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HEPATOPROTECTIVE ACTIVITY OF ROOT BARK OF AZIMA
TETRACANTHA LAM. AGAINST CARBON TETRA CHLORIDE
(CCL4)-INDUCED HEPATOTOXICITY IN WISTAR MALE
ALBINO RATS
M. Balakrishnan*, R. Dhanapal1, K.B. Chandra Sekhar2
*l
Principal, Rosory College of Pharmacy,
yellapur (v), Hasanparthy (M), Warangal, Andhra Pradesh, India.-506 371.
1
Department of Pharmaceutics, Kakatiya Institute of Pharmaceutical Sciences,
Pembarthi (V), Hasanparthy (M), Warangal, Andhra Pradesh, India-506 371.
2
Professor of Chemistry, Director of Evaluation, JNT University, Anantapur, Andhra Pradesh, India-515 002.
ABSTRACT
The present study to evaluate the hepatoprotective activity of ethanol (50%) extract of Azima tetracantha Lam (EEAT)
root bark using CCl4 induced hepatic damage in male Wistar albino rats. The EEAT root bark at doses of 40, 80 and 120mg/kg,
p.o and the standard drug Liv.52 (40mg/kg,p.o) were administered orally for 7 days in CCl4 intoxicated rats. The
hepatoprotective activity was assessed by using various biochemical parameters like SGOT, SGPT, alkaline phosphatase (ALP)
and acid phosphatase (ACP), also total bilirubin along with histopathological studies of liver tissue. The biochemical changes
and histopathological studies were observed on 8 th day. There was a significant increase in serum levels of SGOT, SGPT,
alkaline phosphatase (ALP) and acid phosphatase (ACP), also total bilirubin in the CCl 4 treated animals, reflecting liver injury.
The administration of EEAT root bark at doses of 40, 80 and 120mg/kg, p.o for 3days resulted in significant decrease (P<0.001)
the CCl4-induced elevated levels of the hepatic enzymes SGOT, SGPT, alkaline phosphatase (ALP) and acid phosphatase
(ACP) also total bilirubin in a dose dependent manner. Whereas the extract at tested doses decreases the CCl4-induced elevated
level of hepatic enzymes, and also total bilirubin in rats, and its subsequent return towards near normalcy after 7days indicating
the recovery of hepatic cells. The histological studies were also carried out to support the above parameters. In the liver sections
of the rats treated with EEAT root bark extract for 7 days, the normal cellular architecture was retained as compared to Liv.52,
there by further confirming the potent hepatoprotective effect of EEAT root bark. The ethanol (50%) extract of Azima
tetracantha Lam (EEAT) root bark afforded significant protection against CCl 4 induced hepatocellular injury. To our
knowledge, this report is the first that shows hepatoprotective effect of Azima tetracantha Lam. (Salvodaraceae) root bark
against CCl4 induced liver damage.
Keywords: CCl4, Hepatotoxicity, Hepatic enzymes, Azima tetracantha, Hepatoprotective, EEAT root bark.
Corresponding Author
M. Balakrishnan
E-mail: [email protected]
INRODUCTION
Liver is considered as the key organ in the
metabolism, detoxification, and secretory functions in the
body, and its disorders are numerous with no effective
remedies, however, the search for new medicines is still
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Balakrishnan M. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 366-372.
ongoing. Hepatitis is a common disease in the world
especially in the developing countries. Despite, considerable
progress in the treatment of liver diseases by oral
hepatoprotective agents, search for newer drugs continues
because the existing synthetic drugs have several limitations
(Liu GT, 1989; Handa SS et al., 2001). Because liver
performs many vital functions in the human body, damage
of liver causes unbearable problems (Mitra SK et al., 1998;
Chattopadhyay
RR,
2003).
Thus
study
about
hepatoprotective compounds is of importance.
Azima tetracantha Lam. (Family: Salvadoraceae)
locally known as “Mulsangu”, is a rambling spinous shrub
flowering throughout the year found in Peninsular India,
West Bengal, Orissa, African Countries and extends through
Arabia to tropical Asia. The leaves of the plant are elliptical
in shape and are rigid, pale green colored. The flowers are
small, greenish white (or) yellow colored, unisexual in
axillary fasciles. The berries are white in colour; usually one
seeded and edible. The juice of the leaves is said to relieve
the cough phthisis and asthma. In western India juice of the
leaves is applied as eardrops against earache and crushed
leaves are placed on painful teeth. In India and Sri Lanka
the root, root bark and leaves are administered with food as
a remedy for rheumatism (Chopra RN et al., 1956; Kritikar
KR and Basu BD, 1976; Hebbar SS et al., 2004). The plant
is considered as a powerful diuretic and is also used to treat
rheumatism, dropsy, dyspepsia, chronic diarrhoea and as a
stimulant tonic for women after confinement (Nadkarni
KM, 1976). The leaves of this plant have been reported to
possess anti-inflammatory, wound-healing activity, diuretic
activity and analgesic activity (Syed Ismail T et al., 1997;
Jaswanth A et al., 2001; Nandgude TD et al., 2001). Despite
the traditional uses, the leaves of this plant have been tested
negative in antibacterial and antifungal properties (Vonshak
A et al., 2003).
A number of chemical constituents have previously
been reported from the leaves (Friedelin, Lupeol, Glutinol
and beta-sitosterol), the seed oils (fatty acids such as
myristic acid, palmitic acid, stearic acid, arachidic acid,
oleic acid, linoleic acid, eicosenoic acid and flavonoids), the
seed, root, stem and young leaves (N-methoxy-3indolylmethyl-glucosinolate), roots and the leaves
(terpenoids), also the dimeric piperidine alkaloids azimine,
azcarpine and carpaine have previously been isolated from
all plant parts (Venkata Rao E and Prasada Rao PRS, 1978;
Daulatabad CD et al., 1991; Bennett RN et al., 2004; Rall
GJH et al., 1967; Williams UV and Nagarajan S, 1988;
Dold AP, 2006).
Locally, the traditional healers from Tirunelveli
district of Tamilnadu are using root bark (paste with butter
milk) of this plant as potent remedy for jaundice (Personal
information). However, there are no ethnomedicinal
information and scientific findings for the above said
traditional claim for jaundice. Therefore, to justify the
traditional claims the present study was undertaken to find
out if ethanol extract of Azima tetracantha root bark
demonstrates any hepatoprotective activity against CCl4induced liver damage in vivo in rats.
MATERIALS AND METHODS
Collection of plant material
Fresh root barks of Azima tetracantha were
collected from Wastelands of Kadyanallur, Tirunelveli
(District), Tamilnadu, India. The plant specimen was
authenticated by Dr.V.Chelladurai, Research Officer
Botany, Survey of Medicinal Plants Unit, Tirunelveli. A
voucher specimen (V.No: ATC27/08/2005) has been
deposited at the herbarium unit of the Department of
Botany, Siddha Medical College, Palayamkottai, Tirunelveli
(District), Tamilnadu, India.
Preparation of plant extract
The root barks of Azima tetracantha were chopped,
shade-dried and made into a coarse powder. About 500gms
of the powdered material was taken and dissolved in two
liters of ethanol and distilled water mixture in the ratio of
1:1and subjected to hot maceration process for 24 hours
continuously by using Sohxlet apparatus. Then the extract
was collected and evaporated under reduced pressure then
vacuum-dried. The yield of the extract was 12.4% with
reference to dry powder.
Preliminary phytochemical screening
Various phytochemical tests were carried out on
dry extracts obtained to detect the presence of different
phytoconstituents by using the methods described by Evans
(Evans WC, 1999).
Animals
Wistar albino rats (150-190) of either sex, procured
from National Institute of Nutrition, Hyderabad, were used
for the study. The animals were housed in large
polypropylene cages in a temperature-controlled room
(22±2 ◦C) and provided with standardized pelleted feed
(Hindustan Liver, Bangalore) and clean drinking water ad
libitum. All procedures compiled with the norms of the
Institutional Animal Ethics Committee (Registration No:
129/99/CPCSEA).
Acute toxicity studies
For toxicity studies the EEAT root bark in the
range of doses 50-1000 mg/kg were administered in six
groups of ten rats respectively. The percentage survivals
were observed after 72 hours. The LD50 was determined
using the graphical methods of Reed and Muench (Reed LJ
and Muench HA, 1938).
In vivo hepatoprotective activity studies
The liver protective effect was evaluated using the
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carbon tetrachloride (CCl4) model described by
Visweswaram et al (Visweswaram D et al., 1994).
Thirty six Wistar albino rats (180-200gm) were
divided into six groups of six rats each and were subjected
to the following treatments: Group-I which served as normal
control received distilled water (1 ml/kg; p.o) for 7days.
Group II -VI received 0.75 ml/kg CCl4 administered orally
as single dose. After 36 hours, Groups III-VI received
EEAT root bark with doses of 40, 80 and 120 mg/kg, p.o
and the standard drug Liv.52 with dose of 40mg/kg, p.o,
respectively once daily for 7days. The blood was collected
by puncturing the retro-orbital sinus of three rats from each
group on 8th day after the treatment respectively. From the
collected blood samples, serum was separated to assess
various biochemical parameters.
Biochemical estimation
The separated serum was subjected to estimate
SGOT and SGPT by Reitman and Frankel (Reitman S and
Frankel S, 1957) method, alkaline phosphatase (ALP) and
acid phosphatase (ACP) by Kind and King (Kind PRN and
King EJ, 1954) method, bilirubin by Malloy and Evelyn
(Malloy HT and Evelyn KA, 1937) method and urea by
Bousquet (Bousquet BF et al., 1971) method. The rats were
then sacrificed by bleeding and the liver was carefully
dissected, cleaned of extraneous tissue, and part of the liver
tissue was immediately processed for histopathological
investigation.
Histopathological studies
The tissues of liver were fixed in 10% formalin and
embedded in paraffin wax. Sections of 4-5 microns
thickness were made using rotary microtome and stained
with haematoxylin-eosin and histological observations were
made under light microscope (Luna LG, 1966; Galigher AE
and Kozloff EN, 1971).
Statistical analysis
Results of biochemical estimations are reported as
mean ± SEM of six animals in each group. The data were
subjected to one-way ANOVA followed by Tukey’s
multiple comparison tests. P<0.001 was considered
statistically significant.
RESULTS
Phytochemical Screening
The various chemical tests revealed the presence of
alkaloids, tannins, proteins, and lipids. (Table -1)
Acute toxicity studies
The LD of EEAT root bark was found to be 406
50
mg/kg, b.w. The doses selected for this present study were,
10% (40mg), 20 %( 80mg) and 30% (120mg) of LD .
50
Effect of EEAT root bark on CCl4 – induced hepatotoxicity
The results of Carbon tetrachloride-induced
hepatotoxicity were represented in Table 1. The CCl4 only
treated animals exhibited a significant increase (P<0.001)
the levels of SGOT, SGPT, alkaline phosphatase (ALP) and
acid phosphatase (ACP) and also total bilirubin when
compared to the normal control group on 8 th day, indicating
hepatocellular damage.
The EEAT root bark at tested doses (group III-V)
produced a significant reduction (P<0.001) in the CCl4induced elevated levels of SGOT, SGPT, alkaline
phosphatase (ALP) and acid phosphatase (ACP), also total
bilirubin when compared to the CCl4 only treated animals
(group-II) after 3days of treatment and reduced furthermore
to the normalcy on 8th day although the lowest dose (40
mg/kg) tested could produce significant reduction in
enzymatic levels (Table 1). Overall, EEAT root bark at
tested doses significantly reduced the levels of hepatic
enzymes, total bilirubin in a dose dependent manner. After 7
days, the hepatic enzymes levels were almost restored to the
normal after treating with EEAT root bark at the dose of
120mg/kg, p.o.
A standard drug, Liv.52 at a dose of 40 mg/kg
(group-VI) administered orally produced a significant
reduction (p<0. 001) compared to CCl4 only treated animals
(group-II) on 8th day and these protective effects almost
close to EEAT root bark 120mg/kg,p.o.
Effect of EEAT root bark on histolopathological change
Histopathological examination of liver sections of
control group showed normal cellular architecture with
distinct hepatic cells, sinusoidal spaces and central vein on
8th day (Fig.3). Disarrangement of normal hepatic cells with
centrilobular necrosis, vacuolization of cytoplasm and fatty
degeneration were observed (on 8th day) in CCl4 intoxicated
rats (Fig.4).
Figure 1. Effect of EEATrb and Liv.52 on CCl4-induced
alteration of SGOT, SGPT, ALP and ACP in rat liver after
7 days of treatment
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Table 1. EEAT root bark on CCl4-induced alteration of hepatic enzymes, serum bilirubin in rat liver on 8th
day observation
Design of Treatment
Group-I:
Normal control
(DW-1 ml/kg; p.o)
Group-II: CCl4
(0.75 ml/kg; p.o)
Group-III: EEAT root
bark
(40 mg/kg; p.o)
Group-IV: EEAT root
bark
(80 mg/kg; p.o)
Group-V: EEAT root
bark
(120 mg/kg; p.o)
Group-VI: Liv.52
(40 mg/kg; p.o)
Biochemical parameters
ACP(KA
ALP (KA Units)
Units)
total
Bilirubin(mg/dl)
direct
Bilirubin(mg/dl)
SGOT(U/ml)
SGPT(U/ml)
95.31 ± 1.16
57.61 ± 0.06
142.60 ± 0.92
70.02 ± 1.17
0.62 ± 0.03
c
158.14 ± 1.41* c
255.12 ± 0.91* c
112.02 ±
0.13* c
2.62 ± 0.04* c
1.52 ± 0.02*
260.46 ± 1.15*
92.13 ± 0.25*
191.13 ± 0.04*
105.61 ±
0.04*
2.20 ± 0.05*
1.32 ± 0.03*
161.22 ± .91*
71.07 ± 1.62*
162.01± 0.64*
87.26 ± 0.14*
1.94 ± 0.02*
0.95 ± 0.02*
141.14 ± 1.04*
62.02 ± 0.04*
146.04 ± 0.02*
76.15 ± 0.02*
1.21 ± 0.04*
141.13 ± 0.81*
60.12 ± 0.43*
143.17 ± 0.92*
75.09 ± 0.03*
0.89 ± 0.03*
423.20 ± 1.03*
0.20 ± 0.02*
0.33 ±
0.02*
0.32 ± 0.02*
Values are Mean ± SEM of 6 animals each in a group.
*P<0.001,when compared group II Vs groupI,III,IV,V and VI
EEAT root bark= ethanol (50%) extract of Azima tetracantha Lam (EEAT) root bark, CCl4 = Carbon tetrachloride
Figure 2. Effect of EEATrb and Liv.52 on CCl4-induced
alteration of total and direct Bilirubin in rat liver after 7
days of treatment
Fig 3. Histology of the liver. control group (normal)
(Magnification: 100x)
Fig 4. Histology of the liver. Treated with only CCl4 on 8th
day examination. (Magnification: 100x)
Fig 5. Histology of the liver.treated with CCl 4and
supplemented with EEATrb (120mg/kg, p.o) on 8 th day
examination (Magnification: 100x)
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Fig 6. Histology of the liver.Treated with CCl4 and supplemented with
Liv.52 (40mg/kg, p.o). examined on 8 th day (Magnification: 100x)
The liver sections (on 8th day) of the group-V rats
treated with EEAT root bark (120mg/kg, p.o) showed a sign
of protection as it was evident by the moderate
accumulation of fatty lobules, absence of necrosis and
vacuoles (Fig. 5). Almost similar sign of protection was
shown in the liver sections of Liv.52 at a dose of 40 mg/kg
treated rats (Fig. 6).
DISCUSSION AND CONCLUSION
The present studies were performed to assess the
hepatoprotective activity of ethanol (50%) extract of
A.tetracantha root bark in rats against carbon tetrachloride
as hepatotoxin to prove its claims in folklore practice
against jaundice. It is well documented that carbon
tetrachloride-induced hepatic injury is commonly used as an
experimental method for the study of hepatoprotective
effects of drugs or medicinal plants’ extracts, by in vivo and
in vitro techniques (Kiso Y et al., 1983; Allis JW et al.,
1990; Cornelius CE, 1993). Carbon tetrachloride (CCl4) is a
potent hepatotoxin producing centrilobular hepatic necrosis.
It is accumulated in hepatic parenchyma cells and
metabolized to CCl3 by liver cytochrome P450-dependent
monooxygenases (Recknagel RO et al., 1989).
Usually, the extent of hepatic damage is assessed
by histopathological evaluation and the level of hepatic
enzymes ALT, AST and ALP release in circulation (Plaa G
and Charbonneau M, 1994). The administration of CCl4
resulted in a significant increase in the serum SGOT, SGPT,
alkaline phosphatase (ALP) and acid phosphatase (ACP)
and also total bilirubin within 36 hours (Wachstein M, 1959;
Max Wachstein et al., 1962). The rise in serum levels of
AST, ALT, ALP and ACP has been attributed to the
damaged structural integrity of the liver, because they are
cytoplasmic in location and released into circulation after
cellular damages (Sallie R et al., 1991).
In our study, the biochemical changes were
observed after 7 days. Thereby, it was found that, the
administration of EEAT root bark at doses of 40, 80 and
120mg/kg, p.o. These results indicating the production of
structural integrity of hepatocytic cell membrane or
regeneration of damaged liver cells by the extract. Whereas,
the extract at tested doses decreases the CCl4-induced
elevated level of hepatic enzymes in rats, and its subsequent
return towards near normalcy after 7days. Reduction in the
levels of SGOT and SGPT towards the normal value is an
indication of regeneration process. Reduction of ALP levels
with concurrent depletion of raised bilirubin level suggests
the stability of the biliary function during injury with CCl4.
Bilirubin is the conventional indicator of liver
diseases (Girish S et al., 2004). The rise in the levels of
serum bilirubin is the most sensitive and confirms the
intensity of jaundice (Cavin C et al., 2001). These
biochemical restorations may be due to the inhibitory effects
on cytochrome P450 or/and promotion of its
glucuronidation (Drotman RB and Lawhorn GT, 1978). The
marked elevation of bilirubin level in the serum of group II
CCl4 intoxicated rats were significantly decreased in the
groups III-V EEAT root bark treated animals after 7 days of
treatment, bilirubin level in the serum CCl4 intoxicated rats
subsequently return towards near normalcy in the groups
III-V EEAT root bark treated animals. These results further
substantiate A.tetracantha as a potent hepatoprotective
agent.
It has been reported that Liv.52 protects liver from
the hepatotoxicity of carbon tetrachloride (Karandikar SM et
al., 1963; Meena Kataria and Singh LN, 1997). An
appreciable protective effect was observed during 7 days
treatment using marketed product (Liv.52). The extent of
production by extracts appeared to depend on the duration
of treatment. Overall, these results suggest that the extract
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could protect the liver against damage induced by CCl 4
when comparable with Liv.52.
The attributivity of the observed alterations of
SGOT, SGPT, alkaline phosphatase (ALP) and acid
phosphatase (ACP), serum ALT were confirmed by
histopathological studies of liver sections which reveal that
the normal liver architecture was disturbed by hepatotoxin
(CCl4) intoxication. In the liver sections of the rats treated
with EEAT root bark extract for 7 days, the normal cellular
architecture was retained as compared to Liv.52, there by
further confirming the potent hepatoprotective effect of
A.tetracantha root bark.
Further research is needed to isolate and purify the
active principle involved in hepatoprotection of this plant as
well as to confirm the mechanisms responsible for
hepatoprotective activity. The present finding provides
scientific evidence to the ethnomedicinal use of Azima
tetracantha root bark in treating jaundice.
ACKNOWLEDGEMENT
The authors are thankful to Dr.P.Jayaraman, M.Sc.,
Ph.D, Plant Anatomy Research Centre (PARC), Chennai
and Dr.S.Subramaniam.M.B.BS, Histopathalogist, Vijay
Clinical Laboratory, Madurai, Tamil Nadu, India, for
valuable help in successful completion this research work.
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