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A Saravanakumar et al/ J Biomed Sci and Res.

, Vol 1 (1), 2009, 19 - 26

Hepatoprotective potential of Cordia subcordata Lam. against carbon


tetra chloride (CCl4)-induced hepatotoxicity in Wistar albino rats.
A. Saravana Kumar*, R. Gandhimathi1, K.K. Senthil Kumar 2., Kusuma Praveen Kumar3
*1
Department of Pharmacology and Pharmaceutical Chemistry, Sree Vidyanikethan College of Pharmacy,
Sri Sainathnagar, Chandragiri (M), Tirupati, Andhra Pradesh, India-517102.
2
Department of Pharmaceutical science, St.Peter's College Of Pharmacy, Madikonda, Kazipet, Warangal
Dt, , Andhra Pradesh India-506 001.
3
Department of Pharmaceutical chemistry, Vaagdevi Institute of Pharmaceutical Sciences, Bollikunta,
Warangal Dt, Andhra Pradesh India-506 002.
_____________________________________________________________
ABSTRACT
Aim: To investigate the phytoconstituents, acute oral toxicity and hepatoprotective activity of ethanol
(90%) extract of Cordia subcordata Lam. (EECS) using CCl4 induced hepatotoxicity in male Wistar albino
rats.
Methods: The EECS at doses of 100, 200 and 400mg/kg, p.o and the standard drug Liv.52 (40mg/kg, p.o)
were administered 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 and urea along with histopathological studies of liver tissue. The biochemical
changes and histopathological studies were observed on 4th and 8th day.
Results: EECS at tested doses significantly decrease (P<0.001) the elevated levels of the hepatic enzymes,
total bilirubin and urea in a dose dependent manner after 3days whereas its subsequent return towards near
normal after 7days indicating the recovery of hepatic cells. In the liver sections of the rats treated with
EECS extracts for 7 days, the normal cellular architecture was retained as compared to Liv.52, thereby
furtherly confirming the potent hepatoprotective effect of EECS.
Conclusion: The EECS afforded significant protection against CCl4 induced hepatocellular injury.
KEY WORDS: Hepatotoxicity, CCl4, Hepatic Enzymes, Cordia subcordata Lam. Hepatoprotective,
EECS
________________________________________________________________________

INRODUCTION activity of Cordia subcordata Lam.


The liver regulates many important leaves against carbon tetrachloride-
metabolic functions, detoxification, and induced hepatic damage in albino rats.
secretory functions in the body. Hepatic
injury is associated with distortion of Cordia subcordata Lam. Family:
these metabolic functions [1]. Thus, liver Boraginaceae) is a medium-sized
diseases remain one of the serious health spreading tree to 12m tall with grayish
problems and its disorders are numerous grooved flaking bark. Leaves alternate,
with no effective remedies. Despite, petiolate, the petiole about half as long
considerable progress in the treatment of as blade, broadly ovate and entire, often
liver diseases by oral hepatoprotective wavy-margined, the apex obtuse to
agents, search for newer drugs continues short-pointed, base rounded, the blade
because the existing synthetic drugs up to 20 cm long. Flowers showy,
have several limitations [2-4]. So, the orange, trumpet -shaped, unscented and
search for new medicines is still borne in small axilary or terminal
ongoing. Because liver performs many clusters. Fruit a globose drupe up to 3
vital functions in the human body and cm long, surrounded by the enlarged
damage of liver causes unbearable calyx. Flowers and fruit usually
problems. [5, 6]. Keeping this fact in available throughout the year. The seeds
view, the present study was undertaken float and are highly resistant to salt
to investigate the hepatoprotective water, thus the species is common in

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A Saravanakumar et al/ J Biomed Sci and Res., Vol 1 (1), 2009, 19 - 26

coastal areas. In Tahiti, the leaves are Percentage yield of ethanolic extract of
used in remedies for bronchitis and Cordia subcordata was found to be 16.5
asthma where the leaves probably act as % w/w.
a purgative. The plant is also used in the Preliminary phytochemical screening
treatment of hepatic infections, cirrhosis The phytochemical examination of
of the liver and inflammation of the ethanolic (90%) extract of Cordia
lymph nodes. It is also used to treat subcordata Lam. leaves was performed
albumin present in the urine. Cook by the standard methods [10].
Islanders use the leaves in remedies for
abdominal swellings and urinary tract Animals used
infections [7-9]. Wistar albino rats (150-220g) of either
However, there are no ethnomedicinal sex were obtained from the animal house
information and scientific findings for in C.L. Baid Metha College of
the above said traditional claim for Pharmacy, Chennai. The animals were
hepatic disorders. Therefore, to justify maintained in a well-ventilated room
the traditional claims the present study with 12:12 hour light/dark cycle in
was undertaken to find out if ethanol polypropylene cages. The animals were
extract of Cordia subcordata Lam. fed with standard pellet feed (Hindustan
leaves demonstrates the hepatoprotective Lever Limited., Bangalore) and water
activity against CCl4-induced liver was given ad libitum. Ethical committee
damage in rats. Hence, the present study clearance was obtained from IAEC
was designed to verify the claims of the (Institutional Animal Ethics Committee)
native practitioners. of CPCSEA (Ref No. IAEC / XIII / 01 /
CLBMCP / 2008 - 2009).
MATERIALS AND METHODS
Plant collection Acute Toxicity Study
The Plant material of Cordia subcordata
Lam. leaves was collected from The acute toxicity of 90% ehanolic
Tirunelveli District, in the Month of extract of Cordia subcordata was
August 2008. The plant was determined as per the OECD guideline
authenticated by Dr.V.Chelladurai, no. 423 (Acute Toxic Class Method). It
Research Officer Botany. C.C.R.A.S., was observed that the test extract was
Govt. of India. The voucher specimen not mortal even at 2000mg/kg dose.
(CHE-SA-CS-08) of the plant was Hence, 1/20th (100mg/kg), 1/10th
deposited at the college for further (200mg/kg) and 1/5th (400mg/kg) of this
reference. dose were selected for further study [11].

Preparation of plant extract Carbon tetrachloride-induced


The leaves of Cordia subcordata Lam. hepatotoxicity in rats
were dried in shade, separated and made The liver protective effect was evaluated
to dry powder. It was then passed using the carbon tetrachloride (CCl4)
through the 40 mesh sieve. A weighed model described by Visweswaram et al.
quantity (80gm) of the powder was [12]. Wistar albino rats (150-220gm)
subjected to continuous hot extraction in were divided into six groups of six rats
Soxhlet Apparatus. The extract was each and were subjected to the following
evaporated under reduced pressure using treatments: Group-I served as normal
rotary evaporator until all the solvent has control received distilled water (1 ml/kg,
been removed to give an extract sample. p.o) for 7days. Group II -VI received

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0.75 ml/kg CCl4 administered orally as RESULTS


single dose. After 36 hours, Groups III-
The results of preliminary
VI received EECS with doses of 100,
phytochemical screening of the
200 and 400mg/kg, p.o and the standard
ethanoloic extract of Cordia subcordata
drug Liv.52 with dose of 40mg/kg, p.o,
Lam. revealed that presence of alkaloids,
respectively once daily for 7days. The
flavonoids, carbohydrates, glycosides,
blood was collected by puncturing the
tannins, terpeniods and absence of
retro-orbital sinus of three rats from each
saponins and steroids.
group on 4th day of treatment
and 8th day after the treatment Acute toxicity study
respectively. From the collected blood Acute toxicity study in which the
samples, serum was separated to assess animals treated with the EECS at a
various biochemical parameters. higher dose of 2000 mg/kg did not
manifest any significant abnormal signs,
Biochemical estimation behavioral changes, body weight
The separated serum was subjected to changes, or macroscopic findings at any
estimate SGOT and SGPT by Reitman time of observation. There was no
and Frankel method, alkaline mortality in the above-mentioned dose at
phosphatase (ALP) and acid phosphatase the end of the 14 days of observation.
(ACP) by Kind and King method, Effect of EECS on CCl4 induced
bilirubin by Malloy and Evelyn method hepatotoxicity
and urea by Bousquet method [13-16]. The results of EECS on carbon
The rats were then sacrificed by bleeding tetrachloride-induced hepatotoxicity
and the liver was carefully dissected, were represented in Table 1 and Table-
cleaned of extraneous tissue, and part of 2. The CCl4 only treated animals
the liver tissue was immediately exhibited a significant increase
processed for histopathological (P<0.001) the levels of SGOT, SGPT,
investigation. alkaline phosphatase (ALP) and acid
phosphatase (ACP) and also total
Histopathological studies bilirubin and urea when compared to the
The tissues of liver were fixed in 10% normal control group on both 4th and 8th
formalin and embedded in paraffin wax. day, indicating hepatocellular damage.
Sections of 4-5 microns thickness were The EECS at tested doses (group III-V)
made using rotary microtome and produced a significant reduction
stained with haematoxylin-eosin and (P<0.001) in the CCl4-induced elevated
histological observations were made levels of SGOT, SGPT, alkaline
under light microscope [17, 18]. phosphatase (ALP) and acid phosphatase
(ACP), also total bilirubin and urea
Statistical analysis when compared to the CCl4 only treated
The data were expressed as mean animals (group-II) after 3days of
standard error mean (S.E.M).The treatment and reduced furthermore to the
Significance of differences among the normalcy on 8th day although the lowest
group was assessed using one way and dose (100 mg/kg) tested could produced
multiple way analysis of variance significant reduction even after 3days of
(ANOVA). The test followed by treatment (Table 1). Overall, EECS at
Dunnetts test p values less than 0.05 tested doses significantly reduced the
were considered as significance. levels of hepatic enzymes, total bilirubin
and urea in a dose dependent manner.

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cells, sinusoidal spaces and central vein


After 7 days, the hepatic enzymes levels on both 4th and 8th day (Fig.1a and 1b).
were almost restored to the normal after
treating with EECS at the dose of Disarrangement of normal hepatic cells
400mg/kg, p.o. with centrilobular necrosis,
A standard drug, Liv.52 at a dose of 40 vacuolization of cytoplasm and fatty
mg/kg (group-VI) administered orally degeneration were observed (on both 4th
produced a significant reduction (p<0. and 8th day) in CCl4 intoxicated rats
001) compared to CCl4 only treated (Fig.2a and 2b).
animals (group-II) on both 4th and 8th The liver sections (on both 4th and 8th
day and these protective effects almost day) of the group-V rats treated with
close to EECS 400mg/kg, p.o. EECS (400mg/kg, p.o) showed a sign of
protection as it was evident by the
Effect of EECS on histolopathological moderate accumulation of fatty lobules,
change: absence of necrosis and vacuoles (Fig.
Histopathological examination of liver 3a and 3b). Almost similar sign of
sections of control group showed normal protection was shown in the liver
cellular architecture with distinct hepatic sections of Liv.52 at a dose of 40 mg/kg
treated rats (Fig. 4a and 4b).

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Fig. 1 (a): Normal control treated group Fig. 1(b): normal control treated group
on 4th day (100x) on 8th day (100x)

Fig. 2(a): CCl treated group on 4th day (100x) Fig. 2(b):CCl treated group on 8th day (100x)
4 4

Fig. 3 (a): CCl supplemented with EECS 400 Fig. 3 (b): CCl supplemented with EECS 400
4 4
treated group on 4th day (100x) treated group on 8th day (100x)

Fig. 4(a): CCl with Liv.52 (40mg/kg, p.o) Fig. 4(b): CCl with Liv.52 (40mg/kg, p.o)
4 4
treated group on 4th day (100x) treated group on 8th day (100x)

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DISCUSSION AND CONCLUSION membrane or regeneration of damaged


The present studies were performed to liver cells by the extracts. Whereas, the
assess the hepatoprotective activity of EECS extracts at tested doses decreases
ethanol (90%) extract of Cordia the CCl4-induced elevated level of
subcordata leaves in rats against carbon hepatic enzymes in rats, and its
tetrachloride as hepatotoxin to prove its subsequent return towards near normalcy
claims in folklore practice against liver after 7days. Reduction in the levels of
disorders. SGOT and SGPT towards the normal
It is well documented that carbon value is an indication of regeneration
tetrachloride-induced hepatic injury is process. Reduction of ALP levels with
commonly used as an experimental concurrent depletion of raised bilirubin
method for the study of hepatoprotective level suggests the stability of the biliary
effects of drugs or medicinal plants function during injury with CCl4.
extracts, by in vivo and in vitro Bilirubin is the conventional indicator of
techniques [19-21]. Carbon tetrachloride liver diseases [27]. The rise in the levels
(CCl4) is a potent hepatotoxin producing of serum bilirubin is the most sensitive
centrilobular hepatic necrosis. It is and confirms the intensity of jaundice
accumulated in hepatic parenchyma cells [28]. These biochemical restorations
and metabolized to CCl3 by liver may be due to the inhibitory effects on
cytochrome P450-dependent cytochrome P450 or/and promotion of
monooxygenases [22]. its glucuronidation [29]. The marked
Usually, the extent of hepatic damage is elevation of bilirubin and urea level in
assessed by histopathological evaluation the serum of group II CCl4 intoxicated
and the level of hepatic enzymes ALT, rats were significantly decreased in the
AST and ALP release in circulation [23]. groups III-V EECS treated animals after
The administration of CCl4 resulted in a 3days. Whereas, after 7 days of
significant increase in the serum SGOT, treatment, bilirubin and urea level in the
SGPT, alkaline phosphatase (ALP) and serum CCl4 intoxicated rats subsequently
acid phosphatase (ACP) and also total return towards near normalcy in the
bilirubin and urea within 36 hours [24, groups III-V EECS treated animals.
25]. The rise in serum levels of AST, These results further substantiate Cordia
ALT, ALP and ACP has been attributed subcordata as a potent hepatoprotective
to the damaged structural integrity of the agent.
liver, because they are cytoplasmic in It has been reported that Liv.52 protects
location and released into circulation liver from the hepatotoxicity of carbon
after cellular damages [26]. tetrachloride [30, 31]. An appreciable
In our study, the biochemical changes protective effect was observed even after
were observed after each 3 and 7 days. 3 days compared with 7 days treatment
Thereby, it was found that, the using marketed product (Liv.52). The
administration of EECS at doses of 100, extent of production by extracts
200 and 400mg/kg, p.o for 3 days appeared to depend on the duration of
resulted in significantly decreases the treatment. Overall, these results suggest
CCl4-induced elevated levels of the that the EECS could protect the liver
hepatic enzymes SGOT, SGPT, alkaline against damage induced by CCl4 when
phosphatase (ALP) and acid phosphatase comparable with Liv.52.
(ACP) in a dose dependent manner. The attributivity of the observed
These results indicating the production alterations of SGOT, SGPT, alkaline
of structural integrity of hepatocytic cell phosphatase (ALP) and acid phosphatase

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