Comparative evaluation of Hepatoprotective activity in male albinorats
Transcript of Comparative evaluation of Hepatoprotective activity in male albinorats
9
International Journal of Pharmacy Education and Research
Jan-Mar 2014; 1(1): 9-15.
Available online: www.ijper.net
Research Article
Comparative evaluation of Hepatoprotective activity in male
albino rats
Sandiri RADHIKA1, Rodda HARISH CHANDRA*2
1Department of Pharmacology, Vaagdevi College of Pharmacy, Hanamkonda, Warangal – 506 001,
Andhra Pradesh, INDIA. 2Department of Pharmacognosy, Vaagdevi College of Pharmacy, Hanamkonda, Warangal - 506 001,
Andhra Pradesh, INDIA.
INTRODUCTION
Liver disease is still a worldwide health
problem[1]. The statistical data reveals that there are
more than 900 drugs which can cause liver injury.
Hepatotoxicity is considered to be the most common
reason for a drug to be withdrawn from the market
basing on the post market surveillance reports[2,3]. In
spite of the severity of hepatotoxicity there are no best
remedies for treating it. The efforts for the search of
safe and efficacious hepatoprotective agents is still
continuing. Herbal remedies have always been in the
forefront of treating liver disorders and Silymarin,
since its introduction in 1960 is considered as gold
standard for treating the liver disorders[4]. Several
plants have been used in the various alternative
systems of medicine for treating liver ailments such as
Andrographis paniculata, Silybum marianum,
Coccinia grandis, Annona squamosa, Ficus carica,
Lepidium sativum, Sargassum polycystum, Solanum
nigrum, Swertia chirata, Phyllanthus emblica,
Curcuma longa, Picrorrhiza kurroa, Azadirachta
indica, Aegle marmelos, Cassia roxburghii, etc.[5]. The
present study was aimed at carrying the comparative
hepatoprotective activity of Picrorrhiza kurroa,
Phyllantus amarus and Andrographis paniculata in
two different models of hepatotoxicity.
Picrorrhiza kurroa (Scrophulariaceae) is a
perennial herb, which is well distributed at high
altitudes (2700-4500m) of Himalayas and China. It is
also found in Punjab, Uttar Pradesh, Himachal
Received on: 21 March, 2014
Revised on: 25 March, 2014
Accepted on: 27 March, 2014
*Corresponding author:
R. Harish Chandra
Vaagdevi College of Pharmacy,
Ramnagar, Hanamkonda,
Warangal – 506 001,
Andhra Pradesh, INDIA.
Mobile #: +91-98495-11419
Email id: [email protected]
ABSTRACT
Phyllanthus amarus, Picrorrhiza kurroa and Andrographis
paniculata have prominent place in the management of liver
diseases. Effects of aqueous extracts of Phyllanthus amarus,
Picrorrhizha kurroa and ethanolic extracts of Andrographis
paniculata on the liver of paracetamol and carbon tetrachloride
(CCl4) induced hepatotoxic rats were studied. High levels of liver
enzymes, MDA and reduced levels of glutathione were observed in
paracetamol and CCl4 induced rats which were significantly reduced
after administration of Phyllanthus, Picrorrhiza and Andrographis
extracts at the concentration of 250mg/kg b.w. Of the three plants
studied in two different experimental models Picrorrhiza kurroa
exhibited prominent activity than the other two plants.
Key words: Phyllanthus amarus, Andrographis paniculata,
Picrorrhiza kurroa, Hepatotoxicity, paracetamol, carbon
tetrachloride.
Harish Chandra et al. IJPER | Jan-Mar, 2014; 1(1): 9-15.
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Kashmir and Sikkim. It is a popular herb in the
Ayurveda and it is used in the treatment of liver
disorders[6]. It is one the main ingredient of various
herbal preparations used for liver ailments. It contains
60% of 1:1.5 mixture of picroside I and kutkoside,
which contributes to the hepatoprotective activity[7].
Picroliv is a purified iridoid glycoside obtained from
the roots of Picrorhiza kurroa is reported to have
hepatoprotective, anti-inflammatory and antioxidant
properties[8-10]. Picrorrhiza is also reported to exhibit
anticancer and antiviral activity[11,12].
Phyllanthus amarus (Euphorbiaceae) is
found in India, mainly in the states of Maharashtra,
Uttar Pradesh, Andhra Pradesh and Madhya Pradesh.
It contains, leucodelphinidin alkaloids, flavonoids
such as quercetin, astralgin, quercitrin, isoquercitin,
rutin, hypophyllanthine, and phyllanthine[13]. This
plant has prominent use in the treatment of viral
hepatitis and other liver disorders. It also used as
diuretic in oedema and externally it is used to relieve
inflammation[14]. It is also used as good appetizer[15].
Andrographis paniculata (Acanthaceae) is
native to India and Sri Lanka. It is an annual erect plant
with lanceolate leaves. It grows to a height of 60-
70cm. It is considered as “King of Bitters”. Leaves
and aerial parts of the plant have been used in
treatment of liver disorders in various alternative
systems of medicine[16]. Andrographolide is the main
active principle of the plant. In addition it also
contains 14-deoxy-11,12-didehydroandrographolide
and 14-deoxyandrographolide. Andrographis is
reported to exhibit hepatoprotective, hypotensive,
antiangiogenic and antihyperglycemic activities[17-20].
MATERIALS AND METHODS
PLANT MATERIAL
The Andrographis paniculata, Picrorrhiza
kurroa, Phyllanthus amarus powders were purchased
from the local retailer, Maharshi Ayurveda,
Hanamkonda, Warangal (AP), India.
PREPARATION OF EXTRACTS
The rhizome powder of Picrorrhiza was
serially extracted with methanol and water using a
Soxhlet apparatus in a ratio of 1:6. The extract
obtained was evaporated to dryness and stored in a
vacuum desiccator at room temperature until further
use[21].
A decoction of Phyllanthus amarus was
prepared by boiling in water (1:4, w/v) till the water
level reduced to half. The extract obtained was filtered
through fine muslin cloth. The extract was refrigerated
at 4˚C until use[22].
The coarse powder of Andrographis
paniculata was extracted with ethanol:water (1:1) by
using soxhlet apparatus. The solvent were removed
under reduced pressure to get semisolid mass and the
obtained extract was stored in desiccator until further
use[23].
ANIMAL EXPERIMENTATION
Healthy Albino rats, weighing between 150-
200g of male were used for the study. The rats were
acclimatized to laboratory conditions and fed with
pellet food and given tap water ad libitum. They were
housed in standard metal cages. The study protocol
was approved by Institutional Animal Ethical
Committee of Vaagdevi College of Pharmacy,
Hanamkonda, Warangal (AP), India. Register No.
(IAEC NO: 1047/ac/07/CPCSEA).
STUDY DESIGN
Paracetamol induced hepatotoxic model
Five groups with 6 rats in each were chosen.
The period of study was for 8 days. All treatments
were administered orally by means of oral gavage.
Group-I (Normal Control) was administered with
5ml/kg distilled water p.o. daily for 7 days. Group-II
(Toxic Control) was administered with Paracetamol
250mg/kg b.w. Group-III (Standard) was administered
with Paracetamol 250mg/kg b.w. with simultaneous
administration of silymarin 100 mg/kg. Group-IV was
administered with Paracetamol 250mg/kg b.w. with
simultaneous administration of Picrorrhiza kurroa
250mg/kg. Group-V was administered with
Paracetamol 250mg/kg b.w. with simultaneous
administration Phyllanthus amarus 250 mg/kg.
Group-VI was administered with Paracetamol
250mg/kg b.w. with simultaneous administration
Andrographis paniculata 250mg/kg. On day 8, all
animals were sacrificed for experimental studies.
CCl4 induced hepatotoxic model
Five groups with 6 rats in each were chosen.
The period of study was for 8 days. All plant extracts
were administered orally by means of oral gavage.
CCL4 was administered intraperitoneally (i.p.).
Group-I received 0.5ml olive oil /kg body wt., ip.
Group-II (Toxic control) received 1ml CCl4/kg b.w,
i.p. suspended in olive oil (1:1). Group-III (Standard)
was administered with 1ml CCl4/kg b.w., i.p. with
simultaneous administration of silymarin 100mg/kg,
p.o. Group-IV was administered with 1ml CCl4/kg
b.w., i.p. with simultaneous administration of
Picrorrhiza kurroa 250mg/kg. Group-V was
administered with 1ml CCl4/kg b.w., i.p. with
simultaneous administration of Phyllanthus amarus
250mg/kg., p.o. Group-VI was administered with 1ml
CCl4/kg b.w., i.p. with simultaneous administration of
Andrographis paniculata 250mg/kg. On day 8, all
animals were sacrificed for experimental studies.
Harish Chandra et al. IJPER | Jan-Mar, 2014; 1(1): 9-15.
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BIOCHEMICAL STUDIES
On the 7th day rats were subjected to
overnight fasting and on day 8 all the animals were
anesthetized using ether. Blood samples were
withdrawn by puncturing retro-orbital plexus. The
serum was separated by centrifugation at 7000rpm for
15 min at 5°C. The separated serum samples were
used for biochemical estimations. The rats were
sacrificed with over dose of inhalant anaesthesia,
livers were isolated and kept in ice-cold saline. The
liver homogenates were prepared for biochemical
analysis and liver tissues were stored in 10% formalin
and evaluated for histopathological changes.
SERUM ENZYMES
The estimation of the biochemical markers
Aspartate Transaminase (AST), Alanine
Transaminase (ALT), Alkaline Phosphatase (ALP)
and Bilirubin were carried out on Turbo Chem 100
auto analyser using commercially available kits, at
Vaagdevi College of Pharmacy, Warangal (AP), India.
ASSESSMENT OF OXIDATIVE STRESS MARKERS
Malondialdehyde (MDA) levels
The amount of lipid peroxides levels in serum
samples were estimated by the Thiobarbituric acid
reactive substances (TBARS) method, which
measures the malondialdehyde (MDA) reactive
products by using colorimetry[24].
To 0.5 ml of liver homogenate, 0.5 ml of 30%
trichloro acetic acid (TCA) was added to precipitate
the proteins and vortexed for 30 sec. Clear supernatant
was taken after centrifuging at 3000rpm for 10 min.
To the supernatant, 500l of 1%TBA solution and
500l of water was added and this solution was heated
for 1 h at 98C. Cool the solutions to room
temperature and kept them in ice for 5 min. Then read
the pink colour obtained was measured at 532nm using
spectrophotometer.
Glutathione Levels
Glutathione forms a colored complex with
DTNB, which is measured spectrophotometrically[25].
To 0.5ml of liver homogenate, 0.5ml of 5% TCA
solution was added to precipitate the proteins and
centrifuged at 3000rpm for 20 min. To 0.1ml of
supernatant, 1.0ml of sodium phosphate buffer and
0.5ml of DTNB reagent was added. The absorbance
of the yellow color developed was measured at 412nm.
The glutathione content was determined from the
standard graph by using pure glutathione.
Histopathology
The liver tissues were treated with 10%
formalin, paraffin embedded and 5µM thickness
sections were prepared. The sections were stained
with heamotoxylin and eosin. The stained sections
were examined under binocular microscope for the
pathological changes.
RESULTS AND DISCUSSION
PARACETAMOL INDUCED HEPATOTOXIC MODEL
The effect of ethanolic extract of
Andrographis paniculata aqueous extract of
Phyllanthus amarus and methanolic extract of
Picrorhiza kurroa on paracetamol induced
hepatotoxicity rats shown in Table 1 & 2. The data
reveals that Picrorhiza kurroa extract has decreased
SGOT, SGPT, ALP, total bilirubin and total protein by
2.35, 2.39, 1.64 and 1.91 fold when compared to
control. The MDA levels decreased by 2.49 fold when
compared to control. The GSH levels increased by
1.80 level when compared to control. Andrographis
paniculata extract has decreased SGOT, SGPT, ALP,
and total bilirubin by 2.06, 2.07, 1.39 and 1.78 fold
when compared to control. The MDA levels decreased
by 2.78 fold when compared to control. The GSH
levels increased by 1.71 level when compared to
control. Phyllanthus amarus extract has decreased
SGOT, SGPT, ALP, and total bilirubin by 1.73, 1.77,
1.44 and 1.70 fold when compared to control. The
MDA levels decreased by 2.69 fold when compared to
control. The GSH levels increased by 1.51 level when
compared to control.
CCL4 INDUCED HEPATOTOXIC MODEL
The effect of ethanolic extract of
Andrographis paniculata aqueous extract of
Phyllanthus amarus and methanolic extract of
Picrorhiza Kurroa on CCl4 induced hepatotoxicity
rats shown in Table 3 & 4. The data reveals that
Picrorhiza kurroa extract has decreased AST, ASP,
ALP and total bilirubin by 1.87, 1.82, 1.48 and 1.63
fold when compared to control. The MDA levels
decreased by 2.39 fold when compared to control. The
GSH levels increased by 1.87 fold when compared to
control. Phyllanthus amarus extract has decreased
SGOT, SGPT, ALP, and total bilirubin by 1.63, 1.60,
1.39 and 1.53 fold when compared to control. The
MDA levels decreased by 2.29 fold when compared to
control. The GSH levels increased by 1.69 fold when
compared to control. Andrographis paniculata extract
has decreased SGOT, SGPT, ALP, and total bilirubin
by 1.38, 1.36, 1.35 and 1.43 fold when compared to
control. The MDA levels decreased by 2.18 fold
when compared to control. The GSH levels increased
by 1.54 fold when compared to control.
HISTOPATHOLOGY
The histopathological changes in various
groups of rats are shown in Fig. 1 & 2. The sections
reveal that out of the three extracts studied the
Picrorrhiza kurroa extract has produced remarkable
recovery from hepatotoxicity when compared to other
extracts.
Harish Chandra et al. IJPER | Jan-Mar, 2014; 1(1): 9-15.
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Table 1: Assessment of liver damage in Paracetamol induced hepatotoxicity
Treatment Dose
(mg/kg)
SGOT
(IU/L)
SGPT
(IU/L)
ALP
(U/L)
Total Bilirubin
(mg/dl)
Control (Distilled water) - 25.46±1.43 26.65±1.03 140±2.19 1.50±0.27
Toxic control (Paracetamol) 250 128.91±5.75 130±1.63 300.93±2.24 3.74±0.38
Standard (Silymarin) 100 36.57±0.84# 36.35±1.92# 155.5±9.63# 1.75±0.12#
Paracetamol +
Picrorhiza kurroa
250 54.79±0.81# 54.38±1.02# 182.58±1.23# 1.95±0.09#
Paracetamol +
Phyllanthus amarus
250 62.49±1.00# 62.72±2.33# 214.30±2.81* 2.10±0.20#
Paracetamol +
Andrographis paniculata
250 74.30±1.46# 73.3±1.60# 208.12±3.13* 2.19±0.35*
Values were expressed as mean ± SD (n=6) p-value *p<0.01, #p<0.001 significant.
Comparisons were with Toxic Control group vs. Treated group
Table 2: Comparative oxidative stress in Paracetamol induced hepatotoxicity
Treatment Dose
(mg/kg)
MDA
(nmol/g) % of Total GSH level
Control (Distilled water) - 5.69±0.85 28.59±1.11
Toxic control (Paracetamol) 250 19.12±0.93 19.96±4.51
Standard (Silymarin) 100 6.18±0.71# 39.96±4.51#
Paracetamol + Picrorhiza kurroa 250 6.49±0.72# 36.01±0.76#
Paracetamol + Phyllanthus amarus 250 6.87±0.76# 34.21±5.03#
Paracetamol + Andrographis paniculata 250 7.10±1.25# 30.01±4.21#
Values were expressed as mean ± SD (n=6) p-value #p<0.001 significant.
Comparisons were with Toxic Control group vs. Treated group.
Table 3: Assessment of liver damage in Paracetamol induced hepatotoxicity
Treatment Dose
(mg/kg)
SGOT
(IU/L)
SGPT
(IU/L)
ALP
(U/L)
Total
Bilirubin
(mg/dl)
Control (Distilled water) - 25.93±0.54 26.63±2.93 141.0±1.54 1.58±0.32
Toxic control (CCl4) 250 105.91±0.91 106.5±2.30 293.1±14.40 3.22±0.34
Standard (Silymarin) 100 42.2±0.31# 43.96±2.30# 166.8±12.30# 1.78±0.27#
CCl4 + Picrorhiza kurroa 250 56.37±2.20# 58.45±1.04# 197.7±9.96# 1.97±0.34#
CCl4 +Phyllanthus amarus 250 64.7±2.42# 66.28±1.43# 210.7±19.22* 2.10±0.20#
CCl4 + Andrographis paniculata 250 76.38±1.69# 77.83±0.64# 216.6±18.12* 2.25±0.07*
Values were expressed as mean ± SD (n=6) p-value *p<0.01, #p<0.001 significant.
Comparisons were with the Toxic Control group vs. Treated group.
Table 4: Comparative oxidative stress in Carbon tetrachloride (CCl4) induced hepatotoxicity
Treatment Dose
(mg/kg)
MDA
(nmol/g)
% of Total GSH
level
Control (Distilled water) - 5.72±1.12 28.59±1.23
Toxic control (CCl4) 250 15.37±1.39 20.23±1.34
Standard (Silymarin) 100 6.07±0.94# 41.23±4.50 #
CCl4 + Picrorhiza kurroa 250 6.41±0.71# 38.01±0.98#
CCl4 + Phyllanthus amarus 250 6.70±0.98# 34.21±0.56#
CCl4 + Andrographis paniculata 250 7.03±0.85# 31.34±0.45#
Values were expressed as mean ± SD (n=6) p-value *p<0.05, @p<0.01, #p<0.001 significant.
Comparisons were with Toxic Control group vs. Treated group.
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Fig 1: Histopathological sections of livers in Paracetamol induced hepatotoxic rats
Fig 2: Histopathological sections of livers in Carbon tetrachloride induced hepatotoxic rats
Harish Chandra et al. IJPER | Jan-Mar, 2014; 1(1): 9-15.
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CONCLUSION
The results of this study demonstrate that out
of the three extracts studied Picrorrhiza kurroa has
potent hepatoprotective activity action in both
paracetamol and carbon tetrachloride induced
hepatotoxic models.
ACKNOWLEDGEMENTS
The authors wish to thank Principal and
Management of Vaagdevi College of Pharmacy,
Ramnagar, Hanamkonda, Warangal for providing the
necessary facilities to carry out this work.
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