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Indian Journal of Experimental Biology Vol. 45, June 2007, pp.
538-542
Evaluation of hepatoprotective activity of Clerodendrum serratum
L. S M Vidyaa, V Krishnab*, B K Manjunathaa, K L Mankanic, Manzoor
Ahmedc & S D Jagadeesh Singhc
aDepartment of Biotechnology, S.R.N.M.N. College of Applied
Sciences, Shimoga 577 201, India *bDepartment of Studies and
Research in Biotechnology, Kuvempu University, Shankaraghatta 577
451, India
cDepartment of Pharmacology, National College of Pharmacy,
Shimoga 577 201, India
Received 7 June 2005; revised 21 December 2006
The ethanol extract of C. serratum roots and ursolic acid
isolated from it were evaluated for hepatoprotective activity
against carbon tetrachloride induced toxicity in male Wistar strain
rats. The parameters studied were estimation of liver function
serum markers such as serum total bilirubin, total protein, alanine
transaminase, aspartate transaminase and alkaline phosphatase
activities. The ursolic acid showed more significant
hepatoprotective activity than crude extract. The histological
profile of the liver tissue of the root extract and ursolic acid
treated animal showed the presence of normal hepatic cords, absence
of necrosis and fatty infiltration as similar to the controls. The
results when compared with the standard drug silymarin, revealed
that the hepatoprotective activity of the constituent ursolic acid
is significant as similar to the standard drug.
Keywords: Clerodendrum serratum, Hepatoprotective activity, Root
extract, Ursolic acid.
Clerodendrum serratum L. (Verbenaceae) is a deciduous shrub
distributed in the forests of the Western Ghats of India1. In
Indian system of medicine, the plant is well-known as Bharangi
(Sanskrit) and commonly known as Blue glory (English) and Gantu
Bharangi (Kannada). As per the traditional claims2 roots are the
potential source of drugs for ailments such as asthma, bodyache,
bronchitis, cholera, dropsy, eye diseases, fever, inflammations,
malaria, ophthalmia, rheumatism, snakebite, tuberculosis, ulcers
and wounds. Leaves are used as appetizer and expectorant, young
shoots, leaves and flowers are eaten as vegetables. It is one of
the few shrub that antagonizes the effect of histamine3. Ethanolic
extract of the root is reported for antinociceptive,
anti-inflammatory and antipyretic activities4. Phytochemically the
root bark extract contains D-mannitol, stigmasterols and three
triterpenoids⎯ oleanolic acid, queretaric acid and cerratagenic
acid5. Leaf extract contains stigmasterol, α-spinasterol, luteolin,
luteolin-7-0 glucuronide, apigenin, baicalin and scutellarin 7-0
glucuronide6. In the Western Ghats region of Karnataka the roots of
this species are being used by the traditional practitioners for
the treatment
of jaundice. This communication reports the hepatoprotective
activity of the ethanol extract of the roots and the isolated
constituent ursolic acid against CCl4 induced hepatic damage in
rats. Materials and Methods Collection of plant material⎯Roots of
C. serratum were collected from the Lakkavalli reserve forest range
of the Western Ghats region of Karnataka. Taxonomic authenticity
was confirmed by referring to herbarium specimen at Madras
herbarium, Botanical Survey of India, Southern Circle, Coimbatore
and a voucher specimen (FDD-53) is deposited at Kuvempu University
herbaria, Shankaraghatta1. Extraction⎯Roots were shade dried for a
week and powdered mechanically (Sieve No. 10/44). Powdered material
was extracted using soxhlet apparatus with 70% ethanol for about 48
hr. The extract was filtered and concentrated in vacuum under
reduced pressure using rotary flash evaporator (Buchi, Flawil,
Switzerland) till the complete evaporation of the solvent. The
yield was 28.9% (w/w). The active constituent ursolic acid was
isolated from the ethanol extract following the method of Suresh
and Sastry7. Isolation and characterization⎯The ethanol extract was
made alkaline by adding 2% sodium hydroxide and filtered. The
filtrate was acidified with dilute HCl and extracted with diethyl
ether. The ether soluble portion was separated and concentrated.
This
___________ *Correspondent author Phone : (+91)08282 256235,
9448681856 E–mail: [email protected]
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VIDYA et. al.:HEPATOPROTECTIVE ACTIVITY OF CLERODENDRUM SERRATUM
L.
539
compound was recrystallised in ethanol. The melting point was
determined using melting point apparatus (Jindal, NEW Delhi). The
characterization of the compound was done by IR, 1HNMR, 13C NMR and
MASS spectroscopic studies.
Drug formulation⎯Oral suspensions of the root extract (20 mg/ml)
and the isolated constituent ursolic acid (10 mg/ml) were prepared
in gum tragacanth (1% w/v).
Animals⎯Male Wistar albino rats weighing 150-200 g were procured
from the National College of Pharmacy, Shimoga and were maintained
at standard housing conditions. The animals were fed with
commercial diet (Pranav Agro Industries Ltd., Sangli) and water ad
libitum during the experiment. The Institutional Animal Ethical
Committee (Reg.No. 144/1999/CPCSEA/SMG) permitted the study. Acute
toxicity studies were conducted according to "staircase" method8
following OECD guidelines 2002. The LD50 of root extract and
ursolic acid was found to be 200 and 100 mg/kg body weight
respectively. One tenth of these doses (20 and 10 mg/kg, body
weight respectively) was selected as the therapeutic dose for the
evaluation of hepatoprotective activity9.
Evaluation of hepatoprotective activity⎯The animals were divided
into 5 groups of 6 each. The animals of group I (control) received
the vehicle gum tragacanth (1 ml/kg/day; 1% w/v). Carbon
tetrachloride (E-Merck, Mumbai) with olive oil (1:1) was
administered to all the animals of groups II to V in the dose of
0.1 ml/kg/day, ip for 14 days10. Group III animals were treated
with the standard drug silymarin (Ranbaxy Lab, Dewas; 100
mg/kg/day, po). The animals of group IV received ethanol extract
(20 mg/kg/day, po) and the animals of group V received ursolic acid
(10 mg/kg/day, po). The drugs were administered concomitantly for
14 days. The animals of all the groups were sacrificed on 14th day
under light ether anaesthesia. The blood sample of each animal was
collected separately by carotid bleeding into sterilized dry
centrifuge tubes and allowed to coagulate for 30 min at 37°C. The
clear serum was separated by centrifugation at 3000 rpm for 10 min
and subjected to biochemical investigation viz., total bilirubin
(TB)11, total protein12, serum alanine transaminase (ALT),
aspartate transaminase (AST)13 and alkaline phosphatase (ALP)14.
Results of biochemical estimations were reported as mean ± SE of
six animal in each group. The data were subjected
to one way ANOVA followed by Student’s t test. P≤ 0.01 was
considered as statistically significant. Histology⎯The liver
samples were excised from the animals of each group after draining
the blood and washed with the normal saline. Initially the
materials were fixed in 10% buffered neutral formalin for 48 hr.
They were processed for paraffin embedding. The sections were taken
at 5 μm thickness, processed in alcohol-xylene series and were
stained with alum-haematoxylin and eosin15. The sections were
examined microscopically for the evaluation of histological
changes. Results The triterpenoid compound isolated from ethanol
extract of roots of C. serratum showed the following spectral
characteristics. The IR (KBr) spectrum showed peaks at cm-1 3411,
2931, 2862, 1714, 1382 and 1045. The 1H-NMR analysis showed proton
peaks at δ 11.0(1H); 2.0(1H); 1.44(2H); 0.88(3H). 13C NMR showed
ppm related to TMS, shift at 180(C from 1-corboxyl,); 144(C from
1-ethylene); 122-47(CH from cyclohexane); 42(C from cyclohexane);
35(CH2 from cyclhexane); 22(CH3 aliphatic) and in MASS spectral
analysis molecular ion peak observed at m/Z 457 indicating the
molecular weight of the compound and fragments at 412, 382, 365,
270. These spectral data were similar with that of the constituent
isolated by Suresh and Sastry8 and the compound was identified as
ursolic acid (Fig. 1). At the end of 14 days treatment, biochemical
analysis of blood samples of CCl4 treated animals showed
significant increase in the levels of total bilirubin (5.17-fold),
alanine transaminase (16.23-fold), aspartate transaminase
(24.47-fold) and alkaline phosphatase (2.73-fold) activities. But
the total
Fig. 1⎯Structure of Ursolic acid : IR (KBr) cm-1: 3411, 2931,
2862, 1714, 1382, 1045. 1HNMR: δ-11.0 (1H); 2.0 (1H); 1.44 (2H);
0.88 (3H). 13C NMR showed ppm related to TMS, shift at 180(C from
1-corboxyl,); 144(C from 1-ethylene); 122-47(CH from cyclohexane);
42(C from cyclohexane); 35(CH2 from cyclohexane); 22(CH3
aliphatic). MASS: 457[M]+, 412, 382, 365, 270.
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INDIAN J EXP BIOL, JUNE 2007
540
protein level (39%) was decreased reflecting the liver injury
due to the toxic effect of CCl4. The blood samples of the animals
treated with the root extract and the constituent ursolic acid
showed significant reduction in the levels of liver function serum
markers. The effect was more pronounced in the animals treated with
ursolic acid as similar to that of the standard drug silymarin
(Table 1). Histological profile of control animal showed normal
hepatocytes (Fig. 2). The section of liver of the animals treated
with CCl4 exhibited intense centrilobular. necrosis, vacuolisation
and macrovesicular fatty changes (F) (Fig. 3). The liver sections
of silymarin treated animals showed normal hepatic architecture
(Fig. 4). Moderate accumulation of fatty lobules (F) was observed
in the liver sections (Fig. 5) of root extract treated animals. The
liver sections of the animals treated with ursolic acid exhibited
significant liver protection against CCl4 induced liver damage as
evident by the presence of normal hepatic cords, absence of
necrosis and fatty infiltration (Fig. 6). Discussion The injury and
dysfunction of liver caused by CCl4 in experimental animals
simulates the human viral hepatitis16. The toxic effect of CCl4 is
due to its conversion to highly reactive toxic free radical CCl3O-
by cytochrome P450. The free radicals produced locally, cause
autooxidation of polyenic fatty acids present within membrane
phospholipids and oxidative decomposition of lipid is initiated.
The organic peroxides formed after reacting with oxygen leads to
swelling of smooth endoplasmic reticulum and dissociation of
ribosomes from the rough endoplasmic
reticulum. Accumulation of lipids ensues due to inability of the
cells to synthesize lipoprotein from triglycerides and lipid
acceptor proteins leading to the fatty liver. Further, release of
products of lipid peroxidation causes damage to plasma membrane
owing to increased permeability of plasma membrane. This is
followed by progressive swelling of the cell, massive influx of
calcium leading to cell death17. The increase in the levels of AST,
ALT, TB and ALP was the clear indication of cellular leakage and
loss of functional integrity of the cell membrane18. Plant
constituents like triterpenoids and flavonoids are well known for
their antioxidant and hepatoprotective activities19,20.
Phytochemical analysis of ethanol root extract of C. serratum
revealed the presence of flavonoids, glycosides, triterpenoids,
tannins, quinones and saponins. In the present study a triterpenoid
ursolic acid was isolated and characterized. The concomitant
treatment of CCl4 with the root extract or the constituent ursolic
acid showed significant reduction in the level of serum bilirubin
and liverfunction marker enzymes. The test drugs mediated
restoration in levels of AST, ALT and ALP towards respective normal
value is an indication of stabilization of plasma membranes as well
as repair of hepatic tissue due to damage caused by CCl4. In all
the parameters studied, the hepatoprotective activity of ursolic
acid was significant as similar to that of silymarin. However, the
silymarin is slightly effective than ursolic acid. Hence,
hepatoprotective potency of C.serratum may be attributed to ursolic
acid which is known to normalize the disturbed antioxidant status
either by maintaining the levels of glutathione and by inhibiting
the production of malondialdehyde21 or may be due to
Table 1⎯Effect of ethanol extract and ursolic acid isolated from
C. serratum root on CCl4 induced hepatotoxicity in rats [Values are
expressed as mean ± SE from 6 animals in each group]
Group (N) Total bilirubin (mg/dl) Total protein (gm%) AST (IU/L)
ALT (IU/L) ALP (IU/L) Control 0.46±0.02 9.33±0.77 142.23±0.38
57.70±0.33 185.16±1.68 CCl4 2.38±0.14a 5.65±0.25a 2327.84±11.42a
1412.34±3.29a 451.42±1.81a CCl4 +Silymarin 0.53±0.01 b 9.00±0.02 b
199.27±0.69 b 83.03±1.19 b 214.44±1.21b CCl4 + root extract
1.03±0.01b 8.00±0.01b 282.32±3.30b 156.80±2.07b 285.92±1.12b CCl4 +
Ursolic acid 0.89±0.01b 8.23±0.04b 273.98±0.88b 93.57±0.73b
243.67±1.48b ANOVA
F 156.1 149.0 3.10 1.01 5013.0 P 0.01 0.01 0.01 0.01 0.01 df
4,25 4,25 4,25 4,25 4,25
aP≤0.01 indicates significant when compared to control. bP≤0.01
indicates significant when compared to CCl4. AST = Aspartate
transaminase; ALT = Alanine transaminase; ALP = Alakaline
phosphatase
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VIDYA et. al.:HEPATOPROTECTIVE ACTIVITY OF CLERODENDRUM SERRATUM
L.
541
Figs 2-6⎯Section of the liver tissue of 2: control animal
showing normal histology, portal triad showing portal vein (V),
portal artery (arrow head), hepatic duct (arrow);3: animal treated
with CCl4 showing fatty vacuole (F) and central vein (V); 4:
silymarin treated animal showing normal hepatocytes, portal vein
(V), portal artery (arrow head), hepatic duct (arrow) of portal
triad; 5: root extract treated animal showing normal arrangement of
hepatocytes around the central vein (V), portal artery (arrow
head), hepatic duct (arrow), absence of necrosis and few fatty
vacuoles (F);6: ursolic acid treated animals showing normal
arrangement of hepatocytes around the portal triad vein (V), portal
artery (arrow head), hepatic duct (arrow), absence of necrosis and
fatty vacuoles (40× ; H&E)
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INDIAN J EXP BIOL, JUNE 2007
542
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serratum in treating acute jaundice. Acknowledgement The authors
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Rahiman, Department of Biotechnology and Prof. Y.N. Manohara,
National College of Pharmacy, Shimoga for providing facilities to
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