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reason for a drug to be withdrawn from the market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.

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Harish Chandra et al. IJPER | Jan-Mar, 2014; 1(1): 9-15.

10

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.


11

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.


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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


14

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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