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 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4
Hepatoprotective Activity of Luteolin from A.
millefolium in CCl4 Intoxicated Rat Model
Papiya Bigoniya
Department of P.G. Pharmacology,
Radharaman College of Pharmacy,
Bhopal- 462 002, M.P., India.
ABSTRACT
This study was designed to investigate in vivo and in vitro
hepatoprotective activity of luteolin isolated from Achillea
millefolium in CCl4 intoxicated rat. Luteolin, isolated from
A. millefolium was authenticated by HPTLC and HPLC
techniques. Wistar albino rats were treated with vehicle,
silymarin (20 mg/kg) and luteolin at 250 and 500 mg/kg,
p.o. continuously for 14 days. CCl4 was administered on
seventh day afterwards alternate day for a week. Animals
were accessed for thiopental induced sleeping time,
bromosulphalein uptake, biochemical analysis of serum
for marker enzymes, free radical scavenging ability of
liver and histopathology. Silymarin and luteolin (250 and
500 mg/kg) treated group resulted in extremely significant
(p < 0.001) decrease in thiopental induced sleeping time
compared to the CCl4 treated group. Luteolin showed
55.95 and 79.76% hepatoprotection at 250 and 500 mg/kg
doses. Levels of marker enzymes SGOT, SGPT, ALP,
cholesterol, triglyceride, LDL, and bilirubin in plasma
increased severely whereas the level of HDL, protein and
albumin decreased in CCl4 intoxicated animals. The pre
and post treatment with luteolin (250 and 500 mg/kg)
resulted in significant normalization of biochemical
parameters except protein. Luteolin showed dose
dependent reduction in lipid peroxidase and elevation of
glutathione (p < 0.01). The results indicate the protective
and curative effect of luteolin on CCl4 induced
hepatotoxicity, which may be mediated through its
antioxidant, anti-inflammatory and immune modulating
property.
Keywords-Achillea millefolium, Luteolin, Antioxidant,
Hepatoprotective, Bromosulphalein
1. INTRODUCTION
Liver is the second largest organ in human body and
regarded as a principle site for metabolism and excretion.
Liver serves many vital functions such as remove damaged
red blood cells from the blood in co-ordination with
spleen, secretion of bile, clotting factors, stores vitamins,
minerals, protein, fats and glucose from diet. Thus, to
maintain a healthy liver is a crucial factor for overall
health and wellbeing. Continuous and varied exposure to
environmental toxins and chemotherapeutic agents
eventually lead to various liver diseases [1].
Most of the hepatotoxins damage liver cells mainly due to
lipid peroxidation, low glutathione stores and other
oxidative damages. Every drug or toxin introduced in body
is structurally altered by liver, resulting in therapeutically.
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1477
Chandra Shekhar Singh
Department of P.G. Pharmacology,
Radharaman College of Pharmacy,
Bhopal- 462 002, M.P., India.
active and/or inactive metabolites and some of these
metabolites may be far more toxic than their parent
compound and may result in liver injury.
Further, liver has capacity to recover from acute injury by
hepatocellular regeneration with the production of new
cells, which restore liver functions and normal tissue
structure. Chronic liver injury however often leads to
fibrosis, diffuse damage to the hepatic parenchyma cells
with nodular regeneration and disturbances in normal
tissue architecture. In spite of tremendous advances in
modern medicine, there are hardly any drugs that stimulate
liver function, offer protection to the liver from damage or
help regeneration of hepatic cells [2].
Literature survey reveals that a number of herbs and
bioactive have potential to stimulate and regenerate the
damaged hepatic cells. This adds to a deep belief that
remedies from natural origin are safe and fit into the image
of a gentle. Herbal remedies support natural healing
phenomena through blocking the progression of the
degenerative pathological processes. Modern remedy
offers limited opportunity in providing effective and
harmless cure. In the present scenario, there is a great need
to develop new potential drugs that are capable of treating
liver diseases. In traditional systems of medicine,
numerous plants were claimed to be effective and used
successfully to alleviate multiple liver disorder [3].
Literature overview shown that reactive oxygen species
including oxygen free radicals are causative factors in the
etiology of degenerative diseases, including some
hepatopathies. According to in vitro and in vivo studies,
several classical antioxidants from plants represent a
logical therapeutic strategy for treatment of liver
diseases. There are many plant derived chemicals with
potent antioxidant properties which can serve as primary
compounds for development as hepatoprotective drugs.
Achillea millefolium L. (A. millefolium; Family
Asteraceae), a well-known herb in Indian traditional
Ayurveda system of medicine, has been used to treat liver
disorders and is an important ingredient of many
formulations used for the treatment of liver ailments.
Concerning the bioactivity of this plant, recent studies
reported antimicrobial, anti-inflammatory,
hepatoprotective, antispasmodic, choleretic and
antioxidant activity [4], [5]. It is believed that those effects
are mainly attributed to flavonoids and phenolcarbonic
acid complex [6]. The well-known bioactive compounds
of A. millefolium are achilleine, apigenin, luteolin,
 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4
quercetin, rutin, succinic, salicylic, caffeic, chlorogenic
and dicaffeoylquinic acid [5], [7]. Luteolin, a flavonoid
from A. millefolium and various other plants has been
reported to possess anti-inflammatory, antioxidant,
antiallergic, antitumorigenic and hepatoprotective activity
[8].Considering the pharmacological importance of
luteolin, the present study was designed to evaluate effect
of luteolin on functional integrity of hepatic microsomal
enzyme following CCl4 induced hepatotoxicity on rats by
assessing in-vitro bromosulphalein (BSP) uptake;
barbiturate induced sleeping time, serum biochemical
parameters and free radical scavenging ability to establish
the regenerating or liver stimulant activity of luteolin.
2. MATERIALS AND METHODS
2.1 Drugs and Chemicals
Luteolin standard was purchased from Sigma Aldrich,
USA. Bromosulphalein was procured from HiMedia Lab.
Ltd., Mumbai and thiopental was obtained as gift sample
from Neon Lab., Mumbai. Other chemical used were of
analytical grade and were procured locally. Biochemical
kits were obtained from Aspen Diagnostic Pvt. Ltd., India.
2.2 Plant Material
The flowers of A. millefolium were procured from Khari
Bawli market, New Delhi, India. The flowers were
identified with the aid of available literature and
authenticated by taxonomist Dr. H. B. Singh, Chief
Scientist and Head, Raw Materials Herbarium and
Museum, NISCAIR, New Delhi, India (voucher specimen
no. NISCAIR/RHMD/Consult/-2012-13/2091/98).
2.3 Isolation of luteolin from A. millefolium
Dry coarse powder of A. millefolium flowers (4.00 kg) was
extracted three times with ethanol (16 lit). The marcs were
combined and filtered through medical gauze; the filtrates
were collected and concentrated using rotary vacuum
evaporator. The weight of crude extract was 436.29 g, and
the yield was 10.90%. The crude extract (400 g) was
further fractionated two times sequentially with ethyl
acetate (500 ml × 2), n-butanol (200 ml × 2) and water
(200 ml × 2). The dry weights of ethyl acetate, n-butanol
and water fractions were 63.62, 59.02 and 218.56 g
respectively. The yields of these three fractions from crude
extract were 15.90, 14.75 and 54.64% respectively.
On the basis of literature, thin layer chromatography of
ethyl acetate fraction on 10 × 10 cm2 pre-coated silica gel
60 F254 plates (Merck Ltd., Mumbai, India) were
performed using benzene: acetic acid: water (125:72:3 v/v)
as a solvent system in order to identify luteolin [9]. The
developed plate was placed in iodine chamber for
visualization. Three spots were identified. Further ethyl
acetate fraction was subjected to column chromatography
for isolation of luteolin.
A column of 760 mm height, 120 mm in diameter was
packed upto a height of 550 mm with slurry of silica gel-
60 (particle size 40-63 µm) in 90:10 ratio of n- hexane and
ethyl acetate. The column was left for overnight. Next day
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1478
the ethyl acetate fraction (40 g) was triturated with
sufficient quantity of silica gel-60 and loaded on to the
column. The flow rate of the column was maintained 1
ml/min throughout the elution. The sample was eluted
sequentially with a gradient of n-hexane/ethyl acetate
(from 90:10, 80:20-0:100) and followed by ethyl
acetate/methanol (from 90:10, 80:20-0:100). After the
operation, 159 vials each containing 10 ml elute were
collected. All the vial containing elutes were
chromatographed as mentioned above simultaneously with
standard luteolin. The elutes of vial 83-122 showed
identical Rf in reference to the standard luteolin. The vials
containing luteolin were pooled and evaporated to make it
dry (3.31 g).
2.4 Authentication of isolated luteolin by
analytical HPTLC technique
HPTLC analysis was performed on a CAMAG system
equipped with high pressure auto sample injector of 100 µl
capacity, scanner, UV chamber and CAMAG-Linomat 5
software system for data analysis. Standard and sample
solution were prepared by dissolving one mg of standard
luteolin and two mg of isolated luteolin in 5 ml of ethanol
and filtered through the Whatman filter paper. Two µl of
standard and sample solution was spotted by auto injector
as a band onto a 10 × 10 cm2 pre-coated silica gel 60 F254
plate. The plate was run upto a height of 8 cm in saturated
mobile phase of benzene: acetic acid: water in the ratio of
125:72:3. After development, plate was removed, air dried
and finally dried with aid of mechanical drier. The dried
developed plate was observed in UV cabinet at day light
and UV light (254 nm). The scanning of plate was
performed at 254 nm and data was recorded.
2.5 HPLC fingerprinting of isolated luteolin
The chromatography analysis was performed on Shimadzu
liquid chromatography system, equipped with Prominance
(LC-20AD) pump, SPD-M20A photodiode array UV-
visible detector working in the range 190-800 nm, a
quaternary solvent delivery system and degasser (DGU-
20A5). The chromatographic data was recorded and
processed with LC solution integrated software and a
rheodyne injection valve fitted with a 20 μl injection loop.
Baseline resolution of luteolin was obtained at 25 ± 2°C
using stainless steel Luna column (150 mm × 4.6 mm),
packed with octadecylsilane bonded to porous silica (5
μm). An isocratic solvent system consisting of 0.5% v/v of
aqueous glacial acetic acid: methanol in the ratio of 48:52
(v/v) was used. The mobile phase was passed through 0.45
PVDF filter, degassed before use. The flow rate was kept
constant at 1 ml/min and effluents were monitored at 342
nm [10]. Standard and sample luteolin solutions were
prepared by dissolving 1 mg/ml in methanol. From this,
one ml of each was diluted to 20 ml with methanol (50
μg/ml). The solutions were filtered through a 0.45 μm
membrane filter prior to HPLC analysis and the injection
volume was 20 μl.
 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4
2.6 Animals
Laboratory bred Wistar albino rats of either sexes
weighing between 150-200 g were maintained under
standard laboratory conditions at 25 ± 2°C, relative
humidity 50 ± 15% and photoperiod (12 hr-dark and light).
Commercial pellet diet (Hindustan Lever, India) and water
were provided ad-libitum. Animals were allowed to free
access of water and food during the experiment but no
water and food were allowed before and after 1 hr of
dosing. In order to avoid diurnal variation all the
experiments were carried out at same time of day i.e.,
between 10:00 am to 05:00 pm. Ethical committee
approvals was obtained from institutional animal ethical
committee (approved body of Committee for the purpose
of control and supervision of experiments on animals
Chennai, India; Ref. No.IAEC/RCP/01) of Radharaman
College of Pharmacy, (Reg. No. 1169/ac/08/CPCSEA),
Bhopal, before carrying out the experiments.
2.7 Experimental design
The oral LD50 value of luteolin in mice was reported to be
2500 mg/kg [11]. Therefore, we adopted an oral dose of
250 and 500 mg/kg luteolin in this study, which is
relatively safe and can achieve the maximal protective
effect after CCl4 administration. Animals were randomly
divided into 5 groups with 12 rats in each. Group I and III-
V was treated with vehicle control, positive control
(silymarin) and two doses of luteolin continuously for 14
days. On 7th day, all the groups including group II
(negative CCl4 control) were treated with CCl4 (1 ml/kg) 2
hr after drug treatment and afterwards on alternate days for
a week. On the 14th day, 2 hr after drug treatment, 6
animals from each group were used to determine
thiopental induced sleeping time and remaining 6 animals
from each group were used to access biochemical analysis
of serum for marker enzymes, bromosulphalein (BSP)
uptake test, free radical scavenging ability and
histopathology of liver.
Vehicle control group animals were treated with normal
saline (0.2 ml/100 g). Standard drug silymarin was
prepared freshly in 1% gum-acacia in normal saline.
Luteolin was prepared by dissolving in 1% PEG in water
for injection as per the required quantity. All the
treatments were given by orogastric intubation. Treatment
plan was as following:
Fig. 1A HPLC chromatogram of Std. luteolin
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1479
Group I: Vehicle control group: normal saline for fourteen
days
Group II: CCl4 control group (1 ml/kg) on seventh day
afterwards alternate days for one week
Group III: Silymarin (20 mg/kg) + CCl4 (1 ml/kg)
Group IV: Luteolin (250 mg/kg) + CCl4 (1 ml/kg)
Group V: Luteolin (500 mg/kg) + CCl4 (1 ml/kg)
2.8 Assessment of hepatoprotective activity
Body weights of all the animals were recorded on 1 st, 7th
and on 14th day before sacrifice. On the 14th day, 2 hr after
drug treatment six animals of each group were given
thiopental sodium (40 mg/kg) intraperitoneally and the
effects of drug on CCl4 induced prolongation of thiopental
sodium sleeping time were studied. Remaining six animals
of each group were anaesthetized by light ether
anaesthesia and blood was withdrawn by intracardiac
puncture. Blood was allowed to coagulate for 30 min at
room temperature and serum was separated by
centrifugation at 3000 rpm for 5 min (Remi Centrifuge,
Model RM 12 C). The serum was used to estimate serum
SGOT, SGPT, ALP, cholesterol, HDL, LDL, tri-glyceride,
total and direct bilirubin, protein and albumin as per
method described in diagnostic kits (Aspen Diagnostic
Pvt. Ltd., India). The liver, kidney, spleen and heart were
harvested, washed in normal saline, blotted in filter paper
and weighed. Each liver was cut into three parts. From part
one, three slices of 60 mg was weighted and used for BSP
uptake. Percentage hepatoprotection was calculated with
the method described by Ranjan and Subramanyan [12].
Second part of the liver (30% w/v) was homogenized in
0.9% buffered KCl for estimation of glutathione, lipid
peroxidase and superoxide dismutase [13], [14], [15].
Third part of the liver was preserved in 10% formalin
solution for histopathological assessment of liver damage.
Hematoxylene and eosin staining of liver tissues and
permanent tissue slides were prepared by following the
method described by Nanji et al [16].
2.9 Statistical analysis
The results were expressed in term of Mean ± SEM.
Experimental data of various physical and biochemical
parameters were analyzed using one way ANOVA
followed by Turkey-Kramer multiple comparisons using
InStat-3 graph pad version. Differences between compared
groups were considered significant at p < 0.05.
Fig. 1B HPLC chromatogram of isolated luteolin
 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4 1480

Fig. 2A HPLC chromatogram of Std. luteolin

Fig. 2B HPLC chromatogram of isolated luteolin

3. RESULTS 3.1 Effect of luteolin on body and relative

Luteolin was obtained as a yellow powder (m.p. 329°C),
and gave a positive reaction with AlC13 reagent, probably
indicating its flavonoid nature. Its UV spectrum was
consistent with that of flavonoids with maxima at 252, 265
and 342 nm. The isolated luteolin and standard luteolin
was subjected to TLC on silica gel G plates and HPTLC
analysis on silica gel 60 F254 plates using benzene: acetic
acid: water (125:72:3), which showed the single spot
withRf 0.19. The Rf of isolated and standard luteolin was
found to be identical [Fig. 1A and B].
The isolated luteolin was further authenticated by HPLC
fingerprinting [Fig. 2A and B]. The HPLC method
described herein provides single peak in chromatogram
having the retention time of 12.521 min. The retention
time of isolated luteolin was well comparable with that of
standard luteolin (12.529 min).
organ weight
Vehicle control group showed 6.15% increase in body
weight on 7th and 14th day. Negative control group showed
an increase of 6.70% in body weight on 7 th day followed
by a drastic decrement of 5.95% with reduced food
consumption on 14th day. Standard drug silymarin and
luteolin (250 and 500 mg/kg) treatment showed 5.12, 5.88
and 6.05% increase in body weight on 7 th day followed by
a decrease of 1.42, 1.97 and 0.62% on 14 th day [Table 1].
Administration of luteolin (250 and 500 mg/kg) for 14
days resulted in the significant decrease (p < 0.05 and
0.01) in relative weight of liver in comparison to negative
control group. Whereas, both the doses of luteolin showed
non significant change in relative weight of kidney, spleen
and heart [Table 1].

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 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4 1481

Table 1: Effect of luteolin on body weight and thiopental induced sleeping time of CCl4 treated rats.
Liver weight
% change in in g/100 g of % Hepatoprotection in
Thiopental induced sleeping time
body weight body weight BSP uptake (min)
Treatment
(mg/kg, p.o.)
7th 14th Sleeping time %
10 20 30
day day (min) Hepatoprotection

Vehicle
6.15 6.15 52.37 ± 3.02 - - - -
control
CCl4 (1 ml/kg) 6.70 - 5.95 3.01 ± 0.02 205.46 ± 4.63 - - - -
***
Silymarin (20) 5.12 - 1.42 4.11 ± 0.18 56.94 ± 3.52 97.01 26.25 56.32 86.90
Luteolin (250) 5.88 - 1.97 3.27 ± 0.02** 85.40 ± 4.55*** 78.42 12.50 27.58 55.95

Luteolin (500) 6.05 - 0.62 3.29 ± 0.12* 59.74 ± 3.33*** 95.18 21.25 45.97 79.76
The results are expressed as Mean ± SEM. One way ANOVA followed by Turkey’s multiple comparison tests. *p < 0.05,
** p < 0.01, *** p < 0.001 and ns = not significant when compared to the negative control (CCl4 treated) group.

3.2 Thiopental induced sleeping time
CCl4 intoxicated rats showed significant increase in
sleeping time (205.46 ± 4.63 min) compared to that of
vehicle control group (52.37 ± 3.02 min). The treatment
with silymarin and luteolin (250 and 500 mg/kg) resulted
in extremely significant (p < 0.001) decrease in thiopental
induced sleeping time compared to the CCl4 treated group.
Percentage hepatoprotection in concern to thiopental
induced sleeping time was 95.18 and 97.01% for luteolin
(500 mg/kg) and silymarin respectively [Table 1].
3.3 Bromosulphalein uptake test
Luteolin showed dose dependent hepatoprotection of
55.95 and 79.76% respectively at doses of 250 and 500
mg/kg, whereas, silymarin showed a hepatoprotection of
86.90% [Table 1].
3.4 Effect of luteolin treatment on serum
biochemical parameters
change in the protein level whereas both the doses of
luteolin showed dose dependent gain in albumin level in
comparison to CCl4 treated group.
Silymarin treatment decreased serum SGOT, SGPT, ALP,
cholesterol, tri-glyceride and LDL level which was
extremely significant (p < 0.001). Silymarin treatment also
significantly reduced (p < 0.001 and 0.01) the level of total
and direct bilirubin in comparison to CCl4 intoxicated
animals [Table 2].
3.5 Estimation of liver free radical scavenging
ability
Luteolin treatment showed dose dependent elevation of
glutathione (p < 0.01-0.001) at 250 and 500 mg/kg doses
in comparison to the negative control group whereas, a
highly significant decrement (p < 0.001) in the lipid
peroxidase level was observed at both the doses of
luteolin. Luteolin treatment did not have significant
enhancement ability of SOD level [Table 3].

Result of the biochemical parameters revealed that the
levels of marker enzymes SGOT, SGPT, ALP, cholesterol,
tri-glyceride, LDL, total and direct bilirubin in plasma
increased severely whereas, the level of HDL, protein and
albumin decreased considerably in CCl4 intoxicated
animals. In contrast, luteolin (250 and 500 mg/kg) showed
extremely significant (p < 0.001) decrease in SGOT,
SGPT, ALP, cholesterol, tri-glyceride and LDL level in
comparison to CCl4 treated animals. Whereas, serum HDL
level was significantly increased (p < 0.05 and 0.01) at
250 and 500 mg/kg doses of luteolin in comparison to
CCl4 toxicated animals. The level of total and direct
bilirubin was significantly reduced (p < 0.05 and 0.01) by
luteolin. Both the doses of luteolin showed non significant
3.6 Histological parameters
Histopathological assessment of haematoxylin-eosin
stained liver sections showed zonal necrosis, extensive
diffuse vacuolar degeneration engorged with blood and
microvesicular fatty changes in CCl4 intoxicated liver
tissue. Silymarin treated liver tissue showed focal necrosis,
lobular necrosis and slightly altered hepatic parenchyma.
Luteolin (500 mg/kg) pretreatment normalized the CCl4
induced changes having only focal coagulative necrosis,
zonal necrosis and slight fatty changes with normal
hepatocyte [Fig. 3A, B, C, D and E].

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 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4 1482

Table 2: Effect of luteolin on biochemical parameters of CCl4 treated rats

Treatment SGOT SGPT ALP Cholest Tri- HDL LDL Bilirubin g/dl Protein Albumin
(mg/kg, IU/L IU/L U/I -erol glyceride mg/dl mg/dl Total Direct g/dl g/dl
p.o.) mg/dl mg/dl
Vehicle 28.38 24.45 44.92 113.54 139.54 42.64 42.99 1.00 0.28 7.20 4.28
control ± ± ± ± ± ± ± ± ± ± ±
2.95 3.45 3.10 8.66 5.15 4.02 4.34 0.02 0.08 0.22 0.39
CCl4 79.73 80.84 118.70 207.29 239.50 22.43 136.96 2.45 0.69 4.04 2.64
(1ml/kg) ± ± ± ± ± ± ± ± ± ± ±
4.50 5.78 9.29 12.69 11.90 3.02 3.21 0.04 0.08 1.50 0.26
Silymarin 48.73 36.45 42.90 132.55 166.05 48.15 51.19 1.03 0.30 6.28 4.11
(20) ± ± ± ± ± ± ± ± ± ± ±
3.92*** 3.22** 4.45*** 7.89*** 12.30*** 5.53** 4.81*** 0.04*** 0.07** 1.25 ns 0.22**
Luteolin 40.31 39.25 44.70 128.15 170.70 40.06 53.96 2.14 0.40 6.75 4.15
(250) ± ± ± ± ± ± ± ± ± ± ±
2.76*** 4.77*** 3.09*** 7.53*** 12.64*** 3.89* 3.62*** 0.08* 0.05* 1.04 ns 0.12**
Luteolin 39.76 24.29 43.88 116.10 156.01 45.76 39.18 1.28 0.40 7.04 4.25
(500) ± ± ± ± ± ± ± ± ± ± ±
3.40*** 3.36*** 4.37*** 8.54*** 6.77*** 4.03** 3.85*** 0.11** 0.02* 1.44 ns 0.12***
The results are expressed as Mean ± SEM. One way ANOVA followed by Turkey’s multiple comparison tests. *p < 0.05,
**p < 0.01, ***p < 0.001 and ns = not significant when compared to the negative control group.

Table 3: Effect of luteolin on free radical scavenging ability of CCl 4 treated rats
Treatment Glutathione Lipid peroxidase Superoxide dismutase
(mg/kg,p.o.) µg/g of liver nmol/g of protein Unit/mg of protein
Vehicle control 20.19 ± 1.87 3.94 ± 0.67 6.57 ± 0.94
CCl4 (1ml/kg) 6.46 ± 0.71 26.58 ± 1.84 3.61 ± 0.63
Silymarin (20) 18.05 ± 1.34*** 5.59 ± 0.64*** 6.46 ± 0.96ns
Luteolin (250) 12.94 ± 0.74** 6.14 ± 0.69*** 5.99 ± 0.83 ns
Luteolin (500) 16.27 ± 1.13*** 5.13 ± 0.58*** 6.48 ± 0.88 ns

The results are expressed as Mean ± SEM. One way ANOVA followed by Turkey’s multiple comparison tests. **p <
0.01, ***p < 0.001and ns = not significant when compared to the negative control group

4. DISCUSSION
Preclinical studies have shown that luteolin possesses a Luteolin might be a rational candidate for the prevention
variety of pharmacological activities, including of liver injury among individuals exposed to hepatotoxic
antioxidant, anti-inflammatory, antimicrobial and agents. The hepatoprotective potential of a drug depends
anticancer. With reference to these, we attempt to examine upon its ability to normalize the harmful effects caused by
its potential beneficial effects on in vivo and in vitro a hepatotoxin. Herbs produce clinical effects due the
toxicological model of liver injury. presence of multiple phytoconstituents.

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 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4 1483

A B

C D

Fig. 3 Assessment of CCl4 induced hepatotoxicity by

histopathology in haematoxylin-eosin stained liver
sections. (A) Section from normal liver tissue showing
normal hepatic architecture. (B) CCl4 intoxicated liver
tissue showing zonal necrosis, extensive diffuse vacuolar
degeneration engorged with blood and microvesicular fatty
changes in hepatocytes. (C) Silymarin treated liver tissue
showed focal necrosis, lobular necrosis and slightly altered
hepatic parenchyma. (D) Luteolin (250 mg/kg)
pretreatment showed focal coagulative necrosis, zonal
necrosis and mild fatty vacculation. (E) Luteolin (500
E mg/kg) treatment showed mild portal inflammation and
slight fatty changes with normal hepatocyte (10×).

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 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4
Plant derived phytoconstituent such as flavonoids,
terpenoids, carbohydrates, tannins, saponins, steroids,
proteins, amino acids and vitamin C have received
considerable attention in recent years due to their diverse
therapeutic properties including antioxidant and
hepatoprotective activity [17].
Several flavonoids present naturally in food and plants,
have been shown to modify critical reactions that cause
inhibition of chemically induced hepatocarcinogenesis.
Luteolin is a 3′,4′,5,7-tetrahydroxyflavone, belongs to a
group of naturally occurring compounds called flavonoids
that are found widely in the plant kingdom. Flavonoids are
polyphenols that play an important role in defending plant
cells against micro-organisms, insects, and UV irradiation
[18]. Evidence from cell culture, animal and human
population studies has suggested that flavonoids are
beneficial to human and animal health. Because of their
abundance in foods, e.g., vegetables, fruits, and medicinal
herbs, flavonoids are common nutrients that are
antioxidants, estrogenic regulators and antimicrobial
agents [19]. It has been noticed that luteolin protect the
liver from various kind of insults like d-galactosamine,
CCl4 and N-nitrosodiethylamine in animal models [8],
[20], [21]. This study protocol was developed to explore
all the aspects of luteolin on CCl4 induced liver damage viz
effect on cell viability, microsomal enzyme functional
ability, antioxidant capability and cellular regenerating
potency to have an inside view on mechanism of action.
The present study outcome showed that oral
administration of luteolin in rats does not have any toxic
effect on the body weights and relative organ weights.
Luteolin revealed dose dependent gain in body weight and
significant decrease in relative liver weight of CCl4 treated
rats. The observed decrease in body weights and marked
increase in relative weights of liver in the CCl4 group
confirm its toxic effect on rats as reported by other
researchers [22].
Barbiturates are a class of xenobiotics that are extensively
metabolized in the liver. Deranged liver function leads to
delay in the clearance of barbiturates, resulting in a longer
duration of hypnotic effect [23]. In the present study,
administration of thiopental sodium to rats treated with
CCl4 alone resulted in an increased duration of thiopental-
induced sleeping time. Pre-treatment of luteolin followed
by the post-treatment with luteolin and CCl4 resulted in
highly significant decrease in thiopental-induced sleeping
time which is an indirect evidence of its hepatoprotective
effect.
Luteolin at both doses showed highly significant
improvement in capacity of the damaged liver to take BSP
as the level of dye in incubated media were lowered in
luteolin treated liver in comparison to the CCl4 treated rats.
This increased uptake of BSP by the liver slices showed its
enhanced capacity to excrete the dye from the blood and
further ascertain its hepatoprotective potential.
In the present study, liver damage induced by CCl4 has
selected for screening of hepatoprotective activity of drug
under test since, the enzymatic, biochemical and
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1484
histopathological changes associated with the CCl4
induced liver damage are similar to that of viral hepatitis.
CCl4 is a potent hepatotoxin and a single dose of it causes
lipid peroxidative degradation of biomembrane, increase
in levels of serum enzymes followed by severe centrizonal
necrosis and steatosis [24]. In liver, CCl4 is biotransformed
by cytochrome P450 to produce its active metabolite
trichloromethyl free radical, which binds to the lipoprotein
and leads to peroxidation of lipids of the endoplasmic
reticulum rich in polyunsaturated fatty acids [25]. This
leads to the formation of lipid peroxide which in turn gives
toxic aldehyde that causes damage to liver. The
peroxidative products induce hypoperfusion of the
membrane and finally cytosolic enzymes appear in the
blood. Damaged liver cell plasma membrane starts to
release a variety of enzymes SGOT, SGPT, ALP,
cholesterol, tri-glyceride, LDL and bilirubin into the
circulation therefore; these can be measured in serum.
The elevated level of these entire marker enzymes were
observed in CCl4 treated rats corresponded to the extensive
liver damage induced by toxin. The increased SGPT is a
sensitive indicator of acute liver damage and elevation of
this enzyme may due to damage of the tissue producing
acute hepatic necrosis, such as viral hepatitis and acute
cholestatis. SGPT is more selectively a liver parenchyma
enzyme than SGOT to justify the liver damage, since
SGOT also present in kidney and cardiac muscle [26].
Fourteen days treatment of luteolin (250 and 500 mg/kg)
highly significant decrease the elevated levels of SGPT,
SGOT which may be a consequence of the stabilization of
plasma membrane as well as repair of hepatic tissue
damage caused by CCl4 [27]. The elevated level of ALP in
toxicated group may be associated with defective hepatic
excretion or by increased production by hepatic
parenchymal or duct cells [28]. The luteolin showed
extremely significant normalization in serum ALP
indicating an improvement in the secretary mechanism
hepatic parenchyma and duct cells.
Prominent increase in level of cholesterol, serum
triglycerides, LDL and decreased level of HDL was
observed in CCl4 treated rats. Increase in cholesterol level
in toxicated group may attribute to the inhibition or
destruction of triglycerides secretary mechanism by liver.
Oral administration of luteolin (250 and 500 mg/kg)
significantly reduced the level of total cholesterol, serum
triglyceride and LDL that may be due to improvement in
lipoprotein biosynthesis capacity of luteolin treated
groups. In luteolin treated group, it is observed that HDL
cholesterol was increased in dose dependent manner.
Inhibition of cholesterol absorption caused by silymarin
and luteolin could be due to positive changes in plasma
cholesterol lipoprotein profile and in lipid content.
Serum bilirubin is one of the marker test employed in the
diagnosis of hepatic diseases. It provides useful clinical
information on how well the liver is functioning [29].
Bilirubin, a chemical breakdown product of red blood cells
and hemoglobin, is conjugated with glucuronic acid in
hepatocytes to increase its water solubility. Bilirubin
concentration has been used to evaluate chemically
 International Journal of Indigenous Medicinal Plants, ISSN:2051-4263, Vol.46, Issue.4
induced hepatic injury. The marked increase in
concentration of bilirubin level of CCl4 treated rats clearly
indicates the toxic effect of CCl4 on the liver. Luteolin
treatment prevented severity of liver damage caused by
CCl4 as evidenced by the low level of total and direct
bilirubin in the serum. Luteolin also showed remarkable
reduction of sinusoidal dilation, zonal necrosis and fatty
changes in liver induced by CCl4. This revealed the
hepatoprotective role of luteolin having recovery of liver
damage at a significant level.
The site specific oxidative damage of some protein
susceptible amino acids is regarded as the major cause of
metabolic dysfunction during the pathogenesis of
oxidative damage [30]. Hypoalbumaniaemia is most
frequent in presence of chronic liver diseases. Hence
decline in total protein content can be deemed as a useful
index of severity of cellular dysfunction in chronic liver
diseases. The lower level of total protein recorded in
serum of CCl4 treated group reveals the severity of
hepatopathy. Luteolin treatment did not show any
significant effect toward attaining normal protein content
in CCl4 intoxicated animals. This signifies that luteolin
may not have effect regarding improvement of cellular
regeneration of macromolecules. The reduced level of
albumin concentration was also observed in the CCl4
treated group which may be due to decreased hepatic
capacity of synthesize protein. Luteolin along with the
CCl4 showed dose dependent normalization in serum
albumin signifying hepatic cell regenerating effect. The
tendency of these enzymes to return towards normal in
luteolin treated group is a clear manifestation of
antihepatotoxic effect of luteolin.
Glutathione is one of most abundant tripeptide antioxidant
present in liver. It has been suggested that this compound
protect the thiol groups of protein from oxidation by free
radicals. In present study CCl4 depleted the GSH level
with an association of increased lipid peroxidation, which
leads to tissue injury and liver damage. But luteolin
reversed the changes and increased the GSH level
significantly which refers to antioxidant characters of
luteolin. Lipid peroxidation is a destructive process
causing liver injury followed by CCl4 administration. In
this study the level of TBARS in rat liver was increased
suggesting enhanced lipid peroxidation during tissue
damage and failure of antioxidant defense mechanism to
prevent formation of excessive free radicals. Luteolin
treatment significantly inhibited the lipid peroxidation
emphasizing its antioxidant effect.
5. CONCLUSION
From the results of the current investigation, it can be
concluded that luteolin has the ability to change the levels
of glutathione, lipid peroxidase and significantly increase
the endogenous antioxidant defense mechanisms in CCl4
induced hepatotoxicity. Our finding also showed that
luteolin treatment significantly normalizes the changes in
serum markers of CCl4 treated rats. From the results
obtained, we suggested that luteolin may be developed as
an effective hepatoprotective agent. Further studies are
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1485
underway to elucidate the molecular mechanisms involved
to prove luteolin efficacy as a hepatoprotective agent.
ACKNOWLEDGEMENT
The authors are thankful to Mr. Ajay Atre and Mrs.
Shaheen, Food and Drug Administration, Bhopal for
providing the necessary facilities to carry out the HPTLC
analysis.
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