambridge, 1980, pp. 87-88.
[5] S. K. Talapatra, M. K. Chaudhuri and B. Talapatra,
“Coumarins from the Root Bark of Feronia Elephantum,”
Phytochemistry, Vol. 12, 1973, pp. 236-237.
[6] S. R. Gupta, T. R. Seshadri, C. S. Sharma and N. D.
Sharma, “Chemical Components of Feronia Limonia,”
Planta Medica, Vol. 36, 1979, pp. 95-96.
[7] A. Agrawal, I. R. Siddique and J. Singh, “Coumarins
from the Roots of Feronia Limonia,” Phytochemistry,
Vol. 28, No. 4, 1989, pp. 1229-1232.
[8] A. J. Macleod and N. M. Pieris, “Volatile Flavour Com-
ponents of Wood Apple (Feronia Limonia) and a Proc-
essed Product,” Journal of Agricultural and Food Chem-
istry, Vol. 29, No. 1, 1981, pp. 49-53.
[9] A. Ahmad, L. N. Misra and R. S. Thakur, “Composition
of the Volatile Oil from Feronia Limonia Leaves,” Planta
Medica, Vol. 55, 1989, pp. 199-200.
[10] B. Ahmed, T. A. Al-Howiriny and A. B. Siddique, Jour-
nal of Ethanopharmacology, Vol. 7, 2003, p. 237.
[11] S. Reitman and S. Frankel, “In Vitro Determination of
Transaminase Activity in Serum,” American Journal of
Clinical Pathology, Vol. 28, 1957, p. 56.
[12] P. R. N. Kind and E. J. King, “Estimation of Plasma
Phosphatase by Determination of Hydrolysed Phenol
with Amino Antipyrine,” Journal of Clinical Pathology,
Vol. 7, 1954, p. 322.
[13] O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Ran-
dall, “Protein Measurement with Folin-Phenol Reagent,”
Journal of Biological Chemistry, Vol. 193, No. 1, 1951, p.
[14] L. Jendrassik and P. Grof, “Vereinfachte Photo-Metrische
Methoden zur Bestimmung des Bilirubins,” Biochemische
Zeitschrift, Vol. 297, 1938, pp. 81-89.
[15] E. Beutler, O. Duran and B. M. Kelly, “Improved Method
for the Determination of Blood Glutathione,” Journal of
Laboratory Clinical Medicine, Vol. 61, 1963, p. 882.
[16] W. Z. Hassid and S. Abraham, “Chemical Procedures for
Analysis of Polysaccarides,” In: S. P. Colowick and N. O.
Kaplan Eds., Methods in Enzymology, Academic Press
Inc., New York, Vol. 3, 1957, p. 34.
[17] J. T. Rotruck, A. L. Pope, H. E. Ganther, D. G. Hafeman
and W. G. Hoekstra, “Selenium: Biochemical Role as a
Component of Glutathione Peroxidase,” Science, Vol.
179, No. 4073, 1973, pp. 588-590.
[18] A. E. Galigher and E. N. Kozloff, “Essentials of Practical
Microtechnique,” 2nd Edition, Philadelphia, 1971, p. 210.
[19] K. H. Cheeseman, E. F. Albano, A. Thomasi and T. Sla-
ter, “Biochemical Studies on the Metabolic Activation of
Halogenated Alkanes,” Environmental Health Perspec-
tives, Vol. 64, 1985, pp. 85-101.
[20] K. Singh, A. K. Khanna and R. Chandan, “Hepatoprotec-
tive Activity of Ellagic Acid against Carbon Tetrachlo-
ride Induced Hepatotoxicity in Rats,” Indian Journal of
Experimental Biology, Vol. 37, No. 10, 1999, pp. 1025-