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Ethnobotanical
Leaflets 13: 310-315. 2009.
Hepatoprotective Effect of Rheum emodi Roots (Revand chini) and Akseer-e-Jigar Against Paracetamol-induced
Hepatotoxicity in Rats
M. S. Akhtar1*, M.
Amin2, Maqsood Ahmad1 and
Alamgeer1
1Department of Pharmacy, University of Sargodha,
Sargodha-40100, Pakistan
2Department
of Physiology and Pharmacology University
of Agriculture, Faisalabad
Pakistan
*Address for Correspondence: Dr
Muhammad Shoaib Akhtar,
Ph.D. (Munich), Professor of Pharmacology, Department of Pharmacy, faculty of
Science & Technology, University of Sargodha, Sargodha, Pakistan.
Email:
drmsakhtar@gmail.com
Issued 15 February 2009
Abstract
Hepatoprotective effects of Rheum emodi
roots and their aqueous and methanolic extracts
were studied against liver damage induced by paracetamol
in albino rats. In addition, the effects of herbal preparation, Akseer-e-Jigar
and a control drug, silymarin were also studied.
Pretreatment and post-treatment hepatoprotective
effects of all these drugs were determined. The prevention of liver damage
and curative effects of the drugs were judged by changes in serum ALT,
AST, ALP, albumin and bilirubin (total and direct)
levels. Powdered Rheum emodi roots (1 and
1.5 g/kg) and their aqueous extract did not significantly affect serum
enzymes, albumin and bilirubin levels. However,
treatment with powder (2 g/kg), methanolic
extract (0.6 g/kg), Akseer-e-Jigar
(1 g/kg) and silymarin (50 mg/kg) in both pre and
post-treatment studies significantly prevented the paracetamol-induced
rise of serum enzymes and bilirubin levels whereas
serum albumin was raised after treatment with these drugs. It is conceivable,
therefore, that Rheum emodi roots and Akseer-e-Jigar possess hepatoprotective
principles that can prevent and/or treat liver damage due to paracetamol. The study has supported empirical use of the
plant and its compound preparation used in traditional medicine.
Introduction
Liver
diseases have become one of the major causes of morbidity and mortality in
man and animals all over globe and hepatotoxicity
due to drugs appears to be the most common contributing factor [Nadeem, 1997]. The use of natural
remedies for the treatment of liver diseases has a long history and medicinal
plants and their derivatives are still used all over the world in one form or
the other for this purpose. Scientific evaluation of plants has often shown
that active principles in these are responsible for therapeutic success. A
large number of medicinal plants have been tested and found to contain active
principles with curative properties against a variety of diseases [Lewis, 1977]. Recent experience has
shown that plant drugs are relatively non-toxic, safe and even free from
serious side effects [Momin, 1987].
Rheum emodi
root has been claimed to possess purgative, anthelmintic
and hepatoprotective actions [Saeed,1970]
and its tuber as bitter, laxative, appetite stimulant, diuretic,
anti-bilious, and curative for sore eyes and bruises [Alam, 2005 ]. It has also been
reported to have antifungal [Agarwal, 200]
and hypoglycaemic properties [Li J, 1997]. Although Rheum emodi root is being commonly used
in traditional medicine as hepatoprotective yet its
efficacy has not been well documented. Therefore, a study was conducted to
investigate hepatoprotective effect of crude drug
as well as aqueous and methanolic extracts of Rheum emodi
roots and a popular compound preparation containing the roots against paracetamol-induced liver damage in albino rats.
Materials
and Methods
Plant Material
Dried Rheum emodi
roots locally called “Revand chini” were purchased from local herbal market. R. emodi roots
were identified and authenticated by Department of Botany, University
of Agriculture, Faisalabad. The dried roots were
powdered finely with herbal grinder and stored in well-closed cellophane bags
at 4 °C temperature.
Chemicals and Drugs
Paracetamol and methanol were obtained from GSK Pakistan
Ltd., Karachi and Merck,
Germany, respectively. Silymarin was that of Abbott Laboratories, Karachi.
The diagnostic kits for the estimation of ALT,
AST, ALP, albumin and bilirubin were that of Breuer
& Breuer, Germany;
Merck, Germany;
Medi, Italy;
Giesse, Italy;
Human, Germany,
respectively. Akseer-e-Jigar
was supplied by Al-Mansoor Clinics, Medina
Town, Faisalabad which consists of R. emodi
roots, ammonium chloride and potassium nitrate.
Experimental Animals
Healthy adult male albino Wister rats weighing 200-300 g were
procured from National Institute of Health, Islamabad.
Three animals per cage were housed in metal cages and free access to standard
feed and tap water was provided ad libitum. The animals were kept under observation for
one week before experimentation under usual managemental
conditions in the animal room. The animals were kept under stander laboratory
conditions and the study protocol was approved by local ethical committee.
Induction of hepatic toxicity
Hepatic toxicity in rats was induced by oral administration of a single
dose of paracetamol (640 mg/kg) using 1%
methylcellulose (13 ml/kg) solution as vehicle. The test drugs were administered to the treatment
groups orally, using normal saline (10 ml/kg) as vehicle. The control animals
received equal volumes of the vehicle.
Pretreatment Hepatoprotective
Effects
Animals were randomly divided into 9 groups of six
animals each. Group , serving as control, received 4 doses of normal saline
(10 ml/kg) at 12 hrs intervals; 1 hr after the last saline dose, 1%
methylcellulose solution (13 ml/kg) was administered. Group
received 4 doses of normal saline (10 ml/kg) at 12 hrs intervals; 1 hr after
the last saline dose, paracetamol (640 mg/kg) was
administered. Group received 4 doses of powdered Rheum emodi
roots (1 g/kg) at 12 hrs intervals; 1 hr after the last dose, paracetamol (640 mg/kg) was administered. Group received 4 doses of powdered Rheum emodi roots
(1.5 g/kg) at 12 hrs intervals; 1 hr after the last dose, paracetamol
(640 mg/kg) was administered. Group received 4 doses of powdered Rheum emodi roots (2 g/kg) at 12 hrs
intervals; 1 hr after the last dose, paracetamol
(640 mg/kg) was administered. Groups , VII, VIII & IX were treated
similar to the group V except that they received methanolic
extract of Rheum emodi
root (0.6 g/kg), aqueous extract of Rheum
emodi roots (eq. to 2
g/kg), Akseer-e-Jigar (1 g/kg) and Silymarin (50 mg/kg), respectively.
Post-treatment Hepatoprotective
Effects of Test Drugs
Animals were randomly divided into 9
groups of six animals each. Group , serving as control, was
administered the vehicle (1% methylcellulose orally; 13 ml/kg) at 0 hr and 4
doses of normal saline (10 ml/kg) at 6 hours intervals. Group
received paracetamol (640 mg/kg) at 0 hr and 4
doses of normal saline (10 ml/kg) at 6 hrs intervals, Group
received paracetamol (640
mg/kg) at 0 hr and 4 doses of powdered
Rheum emodi
roots (1 g/kg) at 6 hrs intervals, Group was given paracetamol
(640 mg/kg) at 0 hr and 4 doses of
powdered Rheum emodi
roots (1.5 mg/kg) at 6 hrs intervals, Group received paracetamol
(640 mg/kg) at 0 hr and 4 doses of powdered Rheum emodi roots (2 g/kg) at 6 hrs intervals, Groups , VII, VIII & IX were treated
similar to the group V except that they received methanolic
extract of Rheum emodi
roots (0.6 g/kg), aqueous extract of Rheum
emodi roots (eq. to 2
g/kg), Akseer-e-Jigar (1 g/kg) and Silymarin (50 mg/kg), respectively.
Preparation of Methanolic and Aqueous Extracts of Roots:
Methanolic Extract
Methanolic extract was prepared by continuous
extraction technique using Soxhlet’s apparatus.
Slow heating at 40 °C and continuous stirring evaporated the methanolic extract. The process of evaporation was
continued till complete evaporation of methanol was ensured. The dried
extract was dissolved in 1% methylcellulose sol. just before administration
to rats. The methanolic extract eq.
to 2 g/kg b.w. of powdered R. emodi roots i.e. 0.6 g/kg was
administered orally.
Aqueous Extract
A weighed amount of powdered roots was macerated in distilled water at
room temperature and agitated occasionally for one day [Osol,1975]. Filtered through a fine filter paper and
marc was pressed to avoid loss. The extract obtained was in liquid form at
room temperature. The extract was stored in a sterilized PVC bottle at 4 °C.
The extract was used in liquid form by calculating the volume eq. to the extract from 2 g of powdered Rheum emodi roots.
Collection of Blood Samples
Blood samples were collected 24 hrs after the last treatment. Animals
were anaesthetized with ether, and put in a desiccator.
Up to 5-8 ml of blood from each rat was collected by cutting the carotid artery
in centrifuge tubes. These blood samples were kept at 4°C for a period of 12
hrs. Serum was separated by centrifugation (3000 r.p.m.,
for 15 min.) The serum thus obtained was used for determination of levels of ALT,
AST, ALP, albumin and bilirubin.
Determination
of Serum Enzymes, Albumin and Bilirubin
Serum samples were analyzed by following kinetic methods using B.T.S.
330 (Biosystems, Spain).
Diagnostic kits have been used in the above mentioned semi-automated analyzer
for the quantitative estimation of ALT
(SGPT), AST (SGOT) ALP, albumin and bilirubin.
Statistical Analysis
The data obtained were analyzed by using one way analysis of variance
technique. The means were compared for significance by using Least
Significance Difference (LSD) test (Steel and Torrie,
1980).
Results
Pretreatment Hepatoprotective
Effects
Paracetamol-treated
group had ALT level which was
significantly higher than control (Table 1). The powdered R. emodi
roots (2g/kg), methanolic extract, Akseer-e-Jigar and silymarin
pretreatment inhibited the rise of serum ALT,
AST, ALP and bilirubin levels effectively as their
values were non-significantly different from control whereas level of albumin
was decreased. The lower doses of R. emodi roots and their aqueous extract did not
significantly affect the paracetamol-induced rise
of serum enzymes and bilirubin whereas the decline
in albumin level was also not prevented (Table 1).
Table
1. Pretreatment hepatoprotective effects of powdered Rheum emodi roots, methanolic
extract, aqueous extract, Akseer-e-Jigar and Silymarin on paracetamol-induced rise of ALT, AST, ALP, albumin and bilirubin
(total and direct).
|
Group
|
Treatments
|
ALT
(U/L)
|
AST
(U/L)
|
ALP
(U/L)
|
Albumin
(g/dl)
|
Bilirubin
Tot. (g/dl)
|
Bilirubin
Dir. (g/dl)
|
|
1
|
Saline
+ Vehicle
|
36.33
± 2.03 D
|
49.83
± 2.73 F
|
129.2
±
11.7 G
|
3.50
±
0.10 A
|
0.43
±
0.07 E
|
0.25
±
0.04 E
|
|
2
|
Saline+
Paracetamol
|
693.7
± 30.0 A
|
921.7
± 23.6 A
|
369.5
± 15.8 A
|
2.33
± 0.13 E
|
4.70
± 0.11 A
|
3.26
± 0.10 A
|
|
3
|
R. emodi
roots (1 g/kg) + Paracetamol
|
527.8
± 25.2 B
|
608.5
± 44.3 B
|
312.2
± 15.6 B
|
2.78
± 0.06 D
|
3.60
± 0.24 B
|
2.41
± 0.18 B
|
|
4
|
R. emodi roots (1.5g/kg) + Paracetamol
|
194.7
± 23.9 C
|
277.0
± 31.0 CD
|
223.5
± 15.3 CD
|
2.85
± 0.08 CD
|
1.71
± 0.16 C
|
1.15
± 0.09 C
|
|
5
|
R. emodi
roots (2 g/kg) + Paracetamol
|
52.67
± 2.91 D
|
111.0
± 15.1 FG
|
138.5
± 15.2 FG
|
3.28
± 0.14 AB
|
1.11
± 0.11 D
|
0.61
± 0.08 D
|
|
6
|
Methanolic Extract + Paracetamol
|
42.17
± 2.10 D
|
62.50
±4.15 EFG
|
165.8
± 12.7 EFG
|
3.25
± 0.08 AB
|
0.41
± 0.05 E
|
0.28
± 0.04 E
|
|
7
|
Aqueous
Extract + Paracetamol
|
66.17
± 4.61 D
|
172.7
± 12.2 C
|
227.8
± 18.7 C
|
3.15
± 0.14 B
|
1.03
± 0.07 D
|
0.60
± 0.03 D
|
|
8
|
Akseer-e-Jigar
+ Paracetamol
|
54.67
± 5.14 D
|
76.67
± 8.57 DE
|
186.0
± 5.21 DE
|
3.06
± 0.05 BCD
|
0.51
± 0.10 E
|
0.35
± 0.07 DE
|
|
9
|
Silymarin + Paracetamol
|
48.50
± 3.75 D
|
81.00
± 6.94 EF
|
169.8
± 9.65 EF
|
3.13
± 0.06 BC
|
0.51
± 0.08 E
|
0.31
± 0.07 E
|
Means sharing similar letters are statistically
non-significant (P > 0.05), n=6
Post-treatment Hepatoprotective
Effects
Table 2 shows that
treatment with test substances significantly lowered paracetamol-induced
rise of ALT level except the lower doses of R. emodi roots.
The effect was highest with methanolic extract
followed by Akseer-e-Jigar
and silymarin, respectively. The test drugs also
lowered serum AST, ALP and bilirubin levels in paracetamol treated rats (Table 2). The maximum decrease
in the levels of AST and bilirubin were resulted
from methanolic extract while maximum depression in
ALP level was caused by Akseer-e-Jigar. The methanolic
extract proved to be most efficient in increasing the decreased level of
albumin in paracetamol treated rats (Table 2).
Table 2. Post-treatment hepatoprotective
effects of powdered Rheum emodi roots, methanolic
extract, aqueous extract, Akseer-e-Jigar and Silymarin on paracetamol-induced rise of ALT, AST, ALP, albumin, bilirubin
(total and direct).
|
Group
|
Treatment
|
ALT
(U/L)
|
AST
(U/L)
|
ALP
(U/L)
|
Albumin
(g/dl)
|
Bilirubin
Tot. (g/dl)
|
Bilirubin
Dir. (g/dl)
|
|
1
|
Saline
+ Vehicle
|
43.67
±
2.76 E
|
57.67
±
2.54 F
|
141.0
±
9.04 EF
|
3.43
±
0.11 A
|
0.31
±
0.07 G
|
0.21
±
0.04 F
|
|
2
|
Saline+
Paracetamol
|
690.5
± 30.2 A
|
1027.5
± 30.2 A
|
385.5
± 10.0 A
|
2.08
± 0.08 F
|
5.11
± 0.12 A
|
3.43
± 0.11 A
|
|
3
|
R. emodi
roots (1 g/kg) + Paracetamol
|
567.0
± 31.3 B
|
812.8
± 29.6 B
|
312.5
± 14.0 B
|
2.56
± 0.12 E
|
4.36
± 0.21 B
|
2.90
± 0.22 B
|
|
4
|
R. emodi roots (1.5g/kg) + Paracetamol
|
220.5
± 16.6 C
|
354.8
± 26.4 C
|
236.0
± 10.5 C
|
2.85
± 0.08 D
|
2.81
± 0.18 C
|
1.93
± 0.12 C
|
|
5
|
R. emodi
roots (2 g/kg) + Paracetamol
|
66.83
± 5.18 DE
|
136.0
± 17.6 DE
|
173.2
± 17.9 DE
|
3.10
± 0.07 BC
|
0.98
± 0.10 DE
|
0.60
± 0.09 DE
|
|
6
|
Methanolic Extract + Paracetamol
|
51.67
± 2.44 DE
|
74.50
± 3.92 F
|
153.3
±13.6 EF
|
3.26
± 0.09 AB
|
0.58
± 0.07 FG
|
0.33
± 0.06 EF
|
|
7
|
Aqueous
Extract + Paracetamol
|
89.00
± 4.44 D
|
183.3
± 14.1 D
|
204.2
± 18.5 CD
|
2.98
± 0.07 CD
|
1.20
± 0.03 D
|
0.66
± 0.03 D
|
|
8
|
Akseer-e-Jigar + Paracetamol
|
57.50
± 4.99 DE
|
93.83
± 5.76 EF
|
130.0
± 6.89 F
|
3.10
± 0.03 BC
|
0.78
± 0.10 EF
|
0.51
± 0.07 DEF
|
|
9
|
Silymarin + Paracetamol
|
58.83
± 2.87 DE
|
101.0
± 5.79 EF
|
140.83
± 8.21 EF
|
3.08
± 0.04 BCD
|
0.63
± 0.06 FG
|
0.35
± 0.05 EF
|
Means sharing similar letters are statistically
non-significant (P > 0.05), n=6
Discussion
Paracetamol-induced
hepatic damage has been commonly used as experimental model for screening of
newer hepatoprotectives [Plas,1982] and the extent of hepatic damage is assessed by the
circulating levels of released cytoplasmic enzymes
such as ALT, AST and ALP [Singh,1998]. The data obtained in the present study have shown
that pretreatment of animals with test drugs have resulted in prevention of
rise of serum enzymes, bilirubin and decline of
albumin levels (Table 1). It is, conceivable, therefore that powdered R. emodi root,
its methanolic extract and Akseer-e-Jigar contain MDME
inhibitory constituents that provide hepatoprotection
[Agarwal, 2000;
Li J, 1975]. All the test drugs have marked effect, excepting the low doses of R. emodi and
its aqueous extract that have failed to yield significant change in the
curative trials. These results show that the powdered Rheum emodi in high dose, its methanolic extract and Akseer-e-Jigar have hepatoprotective principles. Our data have also supported
empirical use of the plant drug in traditional eastern medicine and the
herbal preparation, Akseer-e-Jigar being used very commonly by Hakims in
the treatment of some liver disorders,
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