Hepatoprotective Potentials of Butea
monosperma
Stem Bark Extract against Carbon Tetrachloride Induced Hepatotoxicity
in Albino Rats
Prashant
Tiwari1*, Kuldeep Kumar2, Rajnikant Panik3,
Alok Pandey4, Ashish
Pandey4 and Pratap Kumar Sahu5
1Pinnacle
Biomedical Research Institute Bhopal, Madhya Pradesh, India.
2Smt. Vidyawati
Collage of Pharmacy, Jhansi (Uttarpradesh)
3Gover. Girls
Polytechnic College, Raipur (Chhattisgarh)
4Raipur Institute
of Technology, Raipur (Chhattisgarh)
5Siksha ‘O’
Anusandhan University, Bhubaneswar, Orissa.
ABSTRACT:
Carbon tetrachloride (CCl4) pharmacological tool to produce liver damage
in rats. Silymarin (100 mg/kg) and extract of Butea monosperma (shown to be
hepatoprotective substances) prevented the CCl4 induced toxicity. Hydroalcholic extract of the stem bark of Butea Monosperma was evaluated for its hepatoprotective. This in
vitro efficacy was reinforced by a significant dose dependent
hepatoprotection (at 100 mg/kg and 200mg/kg dose) by decreasing the activity of
serum enzymes, bilirubin, and lipid peroxidation while it significantly
increased the reduced Glutathione levels of tissue in a dose dependant manner.
The Hepatoprotective activities of the extract are being comparable to
standards Silymarin. The results obtained in the present study indicate that stem bark extract of Butea Monosperma is a potential source of natural hepatoprotective.
The hepatoprotective property may be attributed to the antioxidant potential
and the phytochemical constituents of the plant. The present study justifies
the claim of the native practitioner that the decoction of the plant is useful
in treating jaundice and find out the clinical efficacy of the Butea Monosperma.
KEYWORDS: Carbon tetrachloride, Butea monosperma, Silymarin, Alanine amino transferase,
Glutathione, Serum glutamate oxaloacetate transaminase, Serum glutamate
pyruvate transaminase and Hepatoprotection.
INTRODUCTION:
Hepatic system is
very vital organ system involved in the body’s metabolic activities. As a
result the chemical reactions in the liver may generate several reactive
species like free radicals. These reactive species form covalent bond with the
lipids of the tissue. However inbuilt protective mechanisms combat the
hazardous reactions associated with the free radicals. Due to excessive
exposure to hazardous chemicals, the free radicals generated will be so high
such that they overpower the natural defensive system leading to hepatic damage
and cause jaundice, cirrhosis and fatty liver, which remain one of the serious
health problems. Carbontetrachloride (CCl4) is one such hazardous chemical
which induces hepatopathy through membrane lipid peroxidation by its free
radical derivative, (CCl3 ·, CCl3O2 ·). Excessive production of the reactive
species manifests in tissuethiol depletion, lipid peroxidation, plasma membrane
damage etc., culminating into severe hepatic injury1. The
traditional systems of medicine together with homoeopathy and folklore medicine
continue to play a significant role largely in the health care system of the
population. Butea monosperma (Lam) Taub (Palas) belonging to the family
leguminoceae grown wildly in many parts of India. The plant is regularly used
by the rural and tribal people in curing various disorders2.
The bark of the
plant is an appetiser, lessens inflammation, dysmenorrhoea used in liver
disorders, fractures, and gonorrhoea, topically in piles and hydrocele purifies
the blood. Leaf is appetiser, very astringent, carminative, anthelmintic,
aphrodisiac, tonic, lessen inflammation and lumbago, cures boils and piles. Gum
is acrid, astringent, aphrodisiac, tonic to the liver, used in the diseases of
the chest and lungs useful in syphilis. The flower is bitter, aphrodisiac,
expectorant, and tonic, emmenagogue, diuretic, astringent, and good in
inflammation, burning urine and gonorrhoea. The fruit and seeds are bitter and
oily, anthelmintic, useful in piles, eye diseases and inflammation. The lye is
useful in enlargement of spleen3.
Butea monosperma (Lam.) is commonly known as Flame of forest, belongs to
the family Fabaceae4. It is locally called as palas, palash,
mutthuga, bijasneha, dhak, khakara, chichra, Bastard Teak, Bengal Kino, Nourouc
and is common throughout India, Burma and Ceylon except in very acrid parts.
Generally it grows gregariously on open grasslands and scattered in mixed
forest. Plantations can be raised both on irrigated and dry lands. The pods
should be collected and sown before the commencement of rains, root suckers are
freely produced and help in vegetative propagation. In India, palas ranks next
to kusum (schleichera trijuga) as a host tree for lac insect5,6.
Almost all the parts of the plant are being used since decades in medicine and
for other purposes. These days herbal medicines are more popular than modern
medicine because of their effectiveness, easy availability, low cost and for
being comparatively devoid of side effects. Nature always stands a golden mark
to exemplify the outstanding phenomenon of symbiosis and it has provided the
storehouse of remedies to cure all ailments of mankind, only the thing is that
there is a need to evaluate them scientifically. Stem bark powder is used to
stupefy fishes. Young roots are used for making ropes7. Green leaves
are good fodder for domestic animals. Leaves are used for making platters,
cups, bowls and beedi wrappers7, 8. Leaves are also used for making
Ghongda to protect from rains and are eaten by buffaloes and elephants.
Similarly there were claims from a local native practitioner that the decoction
of the test plant is highly useful in treating jaundice. Since the
pharmacological profile of the plant is not completely established. Therefore
this plant is taken for the present study. With this scientific information,
the present study was designed with an aim to assess the hepatoprotective
activity of the stem bark extract of Butea Monosperma, against CCl4
induced liver damage.
MATERIALS AND METHODS:
Plant source:
Healthy disease free, mature fresh plant root sample
were collected locally from Bilaspur, Chhattisgarh, India. Fresh plants were
washed thoroughly 2-3 times with running tap water and once with sterile water,
shade-dried without any contamination. The dried stem
bark were then powdered using an grinder.
Preparation of
Extracts:
The Butea
Monosperma collected locally from Bilaspur, Chhattisgarh, India. The
fresh plant were detached from the stems and dried at room temperature
(27°C) for a week. They were then weighed several times until the weight
was constant. The dried stem bark then ground
into a fine powder with the help of grinder and the powder kept in an airtight
amber container, for extraction procedure. The dried powder (25 g) stem bark of Butea Monosperma was extracted with a mixture of water and
methanol in the ratio of 50:50 respectively. Extraction was continued at the
temperature of 27°C till clear solvent was observed in siphon tube. Extract was
concentrated in water bath at 40°C. Concentrated extract was dried at 40°C in
hot air oven. Dried extract was packed in an air tight container.
Preliminary
phytochemical investigation:
All the extracts
were subjected to preliminary phytochemical tests9. All the tests
reveal that the plant possesses steroids, glycosides, triterpenoids, tannins
and flavonoids. Since hydroalcholic extract has shown the better results for
the presence of polyphenolic compounds and triterpenoids, this extract was
selected for further study.
Animals:
Male swiss albino
rats (Animal House of Pinnacle Biomedical Research Institute, Bhopal, India.)
weighing between 120-150 g were used. They were housed in polypropylene
cages under standard conditions (23 ± 2 .C, humidity 60–70%, 12 h
light/dark cycles). They were given standard pellet diet (Lipton India Ltd.
pellets) and tap water ad libitum. The experiments were performed during day
(08:00-16:00 h). The institutional animal ethical committee approved to the
study protocol.
Carbon tetra - chloride
induced toxicity:
The method of Ko
et al10 was used for screening the hepatoprotectivity of the test
extract. The animals were randomly assigned into 5 groups of 6 animals. Group I
and II served as normal and intoxicated control and received only the vehicle
(normal saline). Group III served as standard, was treated with Silymarin (100
mg / kg / day BW for 3 days) The animals of Group IV and V received stem bark extract of Butea Monosperma (100 mg/kg BW and 200mg / kg BW respectively)
for 3 days. Twenty-four hours after the last dosing, animals (except Group 1)
were treated orally with CCI4 (11 % v/v in olive oil) at a dose of 1 ml
/ kg BW. Animals were sacrificed 24 hrs, after CCl4 treatment, hepatic tissue
and heparinized blood sample were taken and assessed for serum enzyme and
Glutathione estimation. Serum enzymes, which were assessed, include Serum
gluataic oxaloacetate transaminase (SGOT) and Serum glutamic pyruvic
tranaminase (SGPT)11, Total Bilirubin and Direct Bilirubin12,
and Alkaline phosphate (ALP) content. Tissue Glutathione measurements were
performed using a modification of the Ellman procedure13, 14.
Table No I: Effect of stem bark extract of Butea Monosperma and
CCl4-induced hepatoxicity (n=6)
|
Groups |
SGOT
Levels (U/L)Mean ±SE |
SGPT (U/L) Mean ±SE |
ALP
(mg/dl) Mean ±SE |
Total Bilirubin (mg/dl) Mean ±SE |
Direct Bilirubin (mg/dl) Mean ±SE |
|
Group 1 |
112.9±0.71 |
49.5±0.05 |
217.6±0.12 |
0.890±0.001 |
0.179±0.001 |
|
Group 2 |
281.9±0.61 |
171.2±0.60 |
890.8±0.57 |
8.47±0.061 |
3.91±0.006 |
|
Group 3 |
179.1±0.46 |
80.1±0.55 |
398.1±1.07 |
3.53±0.115 |
0.185±0.002 |
|
Group 4 |
227.2±1.18 |
154.2±0.63 |
520±0.64 |
5.86±0.577 |
0.532±0.002 |
|
Group 5 |
199.2±0.51 |
114.6±1.80 |
669.7±0.55 |
4.52±0.105 |
0.439±0.001 |
Values are the Mean ± SEM of six rats/ treatment.
Group 1-Normal animals
(untreated); Group 2-CCl4 (1ml/kg)
treated animals; Group 3-CCl4 +
Silymarin (100mg/kg BW) treated animals. ; Group
4-CCl4 + Butea Monosperma
(100mg/kg
BW) treated animals. ; Group 5-CCl4
+ Butea Monosperma
(200mg/kg BW) treated animals.
Table II: Effect of stem bark extract of Butea
Monosperma on hepatic GSH status in rats (n=6) and carbon
tetrachloride-induced peroxidation in rats (n=6).
|
Treatment |
Dose
(mg/Kg BW) |
GHS
(Abs412) |
Lipid
Peroxidation (Abs 543) |
|
Normal Saline |
- |
0.122±0.0001 |
0.090±0.002 |
|
Normal Saline-CCl4 |
- |
0.576±0.002 |
0.305±0.001 |
|
Sylamarin- CCl4 |
100 |
0.791±0.003 |
0.072±0.004 |
|
Butea Monosperma -CCl4 |
100 |
0.429±0.002 |
0.214±0.003 |
|
Butea Monosperma -CCl4 |
200 |
0.659±0.001 |
0.147±0.001 |
Tissue samples
were homogenized in ice cold TCA (lgm tissue plus 10 ml 10 % TCA) in a
homogeniser. Briefly after centrifugation at 3000 rpm for 10 minutes, 0.5 ml
supernatant was added to 2 ml of 0.3 M Disodium hydrogen phosphate solution. A
0.2ml solution of DTNB (5, 5 Dithio-bis 2- nitrobenzoic acid) (0.4 mg in 1 ml
of 1 % Sodium nitrate) was added and the absorbance at 412 nm was measured
immediately after mixing. Extent of lipid peroxidation was done by combining
1.0ml of biological sample (0.1 – 2.0 mg of membrane protein or 0.1 – 0.2
μmol of lipid phosphate) with 2.0 ml of TCA-TBA-HCl and thoroughly mixed.
The solution was heated for 15min in a boiling water bath. After cooling, the
flocculent precipitate was removed by centrifugation at 1000rpm for 10min. The
absorbance of the sample is determined at 535nm against blank that contains all
the reagents without the lipid15.
Statistical analysis:
Data were
analyzed by ANOVA followed by Bonferroni’s multiple variance test. Results with
P<0.05 were considered statistically significant.
RESULTS:
The estimated values of serum GOT, GPT, ALP, Total
Bilirubin and Direct bilirubin values in control (saline + vehicle) group of
rats were found to be 112.9±0.71, 49.5±0.05, 217.6±0.12, 0.890±0.001, 0.179±0.001 respectively (Table
I). A remarkable elevation was observed in serum GOT, GPT, ALP, Total
bilirubin and direct bilirubin values in CCl4 intoxicated rats (Toxic Control
group). In the groups treated with 100mg/kg and 200mg/kg of the stem bark of Butea Monosperma extract, the above biochemical markers of
hepatotoxicity were found to be decreased when compared to CCI4 treated control
group. Evidently, the hepatoprotective effects of higher dose of Butea Monosperma (200mg/kg) were
near to that of standard i.e. Silymarin (100mg/kg). Both the doses of stem bark extract of Butea Monosperma used in the study showed significant
protective property than control. However the test extract was found to be less
potent than that of standard drug. The tissue glutathione was found to be
depleted upon CCl4 intoxication, indicate that the tissue damage is due to over
powering the inbuilt free radical scavenger mechanisms. This tissue GSH
depletion was inhibited by the pretreatment with test extract in a dose
dependant manner. Similarly lipid peroxidation induced by CCl4 treatment was
reversed by test extract in a dose dependant manner. The results are compiled
in table II..
DISCUSSION:
Carbon tetrachloride is a pharmacological tool used to
produce liver damage in animal models; its hepatotoxic action begins with
changes in endoplasmic reticulum which results in loss of metabolic enzymes
located in the intracellular structure16-17. The stem bark extract of Butea Monosperma was taken for assessing the in vivo
hepatoprotective properties. Pretreatment with the test extract has reduced the
elevated levels of biochemical markers of hepatoxicity. Further it was also
observed that the tissue GSH depletion due to CCl4 challenge was reversed by
the test extract and also reduced the extent of lipid peroxidation. Most of the
mammals have an effective mechanism to prevent and neutralize the free radical
induced damage, which is accomplished by a set of endogeneous substances such
as superoxide dismutase (SOD) catalase, glutathione peroxidase(GPx/) and
glutathione reductase (GR) . CCl4
undergo hepatic metabolism to give rise to trichloro methyl radicals, which
upon reacting with reactive oxygen species yields trichloromethyl peroxide
radicals, which forms covalent bond with membrane lipids and destroy the
membrane integrity. The observation of increased malonaldehyde (MDA) formation
in hepatic cells after CCl4 challenge is in accordance with the earlier report,
which suggests involvement of trichloromethyl and trichloromethylperoxy
radicals in the propagation of peroxidation process18. The
pretreatment with extract has prevented oxygen free radicals and thereby
prevented the formation of peroxy radicals. This aspect of test extract also
contributes to the hepatoprotectivity.Thus, from the results of the present
investigation it may be concluded that the stem bark
extract of Butea Monosperma
possess significant hepatoprotective activity. It appears that hepatoprotective
activity of the test extract is due to their phytochemical constituent which
contains kino-tannic acid, gallic acid, pyrocatechin. The plant also contains
palasitrin, and major glycosides as butrin, alanind, allophanic acid, butolic
acid, cyanidin, histidine, lupenone, lupeol, (-) - medicarpin, miroestrol,
palasimide and shellolic acid19 and antioxidant potential. The
antioxidant potential may be attributed to the presence of polyphenolic
compounds. Further studies like isolation and characterization of the active
principal(s) responsible for such activity are needed to confirm. However the
present study justifies the claim of the native practitioner that the plant is
used as a hepatoprotective.
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Received on 20.06.2011
Accepted on 29.06.2011
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Research J. Pharmacology and
Pharmacodynamics. 3(5): Sept –Oct. 2011, 281-284