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Effect of Curcumin and its Analogue on Lipids in Carbon Tetrachloride-

Induced Hepatotoxicity: A Comparative Study
N. Kamalakkannan a; R. Rukkumani a; P. Viswanathan b; K. N. Rajasekharan c; Venugopal P. Menon a
a
Department of Biochemistry, Faculty of Science, b Department of Pathology, Faculty of Medicine,
Rajah Muthiah Medical College, Annamalai University, Annamalainagar, Tamil Nadu, India c
Department of Chemistry, Faculty of Science, Kerala University, Kariavattom, Kerala, India

To cite this Article Kamalakkannan, N., Rukkumani, R., Viswanathan, P., Rajasekharan, K. N. and Menon, Venugopal
P.(2005) 'Effect of Curcumin and its Analogue on Lipids in Carbon Tetrachloride-Induced Hepatotoxicity: A
Comparative Study', Pharmaceutical Biology, 43: 5, 460 — 466
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 Pharmaceutical Biology
2005, Vol. 43, No. 5, pp. 460–466

Effect of Curcumin and its Analogue on Lipids in Carbon

Tetrachloride–Induced Hepatotoxicity: A Comparative Study

N. Kamalakkannan1, R. Rukkumani1, P. Viswanathan2, K.N. Rajasekharan3, and Venugopal P. Menon1

1
Department of Biochemistry, Faculty of Science, and 2Department of Pathology, Faculty of Medicine, Rajah Muthiah
Medical College, Annamalai University, Annamalainagar, Tamil Nadu, India; 3Department of Chemistry, Faculty of
Science, Kerala University, Kariavattom, Kerala, India

Abstract
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Curcumin and its analogue (bisdemethoxy curcumin
analogue [BDMC-A]) were studied for their possible
lipid-lowering properties in carbon tetrachloride (CCl4)-
induced hepatotoxicity in rats. Carbon tetrachloride
requires biotransformation by hepatic microsomal P450
to produce the hepatotoxic metabolite, trichloromethyl

radical (CCl3) which, in the presence of oxygen, is further
converted to a peroxy radical (CCl3OO ). These radicals


(3 ml kg
1 wk
1) administration to albino Wistar rats
increased the levels of hepatic marker enzymes such as
aspartate transaminase (AST), alkaline phosphatase
(ALP), and c-glutamyl transferase (GGT) in the plasma.
The levels of lipids cholesterol, triglycerides, and free
fatty acids were also increased in plasma and tissues
(liver, kidney, heart, and brain). Phospholipid levels
increased in plasma, heart, and brain but decreased in
liver and kidney. Curcumin (80 mg=kg) and BDMC-A
(80 mg=kg) administration to CCl4-treated rats for a per-
iod of 3 months significantly decreased the lipid levels.
The effect exerted by BDMC-A was more prominent
than that of curcumin. Studies on the histopathology
of the liver are also in line with the biochemical
parameters studied. These observations show the lipid-
lowering efficacy of curcumin and its analogue in CCl4-
induced hepatotoxicity.
Keywords: bisdemethoxy curcumin analogue, carbon
tetrachloride, curcumin, lipids.
may interact with membrane lipids leading to peroxi-
dation (Muriel, 1997). CCl4 causes an imbalance between
the synthesis and degradation of lipids (Boll et al., 2001).
Curcuma longa Linn. (Zingiberaceae) is a medicinal
plant widely cultivated in tropical regions of Asia. Cur-
cumin (diferuloyl methane) (Figure 1) is extracted from
the rhizomes of Curcuma longa. Curcumin possesses a
wide variety of pharmacological properties (Quiles et al.,
2002). The lipid-lowering effect of curcumin in CCl4-
induced hepatotoxicity (Akila et al., 1998) and in alcohol
and polyunsaturated fatty acid–induced hyperlipidemia
(Rukkumani et al., 2002) were reported. bisdemethoxy-
curcumin (BDMC) is a natural curcuminoid that also
possess lipid-lowering properties in high fat diet–induced
lipid accumulation (Asai & Miyazawa, 2001). An ana-
logue of bisdemethoxycurcumin (BDMC-A) (Figure 2)
has been reported for its hypolipidemic properties
(Rukkumani et al., 2004a) along with its antioxidant
(Rukkumani et al., 2004b) and antidiabetic (Anusuya
et al., 2003) properties.

Introduction
Carbon tetrachloride (CCl4) is an important model agent
to study the pathogenesis of liver injury (Boll et al.,
2001). It is known to cause damage to the liver, lungs,
adrenals, and central nervous system in humans and
experimental animals (McGregor & Lang, 1996). CCl4 Figure 1. Curcumin.

Accepted: April 1, 2005

Address correspondence to: Dr. Venugopal P. Menon, Professor and Head, Department of Biochemistry, Annamalai University,
Annamalainagar 608 002, Tamil Nadu, India. Tel: þ 91 4144 238343; Fax: þ 91 4144 238343; E-mail: biocmr@sify.com

DOI: 10.1080/13880200590963880 # 2005 Taylor & Francis Ltd.

 Curcumin and its analogue on lipids 461

Figure 2. BDMC-A.

There are no available reports on the hypolipidemic

properties of BDMC-A in CCl4-induced hepatotoxicity.
Hence, we made an attempt to study the effect of Figure 3. Liver section of normal rat showing normal
BDMC-A and to compare it with curcumin on lipid pro- parenchymal architecture (H&E, 10).
file in CCl4-induced hepatotoxicity in rats.

administered BDMC-A (80 mg=kg body weight)

(Anusuya et al., 2003).
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Materials and Methods Group IV Rats subcutaneously injected with CCl4

Drugs and chemicals
Curcumin was obtained from Sigma Chemical Company
(St. Louis, MO, USA). BDMC-A was synthesised as
described by Babu and Rajasekharan (1994). CCl4 was
purchased from Merck Ltd. (Mumbai, India). All other
chemicals and biochemicals used in our study were of
high analytical grade.
(3 ml=kg body weight per week) (Akila et al., 1998)
and orally administered saline.
Group V Rats orally administered curcumin (80 mg=kg
body weight) along with subcutaneous injection of
CCl4 (3 ml=kg body weight per week).

Experimental animals
Male albino Wistar rats of body weight 150–180 g were
obtained from the Central Animal House, Rajah
Muthiah Medical College and Hospital, Annamalai
University. The rats were housed in polypropylene cages
lined with husk. They were fed on a standard pellet diet
(Agro Corporation Private Ltd., Bangalore, India), and
water was freely available. The standard pellet diet com-
prised 21% protein, 5% lipids, 4% crude fiber, 8% ash,
Figure 4. No histological alterations were observed in rats
1% calcium, 0.6% phosphorus, 3.4% glucose, 2%
treated with curcumin or BDMC-A (H&E, 10).
vitamin, and 55% nitrogen-free extract (carbohydrates).
It provides metabolizable energy of 3600 kcal=kg.

Experimental design
A total of 36 rats were used in our study. The rats were
divided into 6 groups of 6 rats each.

Group I Normal control rats injected with saline subcu-

taneously (3 ml=kg body weight per week) and orally
administered with saline.
Group II Normal rats injected with saline subcu-
taneously (3 ml=kg body weight per week) and orally
administered curcumin (80 mg=kg body weight)
(Rukkumani et al., 2002).
Group III Normal rats injected with saline subcuta- Figure 5. No histological alterations were observed in rats
neously (3 ml=kg body weight per week) and orally treated with curcumin or BDMC-A (H&E, 10).
 462 N. Kamalakkannan et al.
Figure 6. Thickening of blood vessels caused by CCl4 admin-
istration (H&E, 10).
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Figure 7. CCl4 þ curcumin administration showing mild sinus-
oidal dilatation (H&E, 10).
Group VI Rats orally administered BDMC-A (80 mg=kg
body weight) along with subcutaneous injection of
CCl4 (3 ml=kg body weight per week).
The experiment was carried out for a period of 3
months. All the experimental protocols were approved
by the Ethical Committee of Annamalai University.
Figure 8. Mild portal inflammation seen in CCl4 þ BDMC-A
treated rats (H&E, 10).
After the last treatment, the animals were fasted over-
night and sacrificed by cervical dislocation. Blood was
collected in heparinized tubes. Plasma was separated
and used for various biochemical estimations. Liver,
kidney, heart, and brain were collected in ice-cold
containers, washed with saline, homogenized with appro-
priate buffer, and used for various estimations.
Biochemical estimations
In plasma, the levels of marker enzymes such as AST
(aspartate transaminase), ALP (alkaline phosphatase),
and GGT (c-glutamyl transferase) were estimated by
the methods of Reitman and Frankel (1957), King and
Armstrong (1988), and Fiala et al. (1972), respectively.
Lipids were extracted using a chloroform-methanol
mixture (2:1 v=v) by the method of Folch et al. (1957).
Total cholesterol (Zlatkis et al., 1953), triglycerides
(Foster & Dunn, 1973), free fatty acids (Falholt et al.,
1973), and phospholipids (Zilversmit et al., 1950) were
estimated in plasma and tissues.
For histopathological studies, livers from animals of
different groups were perfused with 10% neutral forma-
lin solution. Paraffin sections were made and stained
using hematoxylin-eosin (H&E) stain. After staining,
the sections were observed under a light microscope
and photographs were taken.
Statistical analysis
Statistical analysis was performed using one-way analysis of
variance (ANOVA) followed by Duncan’s multiple range
test (DMRT). The values are mean SD for 6 rats in
each group. p values < 0.05 were considered as significant.
Results
The effect of oral administration of curcumin and
BDMC-A on plasma AST, GGT, and ALP levels in
normal and CCl4-induced rats is presented in Table 1.
A significant increase in the levels of these marker
enzymes was observed in CCl4-treated rats. On treatment
with both curcumin and BDMC-A, the levels of these
enzymes were found to be significantly decreased.
Table 2 presents the levels of cholesterol, triglycerides,
free fatty acids, and phospholipids in normal and CCl4-
treated rats in plasma. The levels of these lipids signifi-
cantly increased in CCl4-treated rats. Administration of
curcumin and BDMC-A to these rats significantly
decreased the lipid levels.
The levels of cholesterol, triglycerides, free fatty
acids, and phospholipids in the tissues of normal and
CCl4-treated rats are presented in Tables 3–6. In CCl4-
administered rats, the levels of these lipids significantly
increased while phospholipids decreased in liver and
 Curcumin and its analogue on lipids 463

Table 1. Effect of curcumin and BDMC-A on ALP, GGT, and AST in normal and CCl4-treated rats.

Groups ALP (IU=l) GGT (IU=l) AST (IU=l)

a a
Normal 73.10  4.13 0.57  0.04 73.13  5.47a
Curcumin 74.66  6.12a 0.55  0.03a 74.89  7.02a
BDMC-A 74.36  5.80a 0.57  0.05a 73.61  6.71a
CCl4 193.13  13.28b 1.65  0.11b 139.02  9.42b
CCl4 þ curcumin 117.30  6.54c 0.91  0.04c 94.62  4.18c
CCl4 þ BDMC-A 98.85  5.68c 0.72  0.04d 83.91  4.76d

ALP, alkaline phosphatase; GGT, c-glutamyl transferase; AST, aspartate transaminase.

Each value is mean SD for 6 rats in each group.
Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05.

kidney. Treatment with curcumin and BDMC-A exerted Radical formation and lipid peroxidation are the pre-
a significant effect on the lipid levels. dominant cellular mechanisms involved in the develop-
In all the parameters studied, administration of curcu- ment of fatty liver caused by CCl4 (Tribble et al.,
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min and BDMC-A to normal rats did not show any sig-
nificant effect. Results obtained with BDMC-A were
found to be more effective than those of curcumin.
Discussion
Lipids are more easily attacked by the activated metabo-
lites of CCl4 resulting in damage to intracellular mem-
branes and the plasma membrane (Cheeseman et al.,
1985). As a result of membrane damage, the levels of
marker enzymes such as AST, ALP, and GGT increase
in plasma. This increase is mitigated by treatment with
curcumin and BDMC-A. Curcumin, by scavenging or
neutralizing free radicals, inhibits peroxidation of mem-
brane lipids and maintains cell membrane integrity and
their function (Rukkumani et al., 2003). The presence
of hydroxyl group in the ortho position of the aromatic
ring in BDMC-A may be responsible for the inhibitory
effect (Anto et al., 1996). The o-hydroxyl group, because
of its resonance property, easily donates e
to free
radicals and effectively neutralizes them. Curcumin and
BDMC-A stabilize cell membrane integrity and prevent
the increase of these marker enzymes.
1987). Extensive accumulation of lipids is regarded as a
pathological condition, and when the accumulation
becomes chronic, fibrotic changes occur in the cells that
progress to cirrhosis and impaired liver function (Murray
et al., 1993). An increase in the levels of cholesterol, tri-
glycerides, and free fatty acids were noted in plasma and
tissues. CCl4 increases the synthesis of fatty acids and tri-
glycerides from acetate. This could be due to the trans-
port of acetate into the liver cell, resulting in increased
substrate (acetate) availability. In CCl4 toxicity, the syn-
thesis of cholesterol is also increased (Boll et al., 2001).
On the other hand, CCl4 lowers b-oxidation of fatty
acids and hydrolysis of triglycerides. This increases the
availability of fatty acids to esterification (Lieber, 2000).
Reports have also shown that during CCl4 toxicity, fat
from the peripheral adipose tissue is translocated to the
liver and kidney leading to its accumulation (Devarshi
et al., 1986). Moreover, the synthesis of apolipoproteins
is inhibited by CCl4 (Honma & Suda, 1997) subsequently
resulting in the decreased synthesis of lipoproteins.
A decrease in the secretion of bile acids is also reported
(Boll et al., 2001).
Phospholipids are the vital components of biomem-
branes. They are more susceptible to CCl4-induced lipid

Table 2. Effect of curcumin and BDMC-A on plasma lipids in normal and CCl4-treated rats.

Cholesterol Triglycerides Phospholipids Free fatty acids

Groups (mg=dl)
a a
Normal 78.44  5.43 106.90  9.52 97.08  6.18a 71.56  8.06a
Curcumin 80.15  6.08a 108.71  10.81a 100.13  8.40a 70.28  5.38a
BDMC-A 80.30  9.36a 106.55  9.60a 101.38  8.26a 70.86  6.70a
CCl4 186.75  17.61b 266.54  23.00b 192.35  20.67b 160.45  14.18b
CCl4 þ curcumin 117.10  9.84c 171.22  18.62c 147.31  13.40c 94.06  9.64c
CCl4 þ BDMC-A 88.33  7.32a 113.86  9.36a 108.60  6.74d 77.30  7.10a

Values are mean SD from 6 rats in each group.

Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05.
 464 N. Kamalakkannan et al.

Table 3. Effect of curcumin and BDMC-A on cholesterol level in tissues of normal and CCl4-treated rats.

Liver Kidney Heart Brain

Groups (mg=100 g tissue)

a
Normal 281.16  22.04 344.61  18.20a 162.74  10.52a 1106.33  84.30a
Curcumin 286.70  16.50a 348.50  13.80a 163.41  5.41a 1109.20  66.18a
BDMC-A 284.33  20.62a 342.36  22.96a 161.26  7.90a 1120.36  70.02a
CCl4 442.40  60.51b 586.64  60.88b 414.60  22.18b 1432.81  98.42b
CCl4 þ curcumin 355.08  34.16c 404.02  12.50c 268.43  8.72c 1258.92  96.55c
CCl4 þ BDMC-A 302.42  22.16d 372.33  18.17d 196.20  12.55d 1132.18  74.39d

Values are mean  SD from 6 rats in each group.

Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05.

Table 4. Effect of curcumin and BDMC-A on triglycerides level in tissues of normal and CCl4-treated rats.

Liver Kidney Heart Brain

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Groups (mg=100 g tissue)

Normal 310.08  16.73a 462.36  31.55a 302.08  12.36a 340.28  14.20a

Curcumin 312.71  18.55a 471.36  31.50a 306.65  8.57a 336.15  16.55a
BDMC-A 316.54  20.12a 464.52  40.73a 300.59  11.50a 341.34  11.28a
CCl4 573.56  26.80b 660.18  34.20b 566.58  36.24b 610.08  25.41b
CCl4 þ curcumin 397.22  21.60c 521.22  38.66c 379.40  26.55c 428.16  32.66c
CCl4 þ BDMC-A 326.02  20.07d 474.36  32.50a 328.81  28.50d 362.86  20.06a

Values are mean  SD from 6 rats in each group.

Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05.

Table 5. Effect of curcumin and BDMC-A on free fatty acids level in tissues of normal and CCl4-treated rats.

Liver Kidney Heart Brain

Groups (mg=100 g tissue)

Normal 612.88  32.04a 370.48  16.56a 412.50  18.88a 12.48  1.04a

Curcumin 624.31  22.64a 374.21  21.20a 415.52  33.00a 13.06  0.94a
BDMC-A 618.76  20.85a 371.66  30.16a 411.52  12.36a 12.59  1.16a
CCl4 1230.14  86.49b 702.14  77.66b 782.34  62.70b 33.60  2.76b
CCl4 þ curcumin 785.84  46.28c 480.26  31.08c 521.48  40.17c 20.16  1.33c
CCl4 þ BDMC-A 648.08  44.16d 402.82  25.50d 436.26  30.82a 14.65  1.30d

Values are mean  SD from 6 rats in each group.

Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05.

Table 6. Effect of curcumin and BDMC-A on phospholipids level in tissues of normal and CCl4-treated rats.

Liver Kidney Heart Brain

Groups (mg=100 g tissue)

a
Normal 1510.31  122.80 1260.00  78.32a 984.02  62.10a 2310.80  121.07a
Curcumin 1518.35  102.34a 1271.16  87.53a 973.26  52.17a 2328.19  140.57a
BDMC-A 1510.72  131.60a 1342.20  66.12a 981.67  55.69a 2306.10  100.64a
CCl4 1186.10  96.54b 842.18  51.40b 1642.64  112.04b 2816.57  216.92b
CCl4 þ curcumin 1402.30  112.60c 1130.62  105.43c 1220.66  99.18c 2499.60  187.52c
CCl4 þ BDMC-A 1480.65  130.52d 1236.28  100.68a 1091.16  82.58d 2362.71  150.18a

Values are mean  SD from 6 rats in each group.

Values not sharing a common superscript (a, b, c, d) differ significantly at p < 0.05.
 Curcumin and its analogue on lipids
peroxidation than other lipid classes (Morrow et al.,
1992). A decrease in the levels of phospholipids in liver
and kidney is probably due to an increase in phospho-
lipase activity (Lamb et al., 1988). During normal
lipoprotein metabolism, phospholipids are extensively
converted into triglycerides (Wiggins & Gibbons, 1996).
CCl4-induced inhibition of lipoprotein-associated trigly-
ceride export may also result in increased release of
phospholipids from these tissues. An increase in phos-
pholipid levels in brain and heart could be due to
increased phospholipid content of their membranes.
Oral administration of curcumin to CCl4-treated rats
significantly decreased the lipid levels in plasma and
tissues. It has been reported that curcumin possesses
hypocholesterolemic action (Soni & Kuttan, 1992) and
this could be due to a decrease in absorption of choles-
terol (Rao et al., 1970) or an increase in HDL cholesterol
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(Soudamini et al., 1992). This indicates that curcumin
mobilizes excess cholesterol from extrahepatic tissues to
liver where it is catabolized. Curcumin also increases
the activity of 7a-hydroxylase, which converts choles-
terol to bile acids, facilitating the biliary cholesterol
excretion (Babu & Srinivasan, 1997). The decrease in
the levels of triglycerides and phospholipids might be
due to decreased free fatty acid (FFA) synthesis by
curcumin, which may suppress the enzymes involved in
FFA synthesis (Rukkumani et al., 2002).
Treatment with BDMC-A resulted in a significant
decrease in the lipid profile in CCl4-administered rats.
The structure of BDMC-A resembles that of curcumin,
and this may have an impact on lipid metabolism.
BDMC-A is a potent antioxidant, and the lipid-lowering
activity might be due to its antioxidant property. Pheno-
lics have been recognized as a powerful countermeasure
against lipid peroxidation (Schroeter et al., 2000).
Phenolic compounds are capable of scavenging free radi-
cals and quenching the lipid peroxidative side chain. Pan
et al. (1999) have proposed that hydroxy and hydroper-
oxy radicals initiate Hþ abstraction from a free phenolic
substrate to form phenoxy radical that can rearrange to
quinone methide radical intermediate, which is excreted
via bile. BDMC-A inhibits lipid peroxidation, as well
membrane damage and thus prevent the release of free
fatty acids. A decrease in the levels of FFA might result
in the decreased levels of other lipids.
Histopathological observations of the liver of CCl4-
administered rats showed thickening of blood vessels
(Figure 6). CCl4-administered rats when treated with
curcumin showed mild sinusoidal dilatation (Figure 7),
and treatment with BDMC-A showed a near normal his-
tology of the liver (Figure 8). These observations show the
protective effect of both curcumin and BDMC-A in CCl4-
induced hepatotoxicity in rats. No histological alterations
were observed in liver of normal rats and curcumin or
BDMC-A treated rats (Figures 3–5)
465
Curcumin and BDMC-A effectively reduced the lipid
levels altered by CCl4 metabolism. The effect exerted by
BDMC-A was more pronounced than curcumin. This
could be due to the presence of hydroxyl group at the
ortho position. Because of its positional isomerism, the
ortho hydroxyl group easily donates the e
to the free
radicals and effectively neutralizes them. Hence,
BDMC-A is more effective than curcumin in treating
hyperlipidemia.
Acknowledgments
We thank UGC for sanctioning the project. The first
author is a junior research fellow in the project.
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