Indian Journal of Experimental Biology
Vol. 43, May 2005, pp. 430-436
Effect of Emblica officinalis (Gaertn) on CC14 induced hepatic toxicity and DNA
synthesis in Wi star rats
Sarwat Sultana, Salahuddin Ahmad, Naghma Khan & Tamanna Jahangir
Section of Chemoprevention and Nutrition Toxicology, Department of Medical Elementology and Toxicology, Jamia Hamdard
(Hamdard University), Hamdard Nagar, New Delhi 110 062, India
Received 15 September 2004; revised 21 February 2005
A single dose of CCl 4 (1 mllkg body weight, po in corn oil) increased the levels of SGOT (serum glutamate oxaloacetate
transaminase), SGPT (serum glutamate pyruvate transaminase), LDH (lactate dehydrogenase), glutathione-S-transferase and
depletion in reduced glutathione, glutathione peroxidase and glutathione reductase. It also caused enhancement in the levels
of lipid peroxidation (LPO) and DNA synthesis. There was also pathological deterioration of hepatic tissue as evident from
multi vacuolated hepatocytes containing fat globules around central vein. The pretreatment of E. officinalis for 7 consecutive
days showed a profound pathological protection to liver cell as depicted by univacuolated hepatocytes. Pretreatment with
E. officinalis at doses of 100 and 200 mg/kg body weight, prior to CCl 4 intoxication showed significant reduction in the
levels of SGOT, SGPT, LDH, glutathione-S-transferase , LPO and DNA synthesis. There was also increase in reduced
glutathione, glutathione peroxidase and glutathione reductase. The results suggest that E. officinalis inhibits hepatic toxicity
in Wi star rats.
Keywords: CCI 4 ; Emblica officillalis; Hepatic toxicity.
Liver is a versatile organ of the body that regulates
internal chemical environment. Liver injuries induced
by various hepatotoxins have been recognized as a
major toxicological problem for years 1• Carbon
tetrachloride (CCI 4 ), an established hepatotoxicant for
both animals and humans and has been used as
solvent to coal tar, oil, resins and raw material of
fluorocarbon for coolant and as fire extinguishing
agent 2 • CCl4 is metabolized in the liver by cytochrome
P450 to trichloromethyl radical that reacts with GSH to
form a GSH containing radical and causes various
pathological and toxicological manifestations 3 . Early
events associated with the cytotoxicity of CCI 4
following activation includes Iipidperoxidation and
covalent binding to proteins and lipids followed
immediately by diminished protein and lipid turnover
and alterations in calcium homeostasis 4 • CCl4 has
been shown to acti vate Kupffer cells by increasing
intracellular Ca2+concentration, causing release of
harmful cytokines that contribute to the death of
hepatocytes and oxidative stress 5 . The cellular
infiltration of activated neutrophillic leukocytes
*Correspondent author:
Phone: 0091-11-26059688 Ext: 5565/5566
Fax: 0091-11-26059663
E-mail: [email protected]
amplifies inflammatory response and cellular injury
or death due to release of superoxide anions and other
toxic mediators 6 . Free radicals bind covalently to
membrane proteins and lipids causing enzyme
inactivation and promotion of lipidperoxidation as a
result of interaction with membrane unsaturated fatty
acids?
Regenerating liver, a model of synchronized
cellular proliferation, provides an opportunity to study
the factors that can suppress or intensify the
manifestations involved in the regenerative process 8 .
Historically plants have been used as folk medicine
against various types of diseases 9 . Experimental work
on several plants has been carried out to evaluate their
efficacy against chemically induced toxicitylo.I' . In
the present study the efficacy of extract of fruits of
Emblica officinalis (Gaertn) has been elucidated
against experimental tissue injury, oxidative stress
and proliferative response induced by hepatotoxic
agent, CCI 4 •
Phytochemical evaluation of the plant E. officinalis
showed the presence of quercetin, ascorbic acid and
ellagic acid. The major principle is quercetin 12. The
polyphenolic components of higher plants reportedly
act as antioxidant or as agents of other mechanisms
contributing to anti carcinogenic action I3. Flavonoid
SULTANA eta/.: EMBLICA OFFICINA LIS & CC1 4 INDUCED HEPATIC TOXICITY IN RATS
431
rotatory evaporator (Buchi Rotavapor, Switzerland).
and other plant phenolics possess multiple biological
The methanol fraction obtained (42 g) was suspended in
activity including anticarcinogenic, anti-inflammatory
water to prepare the required dilutions at the time of
and anti-allergic activity 14. Flavonoids are reported
'5
dosing.
scavanger of superoxide radicals , peroxyl radicals
and inhibitor of lipidperoxidation '6 . Quercetin has
Animals-Male albino Wistar rats (130-1S0 g) were
been shown to mediate the down regulation of mutant
obtained from Central Animal House of ·Hamdard
PS3 in human breast cancer cell lines and modulation
University, New Delhi, India. They were housed in
of TGF in ovarian cells 17. Ellagic acid has been shown
polypropylene cages in groups of six rats per cage and
to inhibit carcinogen-induced neoplasia in mouse
were kept in a room maintained at 2So ± 2°C with a
l8
skin, rat mammary gland and mouse forestomach •
12: 12 hr L:D cycle. They were given free access to
The most typical reaction leading to endogenous
standard laboratory feed. (Hindus tan Lever Ltd."
formation of mutagens is between nitrite and
Bombay, India) and water ad libitum. The animals
nitrosamines or amides, which can be inhibited by , were sacrificed according to the guidelines of the
vitamin C, vitamin E, gallic acid, ferrulic acid and
current laws of CPCSEA (Ethical Committee for the
caffeic acid l9 . The above information and present
purpose of control and supervision of experiments on
status of the dietary intervention prompted the present
animals), India.
work on the evaluation of the efficacy of the fruit
extract of Emblica offlcinalis against CCl4 induced
Experimental protocol
hepatic toxicity and DNA synthesis.
To study the biological, serological and
Materials and Methods
pathological changes 24 rats were divided into 4
groups of (; each. Group I served as saline treated
Chemicals-Oxidized and reduced glutathione
control. Rats in Group II were given CCl4 [1 ml/kg
(GSSG and GSH), NADPH, H20 2, dithionitrobenzene
(DTNB), l-chloro-2, 4-dinitrobenzene (CDNB),
body weight, po, in corn oil (1: 1)]. Groups III and IV
dinitrophenyl hydrazine (DNPH), glutathione reductase,
animals were given E.offlcillalis extract at doses of
reduced nicotinamide adenine dinucleotide (NADPH),
100 and 200 mg/kg body weight, respectively for 7
consecutive days followed by CCl4 intoxication on the
ethylenediamine tetra acetic acid (EDTA), pyridoxal
phosphate, phenyl methyl sulphonyl fluoride (PMSF),
seventh day. Animals in all the groups were sacrificed
24 hr after CCl4 or saline administration. The doses of
2-mercaptoethanol, dithiothreitol, Tween 80, Brij 3S,
ethanolamine, methoxyethanol, citric acid, y-glutamyl
the plant extracts were selected after performing
p-nitroanilide, diethyl nitrosamine, 2-acetylaminoin vitro assays like microtonal lipid peroxidation and
fluorine and quercetin were purchased from Sigma
cytochrome P450 . Serum was separated and stored at
Chemical Co. (St. Louis, Mo. USA). eH] thymidine
4°C for the estimation of serum glutamate
(specific activity 73.0 Ci/m mol) was purchased from
oxaloacetate transaminase (SGOT), serum glutamate
Amersham Corporation, UK. All other chemicals and
pyruvate transaminase (SOPT), lactate dehydrogenase
reagents used were of highest purity commercially
(LDH). Tissue was processed for the estimation of
available.
glutathione (GSH) content, microsomal lipidPlant extractioll----Dried fruits of the plant
peroxidation and actIVities of glutathione-SE. offlcinalis were procured from Jamia Hamdard
transferase (GST), glutathione peroxidase (GPx) and
campus and authenticated at source by Prof. M Iqbal,
glutathione reductase (GR). Sections of liver from
Department of Environmental Botany, Jamia Hamdard,
each group were cut and preserved in buffered
New Delhi, India. Freshly collected fruits were shadeformalin for histopathological studies.
dried and coarsely powdered in a grinder. The
For eH] thymidine incorporation study, grouping of
extraction procedure was followed as described by
animals was same as described above. All the animals
Didry et apo. Powdered dried fruits (SOO g) were
were given intraperitoneal eH] thymidine (25 Ilci/0.2
extracted in round-bottomed flask 'with 2000 ml
mlsaline/l00 g of animal) 2 hr before killing. All
petroleum ether, benzene, ethyl acetate, acetone,
animals were sacrificed 48 hr of CCl4 intoxication and
methanol and double distilled water. The residues
liver sections were quickly excised, rinsed with ice
yielded from each solvent (14, 10, 16,30, 42 and 22 g
cold saline, freed of extraneous material and
processed for th~ quantification of eH] thymidine
respectively) were stored at 4°C. The extracts were
incorporation into the hepatic DNA.
evaporated to dryness under reduced pressure in a
432
INDIAN J EXP BIOL, MAY 2005
Post-mitochondrial supernatant (PMS)
microsome
preparation--The
samples
and
were
homogenized in chilled phosphate buffer (0.1 M, pH
7.4) using a Potter Elvehjen homogenizer. The
homogenate was centrifuged at 3000 rpm for 5 min at
4°C by Eltek Refrigerated Centrifuge. The aliquot
obtained was centrifuged at 12000 rpm for 20 min at
4°C to obtain post-mitochondrial supernatant (PMS).
PMS was centrifuged for 60 min by ultracentrifuge at
34,000 rpm at 4°C. The pellet was washed with
phosphate buffer (0.1 M, pH 7.4).
Biochemical estimations-Tissue processing and
preparation of post mitochondrial supernatant (PMS)
were done immediately after animal sacrifice. For all
biochemical estimations, serum samples were utilized.
All the biochemical estimations were completed within
24 hr of animal sacrifice. SGOT21 , SGPT21 , LDH22,
LP0 23, GST24 , GPX25 , GR26, DNA27.28 were estimated as
per standard procedures.
Protein estimation-Protein content in all samples
was estimated by the method of Lowry et al. 29 using
bovine serum albumin as standard.
Statistical analysis-The level of significance
between different groups is based on Dunnett's t test,
followed by the ANOVA test.
Results and Discussion
The effect of prophylactic administration of the
extract on CCl4 mediated leakage of liver marker
enzymes in serum is shown in Table 1. CC4
administration resulted in a significant rise in the
levels of SGOT, SGPT and LDH in serum by 278%.
349% and 155% of saline treated control respectively.
Pretreatment with E. officinalis extract at higher dose
showed marked alleviation of SGOT, SGPT and LDH
by 94%, 127% and 31% respectively as compared
with CCl4 treated group. Hepatoprotective effects of
the extracts can be explained on the basis of decreased
liver enzyme leakage in serum on pretreatment and
significant recovery of the cell membrane towards
stabilization as evident from pathological recovery30.
CCl4 is metabolized in liver by cytochrome P450
dependent electron transport chain system yielding
trichloromethyl radical that in aerobic condition
rapidly gets converted to its peroxyl radical4. These
radicals bind directly to lipids and proteins through
covalent bonds and also interact with membrane
phospholipids leading to promotion of lipid
peroxidation7. Apart from the studies of mechanism of
CCI 4 induced injury, recent studies have shown the
involvement of Kupffer c-ells, cytokines, and
neutrophilic leukocytes31 . A recent study has also
shown the protective effect of plant against CCl4
induced hepatic damage 32 .
The effect of pretreatment of extract on the GSH
metabolizing enzymes GST, GPx and GR is shown in
Table 2. The administration of CCI4 resulted in
significant (P<O.ool) elevation of GST activity by
168% and a concomitant decrease in the activities of
GPx and GR by 42% and 39% as compared with
saline treated control group. The pretreatment of rats
with E. officinalis resulted in marked down regulation
of GST by 39% at higher dose of the plant. The
concurrent alleviation in the GPx was observed on
pretreatment of E. officinalis by 27% and of GR
activity by 31 % respectively as compared with CCl4
treated group. CC4 administration resulted in increase
in GST activity with concomitant decrease in GPx
and GR activities that were significantly modulated
on prophylactic pretreatment of the extract. Liver
possesses growth ability after cellular loss in
hepatotoxicant induced liver injury. Regenerating
Table 1-Effect of pretreatment of the extract of E. officinalis on CC1 4 induced release of liver damage markers in serum
[Values are mean ± SE from 6 animals in each group]
Treatment groups
SGPT
(IU/litre)
SOOT
(IU/litre)
LOH
(nmol NAOH
oxidized/min/mg protein)
Saline
CCI 4alone
CCI4 +EO (D1)
CCI 4 +EO (02)
27.6 ±0.86
76.8 ± 1.97"
62.28 ± 2.51 b
50.79 ± 2.38 b
20.72 ±0.62
72.28 ± 2.12a
59.18 ± 2.15 b
45.98 ± 2.23 b
356.8 ± 31.2
553.8 ± 37.9"
496.4 ± 22.6 b
443.6 ± 24.9 b
P values: "<0.01 (compared to saline treated group)
b<O.OOl (compared to CCI 4 treated group)
c<O.OOI (compared to CCl 4 treated group)
EO=E. officinalis; 01= 100 mg/kg body weight of E. officillalis; D2= 200 mgikg body weight of E. officillalis.
SULTANA etal.: EMBLICA OFFICINALIS & CCI4 INDUCED HEPATIC TOXICITY IN RATS
liver provides a model to study the factors that
modulates the process of regeneration. Acute hepatic
necrosis on CCl4 administration is followed by
proliferative response that reaches peak at 48 hr, with
scarce mitotic cells.
The effect of pretreatment of animals with crude
extract of E. officinalis plants on CCl4 induced GSH
depletion, enhanced MDA formation and DNA
synthesis is shown in Table 3. Pretreatment of rats
with extract produced a marked inhibitory effect on
GSH depletion with concomitant alleviation of MDA
levels. E. officinalis showed a significant protection of
GSH content by 30% and decrease in MDA formation
by 111 % as compared with CCl4 treated group.
Administration of CCl4 resulted in significant
(P<O.OOI) 263% increase in the rate of eH] thymidine
incorporation into hepatic DNA, which is a marker of
DNA synthesis as compared with saline group. The
pretreatment of rats with E. officinalis showed a
marked (P<O.OI) suppressing effect on the rate
eH]
433
thymidine incorporation into hepatic DNA by 102% at
higher dose of the plant as compared with CCl4 treated
group. Cellular thiol status is an important
consideration for the study of hepatotoxicity,
particularly in conditions of oxidative tissue injury33.
GSH is a tripeptide (y-glutamyl cysteinyl glycine)
non-protein thiol abundant in liver that plays pivotal
role in the detoxification processes including
conjugation
of
reactive
intermediates
and
maintenance of GSH redox cycle as antioxidant
armoury34. The enhancement of hepatotoxicity by
GSH depletion has been noted during the metabolism
of several compounds 35 . CCl4 administration resulted
in marked depletion of tissue GSH content
consequently increasing vulnerability of tissue to free
radical attack causing lipidperoxidation. ProteCtive
agents have shown to exert their action against CCl 4
mediated lipidperoxidation either through decreased
production of free radical derivatives or due to
antioxidant activity of the agent itself36. The
Table 2-Effect of pretreatment of the extract of E.officillalis on glutathione metabolizing enzymes against
CCI 4 induced oxidative stress in rat liver
[Values are mean ± SE from 6 animals in each group]
Treatment
Groups
GST
(nmol conjugate
formed/min/mg protein)
GPx
(nmol NADPH oxidized
min/mg protein)
GR
Saline
CCI 4 alone
CCI 4 +EO (01)
CCl 4 +EO (D2)
865.91± 34.63
1454.78±65.19"
1271.88 ± 34.94c
1096.22 ± 46.68 b
210.56 ± 6.27
122.12 ± 6.42"
154.82 ± 7.93 c
179.27±6.2 1b
189.54 ± 4.92
115.65 ± 3.47"
155.2 ± 6.97 b
b
174.92 ± 7.43
P values: °<0.01 (compared to saline treated group)
b<O.OOI (compared to CCl 4 treated group)
c<O.OOI (compared to CCl 4 treated group)
EO=E. officinalis; Dl= 100 mg/kg body weight of E. officillalis; D2= 200 mg/kg body weight of E. officillalis
Table 3--Effect of pretreatment of the extract of E. officinalis on CCl 4 induced depletion of GSH and in·crease in
the levels of LPO and DNA synthesis
[Values are mean ± SE from 6 animals in each group]
Treatment
Groups
Saline
CCl 4alone
CCI 4 +EO (Dl)
CCI 4 +EO (D2)
GSH
(nmol GSHlg tissue)
LPO
(nmol MDA formedlhr/g
tissue)
DNA synthesis
(dprnlJ-lg DNA)
± 0.05
± 0.04"
± 0.05 b
± 0.03 b
8.42 ± 0.8
24.31 ± 1.0"
18.1 ± 0.9 b
15.4 ± 1.2b
31.3 ± 5.8
82.4 ± 12.9"
65 .9 ± 8.7 b
50.4 ± 6.5 b
0.62
0.32
0.45
0.52
P values: "<0.01 (compared to saline treated group)
b<O.OOI (compared to CCl 4 treated group)
c<O.OOI (compared to CCl 4 treated group)
EO=E. officillalis; Dl= 100 mg/kg body weight of E. officinalis; D2= 200 mg/kg body weight of E. officinalis
434
INDIAN J EXP BIOL, MAY 2005
pretreatment of animals with E. officinalis
significantly ameliorated GSH content with
concomitant decrease in lipid peroxidation. It could
be suggested that antioxidant presence of several
active constituents including quercetin, ascorbic acid
and ellagic acid in E. officinalis may have
counteracted the free radicals through effective
scavenging and blocking the conjugation of reactive
intermediates to GSH as evident from ameliorated
GSH content and decreased MDA formation.
Similarly, Vitamin E has been shown to suppress free
radical mediated chain initiation and consequently
protecting tissue from pathophysiological insult".
Induction of GST activity has been correlated to
increased GSH consumption and 4-hydroxynonenolm
(4-HNE) levels in tissue 37 • The sudden elevation of
hepatocytes growth factor (HGF) in plasma provides
mitogenic signals that trigger DNA synthesis during
liver regeneration 38 . In the present, study CCl4
administration resulted in enhancement in the rate of
DNA synthesis when compared to normal group.
Pretreatment of rats with E. officinalis extracts
significantly suppressed the rate of eH] thymidine
incorporation into hepatic DNA in a dose dependent
manner. The enhancement of DNA synthesis has been
shown as a consequent step to the severe necrosis
induced by hepatotoxicants 39 .
The protective effect of plant E. officinalis on the
basis of histopathological findings is shown in Fig. 1.
Figure 1a shows the normal pathology of liver
section. Administration of CCl4 resulted in
pathological deterioration of hepatic tissue as evident
multi vacuolated hepatocytes containing fat globules
showing fatty changes around central vein and large
vacuolated cells with central nuclei at the periphery of
lobule showing necrosis. (Fig. 1b). The pretreatment
of animals with E. officinalis fruit extract showed a
profound pathological protection to liver cells as
depicted by univacuolated hepatocytes surrounding
multivacuolated hepatocytes, mild infiltration and
fatty changes around vascular channels (Fig. 1c, d). In
summary, the present data suggest that pretreatment
Fig. I-Effect of pretreatment of rats with E. ojJicinalis on CCl 4 induced pathological changes in rat liver. a-d: H & E: (a) saline only
(x 50), (b) only CCI 4 • (c) E. ojJicinalis (01) + CCI 4 and (d) E. ojJicinalis (D2) + CCI 4 • b-d x 250.
SULTANA et al.: EMBLICA OFFICINA LIS & CCI 4 INDUCED HEPATIC TOXICITY IN RATS
of animals with E. officinalis markedly inhibited
oxidative stress and lipidperoxidation induced by
necrogenic dose of CCl 4 and also suppressed tissue
injury and hepatotoxicant post necrotic regeneration
process.
15
16
17
Acknowledgement
The author (S S) is thankful to CSIR, New Delhi,
India for financial assistance.
18
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