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Pak. J. Pharm. Sci., Vol.27, No.1, January 2014, pp.121-128
121
Protective effect of crude Curcuma longa and its methanolic
extract in alloxanized rabbits
Mobasher Ahmad*, Sairah Hafeez Kamran and Afroze Mobasher
Department of Pharmacology, University College of Pharmacy, University of the Punjab (Old Campus), Lahore, Pakistan
Abstract: Curcuma longa (C. longa) is commonly found in different areas of Pakistan. It has been locally utilized as a
traditional medicine. The aim of this study was to evaluate the antidiabetic, hepatoprotective and total antioxidant effect
of the crude drug and its methanolic extract in rabbits. Diabetes was induced with alloxan (180mg/kg). Two major
groups were designed, curative and protective groups. In curative group the crude drug and its methanolic extract was
orally administered to the diabetic animals and acute study was performed. On the other hand in protective group the
crude drug and its methanolic extract were administered for eight days prior to the diabetes induction. Results indicated
that in Curative group the crude and methanolic extract of C. longa significantly improved the levels of serum glucose,
serum transaminases and antioxidant activity (AOA). In protective group, serum glucose, serum transaminases were not
significantly increased by alloxan, in both crude as well as methanolic extract group. This study shows that C. longa acts
as antidiabetic, hepatoprotective and antioxidant in diabetes especially type 1 diabetes.
Keywords: Curcoma longa; Alloxan; Antidiabetic; Hepatoprotective; Antioxidant
INTRODUCTION
Diabetes mellitus is a metabolic disorder characterized by
increased blood sugar, polyuria and polydipsia. Diabetes
mellitus causes immune destruction of beta cells and
causes redox imbalance inside the cells, especially in the
liver and kidney. The destruction of the cells also leads to
decreased antioxidant defense mechanism and increased
free radical production. In diabetes mellitus free radical
production increases due to increased oxidative stress and
antioxidant activity is decreased. Therefore, increased free
radical production could be considered one of the
important complications of diabetes mellitus (Meral et al.,
2001).
Diabetes can be produced in animals by the drugs alloxan
and streptozotocin; the mechanism of action of these two
drugs is different, but both result in the production of
active oxygen species. It was proposed (Lenzen et al.,
1996) that alloxan destroys beta cell function by
inhibiting glucokinase activity through oxidation of two
thiol groups which are in the glucose binding site of the
enzyme. Later (Zhang et al., 2007) proposed that alloxan
also affects glucokinase activity in liver, another major
site of glucokinase expression. Therefore it was
hypothesized that part of diabetogenic effect of alloxan is
due to its effect on liver. The liver enzymes alanine
aminotransferase (ALT) and aspartate aminotransferase
(AST) are used routinely for assessing liver function.
Production of reactive oxygen species also causes
changes in kidney tubular cells, hence disturbing the
albumin-globulin ratio in the kidneys (Tierney, McPhee
and Papadakis, 2002).
C. longa L., which belongs to the Zingiberaceae family, is
an erect perennial herb with thick and fleshy rhizomes
and leaves in sheaths. The rhizome is the portion of the
plant used medicinally. C. longa is valued mainly for its
principal coloring pigment, curcumin, which imparts the
yellow colour to C. longa, besides other nutritive
constituents like potassium (Chempakam and
Parthasarathy, 2008).
C. longa L. possess compounds which are potent
inhibitors of inflammation. C. longa has antiprotozoal,
nematocidal, antibacterial, anti venom, anti HIV and
antitumor activity. C. longa also possess curcuminoids
which have phenolic and enolic structure. These types of
structures have the ability to trap radicals and are good
antioxidants (Araújo and Leon, 2001).
In the present investigation an attempt has been made to
assess the antidiabetic and antihepatotoxic and in vivo
antioxidant effects of crude and methanolic extract of C.
longa found locally, in alloxanized rabbits.
MATERIALS AND METHODS
Animals
Male rabbits weighing from 1.5 to 2.5 kg were purchased
from the local market. The animals were kept in the
animal house of the Faculty of Pharmacy with 12 hr light
dark cycle and maintained on 24±2
o
C normal
temperature. They were retained for acclimatization for a
period of 1 week before starting the experiment. The
rabbits were fed standard fresh green fodder throughout
the experiment and water ad libitum.
Chemical
Alloxan purchased from Sigma Chemical Co., St. Louis
USA was used for the induction of diabetes.
*Corresponding author: e-mail: ahmadmobasher@hotmail.com
Protective effect of crude Curcuma longa and its methanolic extract
Pak. J. Pharm. Sci., Vol.27, No.1, January 2014, pp.121-128
122
Preparation of alloxan solution
10% solution of alloxan was prepared freshly in 0.9%
sodium chloride solution before injection. 0.9g of sodium
chloride was dissolved in 100 ml of water and pH was
checked and maintained at pH 5.5 (5-7) by addition of
hydrochloric acid or sodium hydroxide. 10% solution of
alloxan was prepared and different doses i.e. 70mg/kg,
100mg/kg, 150mg/kg, 180mg/kg and 200mg/kg were
injected to five groups of rabbits (n=5). 180 mg/kg was
selected for the study as the death rate was low and stable
glucose level (200-300mg/dl) was obtained within eight
days.
Induction of diabetes
The rabbits were fasted for 12 hr prior to the induction of
diabetes mellitus. Blood was collected for zero hour
determination of serum glucose and serum transaminases.
Rabbits were injected intraperitoneally with 180 mg/kg
body weight (b.w.) of freshly prepared 10% alloxan
monohydrate (Sigma Chemical Co., St Louis USA)
dissolved in isotonic NaCl to induce Diabetes Mellitus at
the start of the experiment. Diabetes was developed and
stabilized over a period of eight days. The rabbits with
fasting glucose range of 200 to 350 mg/dl were
considered diabetic and included in the study.
Plant material
Crude C. longa Linn. (Zingeberaceae) rhizomes were
collected from Kasur district, Punjab, Pakistan. The
species was identified by Department of Botany,
Government College, Lahore. The rhizomes were peeled,
cut into small pieces and shade dried. Crude drug was
powdered. In order to obtain content uniformity the crude
powder was passed through sieve of 42 mesh size. The
crude powder was then preserved in amber colored air-
tight glass jars and was placed in refrigerator.
Preparation of crude C. longa suspension
Fresh C. longa suspension was freshly prepared daily.
Acacia was used with C. longa in ratio of 1:4 as
suspending agent. A small amount of C. longa was
triturated with acacia in a pestle and mortar using distilled
water as a vehicle. C. longa and acacia were triturated
until a smooth paste was formed then water was added to
make up the volume. C. longa suspension was given to
rabbits orally using a soft tube.
Preparation of methanolic extract of C. longa
100g of finely powdered crude C. longa was soaked in
500ml of methanol for about 15 days. The methanolic
extract was stirred at room temperature with electric
stirrer for one hour. The extract was filtered and
evaporated by using rotary evaporator at temperature less
than 40°C. The residue obtained was orange-red colored
sticky gummy material because C. longa contains
curcuminoids mainly curcumin which is an oily residue.
The yield was 7.2%. The methanolic extract suspension
was freshly prepared daily with acacia for experimental
purpose.
Analytical methods
Serum glucose and serum transaminases were determined
by using the kit supplied by Randox Lab., U.K. The total
antioxidant activity in vivo was measured in serum by
using method of Koracevic et al. (2001). In this method a
standardized solution of Fe-EDTA complex reacts with
hydrogen peroxide by a fenton type reaction, leading to
the formation of hydroxyl radicals (OH). These reactive
oxygen species degrade benzoate, resulting in the release
of TBARS. Antioxidants from the added sample cause
suppression of the production of TBARS. This reaction
can be measured spectrophotometrically and the
inhibition of color development defined as the antioxidant
activity.
Experimental design and treatment schedule
The animals were divided into 6 groups containing 6
rabbits in each group. Initially, all the parameters of the
rabbits were checked and only healthy rabbits were
selected for the study. The experimental groups were as
follows:
Curative group
Group 1 Control Diabetic Group (CDG): Alloxan
180mg/kg was injected intraperitoneally and then
the rabbits were kept on vehicle (2% gum acacia
solution) throughout the experiment.
Group 2 Crude drug Treated group (CTG): The effect of
crude C. longa (2g/kg of b.w) was determined on
serum glucose, ALT and AST in alloxan treated
diabetic rabbits 4 hrs, 8 hrs and 24 hr after the
administration of crude drug.
Group 3 Methanolic extract Treated group of C. longa
(MTG): The effect of methanolic extract of C.
longa equivalent to 2g/kg b.w of crude drug
powder was determined on the activity of serum
glucose, ALT and AST in alloxan treated rabbits
4 hrs, 8 hrs and 24 hrs after the administration of
methanolic extract
Protective group
Group 4-Protective Control group (PCG): The animals
were kept on vehicle (2% gum acacia solution)
initially for 8 days. Alloxan (180mg/kg) was
injected on 9
th
day along with other two groups
and observed for next 15 days for the
development of diabetes and hepatic damage.
Group 5-Protective Crude drug group (PCD): Crude drug
2g/kg was given for 8 days initially. Then rabbits
were injected with alloxan monohydrate
(180mg/kg) and observed for next 15 days for
the development of diabetes and hepatic damage.
Group 6 Protective Methanolic extract group of C. longa
(PMG): Methanolic extract equivalent to 2g/kg
Mobasher Ahmad et al
Pak. J. Pharm. Sci., Vol.27, No.1, January 2014, pp.121-128
123
of crude drug was given for 8 days initially. Then
rabbits were injected with alloxan monohydrate
(180mg/kg) and observed for the next 15 days
for the development of diabetes and hepatic
damage.
STATISTICAL ANALYSIS
All values were expressed as ±SEM and analyzed using
Students t test. P values<0.05 were considered significant.
P values were obtained from distribution of t probability
chart.
RESULTS
Curative group
The effect of crude and methanolic extract of C. longa on
serum glucose was observed in alloxanized diabetic
rabbits and was compared with the group injected only
with alloxan (180 mg/kg of b.w). The results are shown in
table 1. The mean glucose level in CDG 8 days after
alloxan injection was 242.17 mg/dl and at 8 hrs without
any drug was 289.5 mg/dl (↑ 165.18%) whereas the mean
glucose level in CTG 8 days after alloxan injection was
218 mg/dl and 8hrs after crude drug administration was
138.8 mg/dl (↓ 36.56 %). The mean glucose level in MTG
8 days after alloxan injection was 290 mg/dl and 8 hrs
after ingestion of methanolic extract of crude drug was
155mg/dl (↓ 46.58%). When CTG and MTG were
compared with the CDG at 8 hrs after drug administra-
tion, a significant decrease in glucose level (P<0.005) was
observed in both CTG and MTG. fig. 1 shows the
comparison between CDG, CTG and MTG at different
time intervals. So, the methanolic extract of C. longa
appeared to have better hypoglycemic effect as compared
to crude C. longa.
Fig. 1: Comparison of effect of crude and methanolic
extract of C.longa with alloxan on serum glucose in
diabetic rabbits.
The mean ALT level in diabetic rabbits 8 days after
alloxan injection was 69.67 U/l (↑ 492.69%) which shows
that there was 5 fold increases in the ALT level in alloxan
treated diabetic rabbits. The effect of crude (2g/kg b.w)
and methanolic extract of crude drug (equivalent to 2g/kg
b.w) can be observed in table 1. The mean ALT level at 4
hrs after drug administration was 70 U/l in CDG (↑
579.28%). There was significant decrease (p<0.005) in
CTG i.e. 39 U/l (↓ 40.53%), when compared with CDG at
the same time interval. In MTG the mean ALT level 4 hrs
after drug administration was 31.5 U/l (↓ 55%), so
significant decrease was observed when compared with
CDG (70 U/l at 244 hrs). fig. 2 shows mean ALT levels at
different time intervals and it could be observed that MTG
reduced ALT more as compared to CTG with the same
dose.
Fig. 2: Comparison of curative effect of crude and
methanolic extract of C.longa with alloxan on serum ALT
in diabetic rabbits.
AST is another important enzyme for assessing liver
function. The normal mean AST level in three groups (i.e.
CDG, CTG and MTG) was 10.17, 10.33 and 8.9 U/l. The
mean AST level in diabetic rabbits in three groups 8 days
after injecting alloxan (180 mg/kg b.w.) was 65.5 (↑
544.05%), 71.1 (↑ 588.96%) and 69.33 (↑ 678.99) U/l as
seen in table 1. It was observed that there was 7 fold
increases in AST levels in diabetic rabbits. The mean AST
level in alloxan treated rabbits (CDG) 24 hrs after drug
administration was 63.33 U/l (↑ 522.71%) and there was
significant decrease (P<0.005) in CTG at same time
interval i.e. 21.67 U/l (↓ 69.55%). In MTG the mean AST
level 24 hrs after drug was 17 U/l (↓ 75.47%). Significant
decrease (P<0.005) in mean AST level in MTG was
observed when compared with the CDG. Fig. 3 depicts the
comparison between CDG, CTG and MTG and significant
decrease (P<0.05) in AST level in CTG and MTG could
be observed.
Fig. 3: Comparison of curative effect of crude and
methanolic extract of C.longa with alloxan on serum AST
in diabetic rabbits.
Protective effect of crude Curcuma longa and its methanolic extract
Pak. J. Pharm. Sci., Vol.27, No.1, January 2014, pp.121-128
124
The mean normal total serum antioxidant level in three
groups assessed was 2.1, 2.07 and 2.13 mmol/l shown in
table 1. After injection of alloxan the antioxidant level
decreased and was 0.713, 0.91 and 0.703 mmol/l in CDG.
CTG and MTG presented in table 1. The antioxidant
activity decreased upto 0.71 mmol/l (↓ 66.19%) in control
group at 8 hrs without any treatment shown in table 1. In
CTG the anti oxidant level 8 hrs after ingestion of crude
C. longa was 1.3 mmol/l (↑42.86%). In MTG group the
AOA level at 8 hrs was 1.35 mmol/l (↑92.86%). fig. 4
shows the comparison between CDG, CTG and MTG.
Fig. 4: Comparison of effect of crude and methanolic
extract of C.longa with alloxan on serum antioxidant
axctivity in diabetic rabbits.
Protective group
The Protective Control Group “PCG” was injected
alloxan intraperitoneally (180 mg/kg b.w.) along with the
Protective Crude Drug Group “PCD” and Protective
Methanolic Extract Group “PMG” after taking the normal
glucose and serum transaminases level. The PCD was
given crude drug suspension (dose: 2g/kg b.w.) prepared
freshly, once daily for 8 days. On the 8
th
day alloxan (180
mg/kg b.w.) was injected intraperitoneally and the
changes in glucose, ALT and AST were observed for the
next 15 days. After alloxan injection serum levels of
glucose and transaminases were noted on 9
th
, 17
th
and 24
th
days. The third group labeled PMG was given methanolic
extract of crude drug suspension prepared freshly for 8
days and treated in same manner as PCD.
The normal glucose level of PCD and PMG observed was
116.3 and 119.5 mg/dl and no significant change in mean
glucose levels were observed after 8 days pretreatment
with crude drug and its methanolic extract. The mean
glucose levels after 8 days pretreatment with crude and
methanolic extract of C. longa were 115.5 and 118.3
mg/dl shown in table 2. The mean glucose level of PCG
was 213 mg/dl (↑ 84.68%) on the 17
th
day whereas the
mean glucose level of PCD and PMG was 166.7 (↑
43.3%) and 137 (↑ 14.6%) mg/dl respectively as can be
seen in table 2. Fig. 5 also shows the comparison of
glucose level in PCD and PMG with the PCG.
Fig. 5: Study of comparison of protective Effect of crude
and methanolic extract of C. longa with alloxan on serum
glucose in rabbits.
Normal mean ALT levels in PCD and PMG observed
were 16.67 and 17 U/l shown in table 2. The mean ALT
levels after 8 days pretreatment with crude and
methanolic extract of crude drug were 12.16 and 14.5 U/l.
No significant changes in mean ALT levels were observed
after 8 days pretreatment with crude drug and its
methanolic extract. The effect of alloxan (180 mg/kg) and
protective effect of crude drug (2 g/kg) and its methanolic
extract (equivalent to 2g/kg) on serum ALT level was
assessed. The mean ALT level of PCG was 65.42 U/l (↑
351.17%) on the 17
th
day whereas the mean ALT of PCD
and PMG was 33.8 U/l (↑ 102.9%) and 31.5 U/l (↑
85.29%) respectively. The comparison among the groups
can be observed in fig. 6.
Fig. 6: Study of comparison of protective Effect of crude
and methanolic extract of C. longa with alloxan on serum
ALT in rabbits.
Normal mean AST levels in PCD and PMG observed
were 14.67 and 15.83 U/l shown in table 2. The mean
AST levels after 8 days pretreatment with crude and
methanolic extract of crude drug were 13.17 and 14.3 U/l.
No significant changes in mean AST levels were observed
after 8 days treatment with crude drug (2g/kg) and its
methanolic extract (equivalent to 2g/kg). The mean AST
level of PCG was 56.17 U/l (↑ 395.76%) on the 17
th
day
whereas the mean AST levels of PCD and PMG were 32.6
U/l (↑ 102.9%) and 30.7 U/l (↑ 85.29%) respectively. The
protective effect of crude C. longa and its methanolic
extract on AST in PCD and PMG in comparison to PCG
can be seen in fig. 7.
Mobasher Ahmad et al
Pak. J. Pharm. Sci., Vol.27, No.1, January 2014, pp.121-128
125
Fig. 7: Study of comparison of protective Effect of C.
longa and its methanolic extract with alloxan on AST in
rabbits.
The total serum antioxidant activity was measured in
PCG, PCD and PMG as shown in table 2. The results are
compared in fig. 8. After injection of alloxan, the
antioxidant activity level in PCG on 8
th
day decreased up
to 0.91 mmol/l (↓ 59.56%) and in PCD was 1.46 mmol/l
(↓ 38.66%) and in PMG the change (1.42 mmol/l) was
non-significant when compared with normal. In PCG,
PCD and PMG the antioxidant level on 17
th
day was 0.91,
1.46 and 1.35 mmol/l respectively. It can be seen in fig. 8
that the antioxidant level in PMG non-significantly
decreased as compared to the PCG.
Fig. 8: Study of comparison of protective effect of C.
longa and its methanolic extract with alloxan on AOA in
rabbits.
DISCUSSION
Diabetes mellitus is an irreversible metabolic disorder
which is characterized by hyperglycemia due to defects in
insulin production and function. In the curative group as
shown in table 1 the statistical comparison of alloxan
treated is done with normal, crude and methanolic extract
of C. longa. Highly significant (P<0.005) results were
obtained. Alloxan resulted in a significant increase
(P<0.005) in serum glucose, ALT and AST enzymatic
activity and decreased total antioxidant activity. In groups
treated with crude and methanolic extract of C. longa the
enzymatic values significantly decreased (P<0.005) and
total antioxidant activity increased.
In the protective group C. longa was first administered for
8 days, alloxan was injected i.p., and then animals were
observed for next 15 days. C. longa (2g/kg) and its
methanolic extract (equivalent to 2g/kg) were
administered as a single dose daily for 8 days and before
the administration zero hr blood sample was collected. On
8
th
day alloxan 180mg/kg was injected intraperitoneally
once. Sample were taken before alloxan injection and
then after 24 hrs, 8 and 15 days. In table no. 2 the
protective effect of crude and methanolic extract of C.
longa is shown on glucose, ALT and AST. Statistical
comparison of normal with alloxan treated is shown in
table 2 and highly significant (P<0.005) results could be
observed.
The mechanism of alloxan has been extensively studied in
experimental animal models and is quite well
characterized now. Alloxan induces chemical diabetes by
destroying the beta cells of the pancreas. The beta cells
rapidly uptake the alloxan and cause formation of reactive
oxygen species. Alloxan is reduced to dialuric acid in the
presence of different reducing agents. It is then reoxidized
back to alloxan establishing a redox cycle for the
generation of super oxide radicals. As a result of this
redox cycle highly reactive hydroxyl radicals are formed.
The reactive oxygen species also damage DNA of the
pancreatic islets and DNA fragmentation takes place in
the beta cells expose to alloxan (Lenzen, 2008; Yali, 2005;
Szkudelski, 2001).
Liver is a vital organ responsible for the metabolism of
drugs and other substances. Liver functions are carried
out by hepatocytes. Damage to the liver cells leads to
altered cell membrane permeability and leaking out of
tissue content into the blood stream. This in turn leads to
increased serum transaminases level (Murugan and Pari,
2007). The changes in serum enzymes are normal in
uncomplicated diabetes. The tissue damage caused by the
metabolic and circulatory disorders causes’ congestion
and metabolic disorders which results into liver damage.
Increased protein catabolism accompanying
gluconeogenesis might be the reason for elevated serum
transaminases in diabetic state. C. longa and its extract
significantly reduced the serum transaminases in both
curative and protective groups by reducing hepatocellular
damage and suppression of gluconeogenesis. It has also
been reported that alloxan destroys beta cell function by
inhibiting the glucokinase enzyme activity. Later it was
also proved that alloxan inhibits liver glucokinase as well,
thus leading to the elevation of serum glucose content that
leads to further descrease in insulin secretion. Zhang et al.
(2007) also demonstrated that liver glucokinase
expression in alloxan induced diabetic mice was only
about 19% of that seen in normal mice. Further
glucokinase enzyme activity was decreased by more than
90% because some immunoreactive glucokinase enzyme
may have abnormal function. Moreover hepatic glycogen
Protective effect of crude Curcuma longa and its methanolic extract
Pak. J. Pharm. Sci., Vol.27, No.1, January 2014, pp.121-128
126
is also decreased in alloxan induced diabetic mice.
Because of the metabolic disorder caused by diabetes,
changes in the serum transaminases may occur, indicating
tissue damage by toxicants. So it could be suggested that
C. longa and its extract improves and protects
glucokinase and inhibits reduction in hepatic glycogen in
liver therefore it reduces serum transaminases (Zhang et
al., 2007; Sakurai and Ogiso, 1995; Takasun et al., 1991;
Munday, 1988; Heikikila et al., 1976).
In the present investigation the studies have been planned
to see the curative and the protective action of the C.
longa and its methanolic extract in alloxanized and
normal rabbits. Tetrahydrocurcumin a major metabolite of
curcumin has also been assessed for its effect on hepatic
and renal markers and proteins in type 2 diabetic rats
(Murugan and Pari, 2007). It was observed that
tetrahydrocurcumin also protects against renal and hepatic
damage in diabetic condition. In the present study serum
glucose, ALT and AST were assessed and it was observed
that C. longa given in a dose of 2g/kg when given to
diabetic rabbits improved these parameters as compared
to the untreated diabetic rabbits. In curative group the
crude and methanolic extract of C. longa reduced glucose
ALT and AST and significant results were obtained. In
both protective groups rabbits pretreated with crude and
methanolic extract of C. longa highly significant results
were obtained in the parameters observed, so it could be
suggested that metabolite tetrahydrocurcumin provides
long term protective effect in liver and pancreas. In
diabetes insulin deficiency results in to reduce synthesis
of proteins and cause glycation which results into the
formation of oxygen derived free radicals. C. longa and
its methanolic extracts reduce the blood glucose level by
reducing the influx of glucose through polyol pathway
(Arun and Nalini, 2000).
Therefore it can be suggested that C. longa and its
methanolic extracts significantly reduced glucose by
increasing glucose utilization in erythrocytes or by
inhibiting or blocking the enzymes that convert the
dietary carbohydrates into sugar. It improves and protects
the level of glucokinase both in liver and pancreas;
therefore it reduces serum transaminases by maintaining
the primary enzyme glucokinase level found both in liver
and pancreas. C. longa reduces glucose level and serum
Table 1: Curative effect of crude and methanolic extract of C. longa on Glucose, ALT, AST and AOA in CDG, CTG
and MTG at different time intervals
CDG CTG MTG CDG CTG MTG
Glucose (mg/dl) AOA (mmol/l)
Normal (a) 109.17±3.44 107.5±3.46 116.83±2.21 2.1±0.124 2.07±0.178 2.13±0.161
8 days after
Alloxan inj
(b)
242.17±6.19***
↑ (121.83%)
218.83±6.25***
↑(103.6%)
290.17±23.49***
↑(148.37%)
0.713±0.053***
↓ (66.05%)
0.91±0.041***
↓(56.04%)
0.70±0.08***
↓(67.14%)
4 hrs (c) 267±6.22***
↑ (144.57%)
163.67±11.72***
↓(25.21%)
183.83±18.48***
↓(36.65%)
0.69±0.061***
↓ (67.14%)
1.9±0.12***
↑(108.79%)
1.12±0.05***
↑(60%)
8 hrs (d) 289.5±6.65***
↑ (165.18%)
138.83±3.98***
↓(36.56%)
155±4.48***
↓(46.58%)
0.71±0.068***
↓ (66.19%)
1.3±0.08***
↑(42.86%)
1.35±0.06***
↑(92.86%)
24 hrs (e) 243.3±6.79***
↑ (122.86%)
129.83±3.79 ***
↓(53.93%)
134.17±3.75***
↓(53.76%)
0.68±0.074***
↓ (67.62 %)
1.03±0.16***
↑(13.19%)
2.84±1.64***
↑(92.86%)
AST (U/l) ALT (U/l)
Normal (a) 10.17±0.87 10.33±1.054 8.9±0.795 10.33±1.48 11.08±10.28 9.03±0.877
8 days after
Alloxan inj
(b)
65.5±7.62***
↑(544.05%)
71.17±7.04***
↑(588.96%)
69.33±6.09***
↑(678.99%)
69.67±7.82***
↑(574.44%)
65.67±8.24***
↑(492.69%)
70±7.87 ***
↑(675.19%)
4 hrs (c) 65.3±7.44***
↑( 542.09(%)
33.82±3.28***
↓(52.48%)
35.33±3.17***
↓(49.04%)
70.17±6.28***
↑(579.28%)
39.05±5.94 **
↓(40.53%)
31.5±4.14 ***
↓(55%)
8 hrs (d) 64.17±7.40***
↑ (530.97%)
25.27±2.65***
↓(64.49%)
21.83±1.64***
↓(68.51%)
67.83±8.09***
↑(556.63%)
25.13±1.68***
↓(61.73%)
25.5±2.43***
↓(63.57%)
24 hrs (e) 63.33±8.37***
↑ (522.71%)
21.67±2.22***
↓(69.55%)
17±2.07***
↓ (75.47%)
68.5±7.65***
↑(563.12%)
22.45±2.09***
↓(65.81%)
18.95±0.72***
↓(72.93%)
CDG: This group was injected alloxan monohydrate, 180mg/kg b.w i.p and then kept orally on 2% acacia solution
CTG: This group was given crude drug suspension, 2g/kg b.w given orally, 8 days after alloxan injection and glucose, AOA,
ALT and AST were observed at 4, 8 and 24 hrs after drug administration.
MTG: This group was given crude drug methanolic extract suspension equivalent to 2g/kg b.w of crude drug orally 8 days
after alloxan injection and glucose, AOA, ALT and AST were observed at 4, 8 and 24 hrs after drug administratio
n.
Values are expressed as mean ± S.E
For CDG b, c, d and e are compared with a for t-test
For CTG and MTG, a is compared with b and c, d and e are compared with b for t test
***Highly significant P<0.005, **Very significant P< 0.01, *Significant P<0.05,
NS
Non-significant P>0.05
% change is in parenthesis
Mobasher Ahmad et al
Pak. J. Pharm. Sci., Vol.27, No.1, January 2014, pp.121-128
127
transaminases to almost 50% with a dose of 2g/kg b.w. It
was also observed that overall methanolic extract
produced better effect as compared to the crude C. longa.
C. longa contains 3 to 4% of curcumin which can
neutralize free radicals formed in the body. With the
prevention of formation of free radicals C. longa prevents
the damage caused by alloxan to pancreas. Turmerin,
water soluble protein found in C. longa exhibits
antioxidant capacity and it was observed that this protein
also inhibits α-amylase and α-glucosidase activities
(Lekshmi et al., 2011). In another study it was observed
that the curcumin diet lowered liver weight and lipid
peroxidation in plasma and urine. C. longa and its extract
may also scavenge superoxide radical production and
inhibit glycosylation of proteins which contribute to the
pathogenesis of cellular dysfunction and hence improve
the metabolic state of diabetic patient. Curcumin, yellow
component in C. longa reduces plasma free fatty acid,
cholesterol, and triglyceride concentrations and increased
the hepatic glycogen and skeletal muscle lipoprotein
lipase in db/db mice. Curcumin also normalized
erythrocyte and hepatic antioxidant enzyme activities
(superoxide dismutase, catalase, gluthathione peroxidase)
in db/db mice that resulted in a significant reduction in
lipid peroxidation (Seo et al., 2008). Hence it can be
concluded that C. longa and its extract improves the
metabolic status of diabetic patient because of its
antioxidant and free radical scavenging properties.
C. longa needs more attention from the researchers to
further evaluate its safety in different diseased conditions
to make its use more common in humans. Natural drugs
are more safe with little side effects hence they should be
made available to common people in proper dosage form
to heal the humanity.
REFERENCES
Araújo CAC and Leon LL (2001). Biological activities of
Curcuma longa L. Mem Ins Oswaldo Cruz. Rio. de
Janerio, 96(5): 723-728.
Arun N and Nalini N (2000). Efficacy of turmeric on
blood sugar and polyol pathway in diabetic albino rats.
Plants Foods Hum. Nutr., 57(1): 41-52.
Chempakam B and Parthasarathy VA (2008). Turmeric.
In: Chemistry of Spices, 6
th
Ed., pp.97-123.
Table 2: Protective effect of crude and methanolic extract of C. longa on Glucose, AOA, ALT and AST in normal and
alloxan treated rabbits.
PCG PCD PMG PCG PCD PMG
Glucose (mg/dl) AOA (mmol/l)
1
st
day (a) 115.33±3.56 116.33±2.38 119.5±2.75 2.25±0.19 2.38±0.06 1.4±0.08
8
th
day (b) 114±5.24
NS
115.5±1.61
NS
118.33±1.73
NS
2.25
±0.18
NS
2.6±0.07** 1.75±0.043***
9
th
day
(c)
106.5±6.35
NS
↓(7.65%)
111.3±7.72
NS
↓(4.32%)
110.67±3.90*
↓(7.39%)
1.53±0.15**
↓(32%)
1.88±0.03***
↓(21%)
1.58±0.04*↓
(12.9%)
17
th
day
(d)
213±7.86***
↑(84.68%)
166.7±11.05***
↑(43.3%)
137±3.79***
↑(14.6%)
0.91±0.05***
↓(59.56%)
1.46±0.03***
↓(38.66%)
1.42
±0.03
NS
24
th
day
(e)
257.4±14.21***
↑(123.19%)
181.33±16.12***
↑(55.85%)
179.67±7.14***
↑(50.35%)
0.44±0.015***
↓(80.35%)
1.33±0.05***
↓(44.12%)
1.28
±0.05
NS
ALT (U/l) AST (U/l)
1
st
day (a) 14.5±1.38 16.67±1.43 17±1.15 11.33±0.88 14.67±1.33 15.83±1.25
8
th
day (b) 15.5±0.96
NS
12.17*±1.58 14.5±1.31
NS
11.33±0.72
NS
13.17±1.01
NS
14.3±0.56
NS
9
th
day (c) 33.88±2.45***
↑(133.7%)
31.67±2.79***
↑(89.98%)
21.83±0.75***
↑(28.41%)
32.7±2.45***
↑(188.61%)
27±1.97***
↑(84.05%)
20.33±0.88**
↑(28.43%)
17
th
day (d) 65.42±7.32***
↑(351.17%)
33.83±4.38***
↑(102.9%)
30.7 ±1.385***
↑(85.29%)
56.17±8.49***
↑(395.76%)
32.67±8.49***
↑(122.7%)
30.67±1.12**
* ↑(93.75%)
24
th
day (e) 69±6.89***
↑(375.9%)
36.5±3.94***
↑(118.96%)
35±1.84***
↑(105.9%)
61.2±6.88***
↑(440.16%)
33.5±1.45***
↑(128.36%)
41±1.39***
↑(159%)
PCG: PCG was kept on 2% acacia solution initially for 8 days and injected alloxan intraperitoneally (180 mg/kg b.w) along
with PCD and PMG and the changes in glucose, ALT and AST were observed on 9
th
, 17
th
and 24
th
day.
PCD: The PCD was given crude drug suspension (dose: 2g/kg b.w) prepared freshly, once daily for 8 days. On the 8
th
day
alloxan (180 mg/kg b.w) was injected intraperitoneally and the changes in glucose, ALT and AST were observed on 9
th
, 17
th
and 24
th
day.
PMG: The PMG was given suspension of methanolic extract of crude drug (dose: equivalent to 2g/kg b.w of crude drug)
prepared freshly, once daily for 8 days. On the 8
th
day alloxan (180 mg/kg b.w) was injected intraperitoneally and the changes
in glucose, ALT and AST were observed on 9
th
, 17
th
and 24
th
day.
Values are expressed as mean ± S.E
For PCG b, c, d, and e are compared with a for t test
For PCD and PMG b is compared with a, and c, d and e are compared with a for t test.
***Highly significant P< 0.005, **Very significant p< 0.01, *Significant P< 0.05,
NS
Non-significant P> 0.05
(% change is in parenthesis)
Protective effect of crude Curcuma longa and its methanolic extract
Pak. J. Pharm. Sci., Vol.27, No.1, January 2014, pp.121-128
128
Heikkila RE, Winston B, Cohen G and Barden H (1976).
Alloxan induced diabetes, evidence for hydroxyl
radicals as a cytotoxic intermediate. Biochem.
Pharmacol., 25: 1085-1092.
Koracevic D, Koracevic G, Djordjevic V, Andrejevic S
and Cosic V (2001). Method for the measurement of
antioxidant activity in human fluids. J. Clin. Pathol.,
54: 356-361.
Lekshmi PC, Arimboor R, Raghu KG and Menon AN
(2011). Turmerin, the antioxidant protein from turmeric
(Curcuma longa) exhibits antihyperglycaemic effects.
Nat. Prod. Res., 1-5, iFirst. (Nat. Prod. Res. 2012;
26(17): 1654-1658.
doi:10.1080/14786419.2011.589386.Epub 2011 Oct 6)
Lenzen S (2008). The mechanisms of alloxan and
streptozotocin-induced diabetes. Diabetologia., 51:
216-226.
Lenzen S, Tiedge M, Jorns A and Munday R (1996).
Alloxan derivatives as a tool for the elucidation of the
mechanism of the diabetogenic action of alloxan. In:
Lessons from Animal Diabetes VI, Shafrir, E.,
Birkhauser, Boston, pp.113-122.
Meral I, Yener Z, Kahraman T and Mert N (2001). Effect
of nigella sativa on glucose concentration, lipid
peroxidation antioxidant defense system and liver
damage in experimentally induced diabetic rabbits. J.
Vet. Med. A. Physiol. Pathol. Clin. Med., 48(10): 593-
599.
Munday R (1988). Dialuric acid autoxidation. Effects of
transition metals on the reaction rate and on the
generation of "active oxygen" species. Biochem.
Pharmacol., 37(3): 409-413.
Murugan P and Pari L (2007). Influence of
Tetrahydrocurcumin on Hepatic and Renal Functional
Markers and Protein Levels in Experimental Type 2
Dibetic Rats. Basic Clin. Pharmacol. Toxicol., 101(4):
241-245.
Sakurai K and Ogiso T (1995). Effect of ferritin on
Lambda DNA strand breaks in the reaction system of
alloxan plus NADPH cytochrome P450 reductase:
Ferritin's role in diabetogenic action of alloxan. Biol.
Pharm. Bull., 18: 262-266.
Seo K, Choi MS, Jung UJ, Kim HJ, Yeo J, Jeon SM and
Lee MK (2008). Effect of curcumin supplementation
on blood glucose, plasma insulin and glucose
homeostasis related enzyme activities in diabetic db/db
mice. Mol. Nutr. Food Res., 52(9): 995-1004.
Szkudelski T, Kandulska K and Okulicz M (1998).
Alloxan in vivo does not only exert deleterious effects
on pancreatic beta cells. Physiol Res., 47: 343-346.
Takasun N, Asawa T, Komiya I, Nagasawa Y and Yamada
T (1991). Alloxan-induced DNA strand breaks in
pancreatic islets. J. Biol. Chem., 266: 2112-2114.
Tierney LM, McPhee SJ and Papadakis MA (2002).
Current medical Diagnosis & Treatment. International
edition, N.Y: Lange Medical Books, McGraw-Hill, pp.
1203-1215.
Yali Z, Meifang X, Gang N and Huanran T (2005).
Mechanism of oleic acid deterioration in insulin
secretion: Role in pathogenesis of type 2 diabetes. Life
Sci., 77: 2071-2081.
Zhang X, Liang W, Mao Y, Li H, Yang Y and Tan H
(2009). Hepatic glucokinase activity is the primary
defect in alloxan induced diabetes of mice. Bio. Med.
Pharmacotherapy, 63: 180-186.
