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Investigation the Effects of Curcumin on Serum Hepatic Enzymes Activity in a Rheumatoid Arthritis Model

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Curcumin (diferuloylmethane) is the most active component of Curcuma longa L. Several clinical trials have found curcumin to be a notable anti- inflammatory and antioxidant compound. The present study evaluates the effects of curcumin and indomethacin treatment on the inflammatory responses and on plasma phosphatase and transaminase activities, employing an adjuvant– induced arthritis model in rat. Arthritis was induced by the subcutaneous injection of a Freund’s complete adjuvant into the plantar surface of right hind paw of rats. Development of the inflammatory response was assessed over a 21-days period. In this study a sample of 32 male Wistar albino rats (180-220 gr) were used which randomly divided into four groups with eight rats per group. Group I served as normal control rats (untreated), group II arthritic rats which received vehicle, group III arthritic rats treated with curcumin (30 mg/Kg orally) and another group, arthritic rats treated with indomethacin (3 mg/Kg reference drug orally). Treatment was started from 7 th day of adjuvant injection and was terminated on day 21. At the end of the experimental period, the animals were anesthetized and killed; blood samples were collected and prepared for measurement of alkaline phosphatase (ALP), aspartate (AST) and alanine aminotransferase (ALT) activities.
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eJBio
Electronic Journal of Biology, 2009, Vol. 4(4): 129-133
Investigation the Effects of Curcumin on Serum Hepatic
Enzymes Activity in a Rheumatoid Arthritis Model
Fatemeh Aghaei Borashan1,*, Mino Ilkhanipoor1, Mohammad Hashemi1,
Farah Farrokhi2
1 Department of Biology, Faculty of Science, Urmia University, Urmia, Iran;
2 Department of Clinical, Faculty of Veterinary, Urmia University, Urmia, Iran.

* Corresponding author. Tel: +98-9144478398; E-mail: Aghaei.fatemeh@yahoo.com

Abstract
1. Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune
Curcumin (diferuloylmethane) is the most active
disease characterized by joint swelling, synovial
component of Curcuma longa L. Several clinical
inflammation
and
cartilage
destruction
and
trials have found curcumin to be a notable anti-
commonly leads to significant disability and a
inflammatory and antioxidant compound. The
consequent reduction in quality of life [1-4].
present study evaluates the effects of curcumin and
Epidemiology of the arthritis in female: male is 3:1
indomethacin treatment on the inflammatory
and afflicts generally 0.5-1.0% of the population
responses and on plasma phosphatase and
around the world [5]. RA caused by number of pro-
transaminase activities, employing an adjuvant–
inflammatory molecules released by macrophages
induced arthritis model in rat. Arthritis was induced
[6]. These include reactive oxygen species and
by the subcutaneous injection of a Freund’s
eicosanoids such as, prostaglandins, leukotrienes
complete adjuvant into the plantar surface of right
and cytokines [7, 8]. The regulation of these
hind paw of rats. Development of the inflammatory
mediators secreted by macrophages and other
response was assessed over a 21-days period. In
immune cells therefore may control the chronic
this study a sample of 32 male Wistar albino rats
inflammatory conditions [9, 10]. Many studies have
(180-220 gr) were used which randomly divided into
demonstrated
the
involvement
of
aberrant
four groups with eight rats per group. Group I
Arachidonic acid metabolism in the inflammatory
served as normal control rats (untreated), group II
response
by
generating
potent
chemical
arthritic rats which received vehicle, group III
messengers known as eicosanoids. Membrane
arthritic rats treated with curcumin (30 mg/Kg orally)
phospholipids, the major source of arachidonic acid
and another group, arthritic rats treated with
are hydrolyzed by phospholipase A
indomethacin (3 mg/Kg reference drug orally).
2 (pLA2); the
released arachidonic acid is further metabolized by
Treatment was started from 7th day of adjuvant
three different types of oxygenases: cyclooxygenase
injection and was terminated on day 21. At the end
(COX), liopxygenase (LOX) and cytochrome P
of the experimental period, the animals were
450.
Modulation of arachidonic acid metabolism by
anesthetized and killed; blood samples were
inhibiting these enzymes has been considered as an
collected and prepared for measurement of alkaline
effective mechanism for chemoprevention [11]. For
phosphatase (ALP), aspartate (AST) and alanine
the treatment of RA, although non-steroidal anti-
aminotransferase (ALT) activities.
inflammatory drugs (NSAIDs), steroidal agents and
The results indicated that inflammation and
immunosuppressants usually used, their side effects
plasma ALP, AST and ALT activities were very
and toxicity call for new and more effective natural
elevated in the arthritic rats and curcumin treatment
drugs.
to similar indomethacin were significantly reduced
Curcumin
(diferuloylmethane)
is
a
major
the swelling of paws and plasma ALP, AST and ALT
constituent found in the spice turmeric, which is a
activities in arthritic rats.
dried powder from the rhizomes of Curcuma longa L.
This spice has been used as a traditional medicine
In conclusion, the present data suggest that
for treatment of inflammation, gastrointestinal
aminotransferases and alkaline phosphatase were
disorders, hepatic disorders, diabetic wounds, skin
significantly reduced in arthritic rats after the
wounds, rheumatism, sinusitis and other disorders
administration of curcumin. This reducing effect may
[12] Curcumin is also a food coloring and flavoring
be related to their anti-inflammatory activity.
agent
approved
by
the
Food
and
Drug
Keywords:
Curcumin;
Aminotransferanses;
Administration [11]. It is believed that curcumin is a
Inflammatory; Arthritis.
potent antioxidant and anti-inflammatory agent [13]
(Figure 1).
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eJBio
Electronic Journal of Biology, 2009, Vol. 4(4): 129-133
surroundings over 1 week to eliminate the effect of
stress prior to initiation of the experiments.
2.2 The induction of arthritis

The method described by Newbould in 1963 was

employed with some modifications [22]. Arthritis was
Figure 1. Chemical structure of curcumin
induced by a single subcutaneous injection of 0.1 ml

Curcumin affects arachidonic acid metabolism by
Freund’s complete adjuvant (FCA), suspension of
blocking the phosphorylation of cPLA
heat-killed Mycobacterium tuberculosis in mineral
2, decreasing
the expression of COX-2 and inhibiting the catalytic
oil, into a footpad of the right hind of male rats. A
activities of 5-LOX. These activities may contribute
glass syringe (1 ml) with a 21 GA needle was used
to the anti-inflammatory actions of curcumin and its
for injection, while, the vehicle control rats were
analogs [11]. Evidence suggests that in some
injected with 0.1 ml normal saline. The rats were
experimental conditions, curcumin can have similar
anesthetized
with
Ketamin
(70mg/kg
anti-inflammatory activity as some of the common
intraperitoneally [I.P]) and Xylazin (5 mg/kg I.P) prior
NSAIDs like indomethacin, Vioxx, Celebrex, and
to and during adjuvant injection.
ibuprofen, but without many of the side effects, such
2.3 Experimental setup
as gastrointestinal distress and cardiovascular
complications [14, 15].
Animals were divided into four groups of eight
Adjuvant induced arthritis (AIA) is experimental
animals in each group as follows:
rats, a chronic inflammatory disease characterized
Group I: Control rats (untreated)
by infiltration of the synovial membrane and
Group II: Adjuvant induced arthritic rats (0.2 ml
associated with destruction of the joints resembles
normal saline)
closely to the human rheumatoid arthritis [16-18].
Group III: Arthritis treated by curcumin (30
Tissue damage was assessed by measuring the
mg/kg/day, orally)
activities of enzyme in the serum and in the
Group IV: Arthritis treated by indomethacin (3
respective organ, since liver impairment is also a
mg/kg/day, orally, reference drug) for 15 days by
feature of adjuvant arthritis [19]. The increase in
gavage starting 7 days after adjuvant injection.
aminotransferases is due to their release from the
The hind paw thicknesses (mm) were measured
cells
of
the
damaged
organ
[20].
using a vernier caliper on days 0, 7, 14, 21 after
Hyperphosphatasamia
has
been
observed
FCA injection. At the end of the experimental period
occasionally in the patients with rheumatoid arthritis,
(on day 22nd), rats were fasted overnight and killed
and it has been suggested that the serum alkaline
after general anesthesia by inhalation of ether.
phosphatase level is related to the activity of the
Blood samples were collected from the abdominal
disease [21]. The present study is designed to
aorta and serum immediately separated.
investigate
the
effects
of
curcumin
and
2.4 Serum biochemistry measurement
indomethacin on inflammation and the relationship
between serum alkaline phosphatase (ALP),
Assay of AST and ALT activities was modified by
aspartate aminotransferase (AST) and alanine
the International Federation of Clinical Chemistry
aminotransferase (ALT) activities and rheumatoid
(IFCC) and become the recommended methods for
arthritis in AIA rats.
measurement of AST and ALT activities in serums
[23]. The activities of the two enzymes are reflected
2. Materials and Methods
by the decrease of NADH in enzyme-coupled
reactions as shown in the following equations:
2.1 Materials

L ? Aspartate (L ? Alanine) + a ? ketoglutarate AST (ALT)
Curcumin, Freund’s complete adjuvant (FCA),
Oxalacetate (pyruvate) + L ? Glutamate (1)
Indomethacin were purchased from Sigma–Aldrich,

Ether was purchased from Merck. Ketamin
Oxalacetate (pyruvate) + NADH + H+ MDH (LDH)

(Germany Rotex Medica), Xylasin (Holland Alfasan
L ? malate (lactate) + NAD+ (2)
woerden). All other chemicals used were of the

analytical reagent grade.
Alkaline phosphatase (ALP) was estimated by
Animals: As sample of 32 male Wistar albino rats
the p-nitropheyl phosphate (P-NPP) method using
(180-220g; departmental animal house; Faculty of
the recommendations of German society for Clinical
Science; Urmia University) were used. They were
Chemistry (optimized IFCC, P NPP-AMP method)
maintained under controlled temperature (22±2ºC)
[24].
and relative humidity (40-60%) with standard

environmental conditions of 12h light/dark cycle in
ALP
the departmental animal house. Food and water
P-Nitrophenyl phosphate
p-Nitrophenol +pi
were
provided
ad
libitum
throughout
the

experiments. Rats were acclimated to their
2.5 Statistical analysis

ISSN 1860-3122
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eJBio
Electronic Journal of Biology, 2009, Vol. 4(4): 129-133
All the results were expressed as mean ± standard
500
error (S.E.M). Data were analyzed using one-way
a
450
ANOVA followed by Tukey MRT test. p<0.05 were
400
considered as statistically significant.
350
b
b
300
3. Results and Discussion
/
l
)

(
u
250
T
L
A
3.1 Effect on adjuvant-induced rat paws edema
200
150
Figure 2 depicts the anti-inflammatory effect of
c
100
curcumin and indomethacin on the changes in paw
50
edema of control and experimental animals.
0
Swelling and redness developed over a 24 h period
A
B
C
D
in the foot injected with adjuvant. This inflammation
Treatment groups
reaction increased over time when disseminated


arthritis appeared and remains constant by the end
Figure 3. Effect of curcumin and indomethacin on serum
of 1 week. After curcumin and indomethacin
alanin aminoteransferase in rats with adjuvant arthritis. A:
treatment the increase in swelling of the injected
control group, B: arthritic rats treated by vehicle, C:
hind paw that occurred with the appearance of poly
arthritic rats treated with 30 mg/kg curcumin and D:
arthritis
was
suppressed
.Upon
curcumin
arthritic rats treated with 3 mg/kg indomethacin .values
administration similar to indomethacin after seven
represent means ± the S.E.M. same words aren't
days of adjuvant injection, the inflammation were
significant in p<0.05 in TUKEY MRT (n=8).
started to decrease significantly when compared to

AIA rats.
600
a
550
12
500
*
*
b
b
450
**

**
10
*
400
350
/
l
)
)
8

(
u
m
300
T
S
(
m

*
A
250
s
s

6
e
*
200
n
k

*
150
c
i
c

4
100

t
h

*
w
a

50
p
2
0
A
B
C
D
0
Treatment groups
0
7
14
21

-2

time (day)
Figure 4. Effect of curcumin and indomethacin on serum

aspartat aminoteransferase in rats with adjuvant arthritis.

A: control group, B: arthritic rats treated by vehicle, C:
Figure 2. Paws thickness in rats with adjuvant arthritis. A:
arthritic rats treated with 30 mg/kg curcumin and D:
control group (-?-), B: arthritic rats treated by vehicle (-?-),
arthritic rats treated with 3 mg/kg indomethacin. Values
C: arthritic rats treated with 30 mg/kg curcumin (-×-) and
represent means ± the S.E.M. same words aren't
D: arthritic rats treated with 3 mg/kg indomethacin (-
-
significant in p<0.05 in TUKEY MRT (n=8).
) .values represent means ± standard error of the mean

(S.E.M).
** p<0.05 compared with arthritic rat treated with vehicle
900
* p<0.05 compared with control rats (n=8).
800
a

700
3.2
Effect
on
Serum
Hepatic
600
b
Aminotransferanses
/
l
)
b
500

(
u
P
L 400
Figures 3, 4 and 5 show the changes in the
A
activities of serum aminotransferases and ALP in
300
c
control and experimental animals. As a result of
200
inflammation induced by adjuvant, the levels of
100
AST, ALT and ALP were increased (p<0.05) in all
0
arthritis rats when compared to control rats. After
A
B
C
D
Treatment groups
curcumin treatment, the levels of these enzymes

were significantly decreased (p<0.05) in group III

rats when compared to AIA rats.
Figure 5. Effect of curcumin and indomethacin on serum

alkaline phosphatase in rats with adjuvant arthritis. A:
control group, B: arthritic rats treated by vehicle, C:
arthritic rats treated with 30 mg/kg curcumin and D:
ISSN 1860-3122
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eJBio
Electronic Journal of Biology, 2009, Vol. 4(4): 129-133
arthritic rats treated with 3 mg/kg indomethacin .values
showed significantly higher serum biochemistry
represent means ± the S.E.M. same words aren't
markers (AST, ALT and ALP) than normal rats [26].
significant in p<0.05 in TUKEY MRT (n=8).
Reddy and Lokesh found that oral administration of
curcumin (30 mg/kg body weight) for 10 days
4 Conclusions
lowered the liver and serum lipid peroxide levels,
In this study, was investigated effects of curcumin
serum ALT, AST enhanced by I.P. injection of iron in
and indomethacin of paw swelling and activity of
rats [36]. Also Arafa reported Curcumin also
hepatic enzymes in adjuvant arthritis rats. Freund’s
decreased the enzyme activities of serum AST and
complete adjuvant is generally used induce arthritis
ALT [37]. The results this study indicated that
in animal models [25]. Adjuvant arthritis in rat shows
inflammatory and plasma ALP, AST and ALT
several clinical and histological similarities to human
activities were very elevated in the arthritic rats
RA [26]. In this study, interplanetary injection of
which
curcumin
treatment
to
similar
that
adjuvant into right hind paw rapidly induced paw
indomethacin were significantly reduced the swelling
swelling and redness.
of paws and plasma ALP, AST and ALT activities in
Spice principle like curcumin was shown to inhibit
arthritic rats.
the synthesis of proinflammatory prostaglandins and
In conclusion, in this study, the development of
leukotrienes [27]. Curcumin has been shown to
inflammation during induced arthritis may be related
possess beneficial effects in alleviating arthritic
to the biochemical changes of hepatic enzymes and
symptoms in both animal and human models [12,
the present data suggest that aminotransferases
28]. Bina Joe et al, have show that curcumin inhibits
and alkaline phosphatase were significantly reduced
the uptake of arachidonic acid by macrophages,
in arthritic rats after the administration of curcumin.
thereby limiting the availability of the substrate for
This reducing effect may be related to their anti-
eicosanoid production [29]. Shimizu et al, have
inflammatory activity.
demonstrated that curcumin also reduces the
conversion of linoleic acid to arachidonic acid [16].
Acknowledgements
Like curcumin, other NSAIDs such as indomethacin
This study was supported by a grant from the
lower the production of potent proinflammatory
research council of Urmia University, Urmia, Iran.
agents such as prostaglandins [29]. The results of
the present study indicated that the curcumin
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Measurement of Catalytic Activity Concentrations of

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