STUDIES ON HEPATOPROTECTIVE EFFECT OF AQUEOUS EXTRACT OF ANNONA SQUAMOSA AND MURRAYA KOENIGII LEAVES ALONE AND INCOMBINATION ON CARBON TETRACHLORIDE INDUCED HEPATOTOXIC RAT MODEL
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Date
2016
Authors
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Journal ISSN
Volume Title
Publisher
AAU, Anand
Abstract
The liver is the main organ where exogenous chemicals are metabolized and
eventually excreted. As a consequence, liver cells are exposed to significant
concentrations of chemicals, which can result in liver dysfunction, cell injury and
even organ failure. The present study was conducted to evaluate hepatoprotective
effects of aqueous extracts of Annona squamosa and Murraya koenigii leaves alone
and incombination following repeated oral administration for 28 days in carbon
tetrachloride induced hepatotoxic rats. The study was conducted on seventy two (72)
male albino Wistar rats dividing them in various groups having six rats in each group.
Group I served as vehicle control and received the normal saline solution
orally for 28 days of dosing period. Group II served as hepatotoxic control and
received 50 % carbon tetrachloride in olive oil at the dose rate of 1 ml/kg body
weight, intraperitoneally twice in a week throughout the study period. Rats of group
III, IV, V, VI, VII, VIII, IX, X, XI and XII also received 50 % carbon tetrachloride in
olive oil at the dose rate of 1 ml/kg body weight, intraperitoneally, twice in a week
throughout the study period for induction of hepatotoxicity. Group III received
standard drug silymarin at dose rate of 50 mg/kg of body weight (p.o.) daily once for
28 days of dosing period. Group IV, V and VI received aqueous extracts of A.
“Studies on hepatoprotective effect of aqueous extract of Annona squamosa and
Murraya koenigii leaves alone and incombination on carbon tetrachloride-induced
hepatotoxic rat model”
squamosa at dose of 100, 200 and 400 mg/kg, group VII, VII and IX received
aqueous extracts of M. koenigii at dose of 100, 200 and 400 mg/kg and group X, XI
and XII received aqueous biherbal extracts at dose of 100, 200 and 400 mg/kg (p.o.)
respectively, daily once for 28 days of dosing period.
Animals were observed daily for clinical signs and mortality, if any. Body
weight and feed consumption were recorded at weekly interval. On 29th day of study,
animals were subjected to blood collection; blood and serum sample were analyzed
for haematological and biochemical parameters respectively. At the end of study
period, animals were sacrificed and necropsy was performed; tissues (liver, kidney,
spleen, heart and intestine) were collected for histopathological studies.
Hepatotoxic rat model was developed by intraperitoneal injection of 50 %
carbon tetrachloride in olive oil at dose rate of 1 ml/kg body weight twice in a week
throughout the study period (28 days). The result showed significant increased serum
concentration of ALT, AST, GGT, ALP, creatinine kinase, bilirubin, serum creatinine
(481.6 ± 2.91 U/l, 338.83 ± 9.13 U/l, 85.91 ± 3.57 U/l, 433.64 ± 4.48 U/l, 1036.33 ±
4.19 U/l, 1.51 ± 0.14 mg/dl and 1.29 ± 0.08 mg/dl, respectively) and significant
decreased albumin, globulin and total protein (3.58 ± 0.11 g/dl, 2.14 ± 0.13 g/dl and
5.72 ± 0.11 g/dl, respectively) in hepatotoxic control rats as compared to rats of
vehicle control group were serum concentration of ALT, AST, GGT, ALP, creatinine
kinase, bilirubin, serum creatinine (66.47 ± 1.88 U/l, 152.17 ± 3.13 U/l, 24.76 ± 1.41
U/l, 168.98 ± 5.69 U/l, 673.02 ± 6.63 U/l, 0.21 ± 0.07 mg/dl and 0.35 ± 0.03 mg/dl)
and albumin, globulin and total protein 4.47 ± 0.10 g/dl, 3.53 ± 0.14 g/dl and 8.00 ±
0.23 g/dl, respectively), also there were significant decrease in hematological
parameters like Hb, TEC, PCV, MCV, MCH, MCHC, WBCs/TLC and platelets (8.62
± 0.70 g/dl, 6.15 ± 0.24 × 106/μl, 26.57 ± 1.33 %, 29.77 ± 1.02 fl, 12.92 ± 0.36 pg,
“Studies on hepatoprotective effect of aqueous extract of Annona squamosa and
Murraya koenigii leaves alone and incombination on carbon tetrachloride-induced
hepatotoxic rat model”
27.61 ± 0.69 g/dl, 4.62 ± 0.41 × 10³/μl and 542.5 ± 4.88 × 10³/μl, respectively) in rats
of hepatotoxic control group as compared to vehicle control group whereas Hb, TEC,
PCV, MCV, MCH, MCHC, WBCs/TLC and platelets (15.56 ± 0.72 g/dl, 8.08 ± 0.37
× 106/μl, 43.87 ± 1.02 %, 54.69 ± 1.66 fl, 19.28 ± 0.36 pg, 42.35 ± 0.70 g/dl, 8.88 ±
0.27 × 10³/μl and 930.83 ± 3.61× 10³/μl, respectively) which were received normal
saline orally for 28 days and significant increase in liver: body weight ratio (48.29 ±
2.68 × 10-3) in hepatotoxic control group as compared to vehicle control group
whereas liver: body weight ratio was 34.92 ± 0.76 × 10-3. It suggests that carbon
tetrachloride is useful substance for successful induction of hepatotoxicity in rats.
In hepatotoxic control rats demonstrated clinical signs like dull, depressed and
anorectic changes along with reduced body weight gain and sluggishness in the last
week of experiment.
There was significant reduction in feed consumption in hepatotoxic control
rats as compared to vehicle control rats at 28th day of experiment. At the end of
experiment, hepatotoxic rats receiving aqueous extracts of Annona squamosa and
Murraya koenigii alone and incombination at doses of 100, 200 and 400 mg/kg and
silymarin at dose rate of 50 mg/kg body weight showed significance increase feed
consumption as compared to rats of hepatotoxic control group. Group of rats which
were administrered aqueous extracts of M. koenigii showed significant increase in
feed consumption during 2nd, 3rd and 4th week of experiment and also group of rats
which were administered aqueous extract of A. squamosa alone as well as biherbal
aqueous extracts of A. squamosa and M. koenigii showed significant increase in feed
consumption during entire experimental period.
Daily oral administration of standard reference compound silymarin at 50
mg/kg body weight significantly reduced serum ALT, AST, GGT, ALP, bilirubin,
“Studies on hepatoprotective effect of aqueous extract of Annona squamosa and
Murraya koenigii leaves alone and incombination on carbon tetrachloride-induced
hepatotoxic rat model”
creatinine kinase and serum creatinine (92.90 ± 1.45 U/l, 165.67 ± 1.48 U/l, 37.71 ±
1.76 U/l, 190.65 ± 4.83 U/l, 0.27 ± 0.03 mg/dl, 676.62 ± 6.58 U/l and 0.51 ± 0.02
mg/dl, respectively) and significant increase in serum albumin, globulin and total
protein level (4.44 ± 0.10 g/dl, 3.14 ± 0.06 g/dl and 7.59 ± 0.05 g/dl, respectively) as
compared to hepatotoxic control rats. Hepatotoxic rats receiving aqueous extracts of
Annona squamosa and Murraya koenigii alone and incombination at doses of 100,
200 and 400 mg/kg body weight for 28 days also showed significant reduction in
serum ALT, AST, GGT, ALP, bilirubin, creatinine kinase and serum creatinine and
significant increase in serum albumin, globulin and total protein level as compared to
rats of hepatotoxic control group in dose dependent manner except A. squamosa (100
mg/kg) and M. koenigii (100 mg/kg) in serum globulin level showed non significant
increase as compared to rats of hepatotoxic control group. However, above parameter
level did not reach the normal level in these rats. The group of rats given standard
reference compound silymarin at 50 mg/kg body weight was slightly more efficacious
than aqueous extracts of Annona squamosa and Murraya koenigii alone and
incombination.
Gross pathological examination of liver collected from rats of hepatotoxic
control group receiving 50 % carbon tetrachloride in olive oil at 1 ml/kg body weight
showed paleness and diffused necrotic foci as compared to liver of vehicle control
group.
Microscopically, group of hepatotoxic control rats receiving intra-peritoneal
injections of 50 % carbon tetrachloride in olive oil at dose rate of 1 ml/kg body weight
twice in a week throughout the study period, showed liver sections showed massive
changes throughout the lobules, with sinusoidal dilatation, cellular vacuolization,
necrosis, distortion of the central venules and ballooning of hepatocytes. Histological
“Studies on hepatoprotective effect of aqueous extract of Annona squamosa and
Murraya koenigii leaves alone and incombination on carbon tetrachloride-induced
hepatotoxic rat model”
examination of sections of kidney of rat from hepatotoxic control group showed
congestion with degeneration, necrosis of renal tubular epithelium and cloudy
swelling of tubular cells. On histopathological examination, section of spleen of
hepatotoxic control rats showed lesions mild congestion and haemorrhage with
multifocal area of necrosis and mild lymphoid depletion. Section of heart of
hepatotoxic rat showed severe congestion.
Treatment of hepatotoxic rats with aqueous extracts of Annona squamosa and
Murraya koenigii alone and incombination at doses of 100, 200 and 400 mg/kg body
weight preserved normal histoarchitecture in dose dependent manner and standard
treatment silymarin at dose rate of 50 mg/kg body weight almost preserved normal
histoarchitecture of all the organs as compared to rats of hepatotoxic control group.
The findings of present study suggest that aqueous extracts of Annona
squamosa and Murraya koenigii alone and incombination has hepatoprotective effect
at dose dependent manner. The findings also suggest the carbon tetrachloride induced
hepatotoxic model is useful for hepatotoxicity study. More studies in other laboratory
and domestic animals are required to establish safety of aqueous extracts of Annona
squamosa and Murraya koenigii alone and incombination and to elucidate mechanism
of hepatoprotective properties. Further investigation to define its clinical efficacy in
clinical cases of animals would be highly desirable.
Description
Keywords
veterinary science, pharmacology, toxicology, study