Received 20 Dec 2012
Accepted 14 Feb 2013
Revista Brasileira de Farmacognosia
Brazilian Journal of Pharmacognosy
Comparative effects of mature coconut water
(Cocos nucifera) and glibenclamide on some
biochemical parameters in alloxan induced
P. P. Preetha,
V. Girija Devi,
Department of Biochemistry, University of Kerala, Kariavattom Campus,
Department of Home Science, Govt. College for Women, Thiruvananthapuram,
Abstract: In the present study, comparative effects of mature coconut water
(Cocos nucifera L., Arecaceae) and glibenclamide in alloxan induced diabetic
rats were evaluated. Diabetes mellitus was induced in Sprague-Dawly rats using
alloxan monohydrate (150 mg kg
body weight). Treatment with lyophilized
blood glucose and glycated hemoglobin along with improvement in plasma
insulin level. Elevated levels of liver function enzymes markers like alkaline
phosphatase, serum glutamate oxaloacetate transaminase and serum glutamate
pyruvate transaminase in diabetic rats were significantly reduced on treatment
with mature coconut water. In addition to this, diabetic rats showed altered
levels of blood urea, serum creatinine, albumin, albumin/globulin ratio which
were significantly improved by treatment with mature coconut water and
glibenclamide. Activities of nitric oxide synthase in liver and plasma
mature coconut water reversed these changes. The overall results show that
mature coconut water has significant beneficial effects in diabetic rats and its
effects were comparable to that of glibenclamide, a well known antidiabetic
Diabetes mellitus is a metabolic syndrome,
resulting from defects in insulin secretion, insulin action
both resulting in impaired glucose metabolism and other
energy-yielding fuels such as lipids and protein (El-Soud
et al., 2007). As per WHO, 346 million people worldwide
have diabetes and it is also projected that death due to this
will be the double between 2005 and 2030 (Rai et al., 2012).
The benefi cial effect of synthetic drugs provide good
glycemic control but long term use have side effects and
thus searching for a new class of compounds is essential
to overcome diabetic problems (Prasad et al., 2009). There
has been increasing demand for the use of plant products
with antidiabetic activity due to low cost, easy availability
and lesser side effects (Sharma et al., 2010). There are
several published reports that plants like Aegle marmelos,
Ficus exasperata, Annona muricata, Syzygium cumini,
Gymnema sylvestre etc. exhibit signifi cant antidiabetic
potential (Anandharajan et al., 2006; Adewole et al., 2012;
Adeyemi et al., 2010; Kang et al., 2012). So many people
often combine the herbal remedies with oral hypoglycemic
agent (Rai et al., 2012). Functional foods with preventive
and therapeutic effects on metabolic disorders are very
helpful for the improvement of lifestyle-related diseases
(Muraki et al., 2011). The bioactive phytochemicals
have become a very signifi cant source for nutraceutical
ingredients (Espín et al., 2007).
Coconut water is a natural nutritious beverage
it contains several biologically active components and
possess cardioprotective, hepatoprotective, hypolipidemic
and antihypertensive properties in experimental animals
(Anurag & Rajamohan 2003; Loki & Rajamohan 2003;
Sandhya & Rajamohan 2008; Bhagya et al., 2010; Prathapan
& Rajamohan 2011). Results of our previous studies have
shown that mature coconut water has hypoglycemic and
antioxidant activities in rats induced diabetes (Preetha et
Rev. Bras. Farmacogn. / Braz. J. Pharmacogn.
Among the various synthetic drugs, glibenclamide
dependent diabetes mellitus (Figueroa-Valverde et al.,
2012). The aim of the present study was to investigate the
effects of lyophilized mature coconut water (LMCW) in
comparison with glibenclamide in alloxan induced diabetic
Coconut water from mature coconuts (Cocos
Coast Tall variety) grown in the University campus were
used for this study. The coconuts were dehusked and liquid
endosperm was collected filtered and pooled. The pooled
mature coconut water was then lyophilized at -4
C in a
lyophilized mature coconut water was stored at 0
distilled water prior to administration to rats.
Male Sprague- Dawley rats weighing between
individually in polypropylene cages in room maintained
C with alternate exposure to light and dark for
(Sai Feeds, Bangalore, India) and water ad libitum prior
to dietary manipulation. The protocol was approved by the
animal ethics committee of the University of Kerala (BC-
Dose response study
For dose response study, rats were divided into
Group B: Diabetic control; Group C: Diabetes+LMCW
(100 mg kg
bw); Group D: Diabetes+LMCW (250 mg
bw); Group E: Diabetes+LMCW (500 mg kg
bw); Group G:
After the optimization of the dose of LMCW,
further experiments were carried out with 24 rats which
were divided into four groups of six rats each.
Group I: Normal control
Group II: Diabetic control
Group III: Diabetes+LMCW (1000 mg kg
Group IV: Diabetes+glibenclamide (0.6 mg kg
Diabetes was induced in rats of groups II, III and
IV by a single intraperitoneal (i.p) injection of alloxan
monohydrate (150 mg kg
bw) after fasting the animals
for the next 24 h to prevent hypoglycemia. After 72 h, rats
with fasting blood glucose more than 250 mg dL
mature coconut water (LMCW) was fed daily using an
intragastric tube for 45 days. After the experimental period,
animals were fasted overnight and they were sacrificed
by sodium pentothal injection. Blood and tissues were
collected for various estimations.
Serum glucose was determined (Trinder,
India. Serum insulin was measured with an automated
immunochemiluminometric (ICL) assay according to
the manufacturer’s instruction and was provided by
Bayer Diagnostics (ADVIA Centaur insulin assay).
Estimation of glycated hemoglobin was done using a
Micromat2 hemoglobin Acc test, using a micromat II
instrument, Catalogue No. 280-00016XI (Biorad). Liver
glycogen was estimated by the method of Carroll et al.,
(1956). Blood urea was estimated by modified Berthelot
method (Wheatherburn, 1967). Serum and urinary nitrate
concentration, was estimated using the Griess reaction
(Green et al., 1982). Serum protein was estimated by the
method of Lowry et al, 1951. Albumin was estimated based
on bromocresol green method using Agappe Diagnostics
Albumin Kit (Doumasa et al., 1971). Serum glutamate
oxaloacetate transaminase (SGPT) and Serum glutamate
pyruvate transaminase (SGOT) was assayed by DNPH
method (Reitman & Frankel, 1957) using the enzyme kit
from CML Biotech (P) Ltd, Ernakulam, India. Quantitative
determination of alkaline phosphatase was done as
described by King & King (1954) using the enzyme kit
procured from Dr.Reddy`s laboratories, Hyderabad, India.
Creatinine in serum was estimated as per Bowers & Wong
(1980). Activity of nitric oxide synthase was estimated by
the method of Salter & Knowles (1997). Concentration
-arginine was estimated as described by
The results are expressed as the mean values
performed by one-way ANOVA followed by Duncan’s
variance. Significance was set at p<0.05.
Concentration of blood glucose
Dose dependant response of LMCW in alloxan
induced diabetic rats were also evaluated and found that
1000 mg kg
of LMCW was effective in reducing blood
Signifi cant increase of blood glucose levels were observed
in alloxan induced diabetic rats (275.32±4.25 mg dL
of blood glucose (129.23±1.95 and 120±2.3 mg dL
respectively) when compared to diabetic control.
Values expressed as mean±SD of six rats. Signifi cance accepted at
indicates values are signifi cantly different from group II.
Figure 1 shows the concentration of plasma
glycogen in control and experimental rats. Alloxan induced
diabetic rats showed signifi cant decrease in plasma insulin
and liver glycogen compared to normal control. On the
other hand, treatment of diabetic rats with LMCW and
glibenclamide increased the insulin level and concentration
of liver glycogen along with reduction of HbA1c level.
Figure 1. Concentration of plasma insulin (µ IUmL
accepted at p<0.05. ‘a’ indicates values are signifi cantly different
from group I. ‘b’ indicates values are signifi cantly different from
Concentration of blood urea, serum creatinine
induced diabetic rats when compared to normal control
rats. On the other hand, diabetic rats treated with LMCW
and glibenclamide reversed these changes when compared
to diabetic rats (Table 2, Figure 2).
Table 2. Concentration of blood urea; serum and urinary nitrite.
Groups Blood urea
I. ‘b’ indicates values are signifi cantly different from group II.
Table 3 shows the concentration of serum protein,
rats. Signifi cant decrease in the concentration of serum
protein, serum albumin and A/G ratio were observed in
alloxan induced diabetic rats when compared to normal
control rats. Treatment of diabetic rats with LMCW and
glibenclamide showed an increase in the concentration
of serum protein, serum albumin and A/G ratio when
compared to diabetic control.
Activities of alkaline phosphatase (ALP),
glutamate oxalo acetate transaminase (SGOT) and
glutamate pyruvate transaminase in serum (SGPT)
Activities of ALP, SGOT and SGPT in serum
rats. LMCW and glibenclamide treated rats resulted in
signifi cant decrease in the activities of these enzymes
when compared to diabetic control (Table 4).
oxaloacetate transaminase (SGOT) and glutamate pyruvate
transaminase in serum (SGPT).
(kA Units L
The activity of nitric oxide synthase in liver and
-arginine were signifi cantly
to normal control rats. Treatment of diabetic rats with
LMCW and glibenclamide showed signifi cant increase
in the activities of nitric oxide synthase in liver and
concentration of plasma
-arginine when compared to
In the preset study, the antidiabetic effects of
standard drug, glibenclamide in alloxan induced diabetic
rats. Concentrations of blood glucose and glycosylated
hemoglobin (HbA1c) levels were found to have reduced
in diabetic rats treated with LMCW. The non-enzymatic,
irreversible covalent bonding of glucose with hemoglobin
in the circulation results in the formation of HbA1c and the
concentration of HbA1c refl ects the average blood glucose
levels over a period of time (Venkatesan & Sorimuthu,
2012). LMCW treated diabetic rats exhibited reduced
level of HbA1c. This may be due to the restoration of
blood glucose levels, thereby reducing the intensity of
hemoglobin glycosylation during the experimental period.
The effects were comparable to that of standard drug
glibenclamide. In addition to this, serum insulin level was
increased by the treatment with LMCW in diabetic rats.
The reduction of blood glucose and HbA1c in LMCW
treated diabetic rats may be due to the increased level of
Figure 3. Activity of nitric oxide synthase (Ug
-arginine (µmol mL
expressed as mean±SD of six rats. Signifi cance accepted at
p<0.05. ‘a’ indicates values are signifi cantly different from group
Liver glycogen level is considered as the best
drug (Ahmed et al., 2012). The increase in liver glycogen of
diabetic treated with natural products and glibenclamide is
due to the increased insulin response which in turn promotes
conversion of inactive form of glycogen synthase to the
active form and enhances conversion of blood glucose into
glycogen (Rawi et al., 2011). The prevention of depletion
of glycogen in the liver is possibly caused by stimulation
of insulin release from existing pancreatic β-cells, which
enhances glycolysis (Ramkumar et al., 2011). Increased
liver glycogen content in MCW treated diabetic rats
suggests the stimulation of insulin release by LMCW
from pancreatic β-cells, which enhances upregulation of
Hyperglycemia induces elevation of the blood
signifi cant markers of renal dysfunction. Degradation of
protein and nucleic acid results in the formation of non-
protein nitrogenous compound such as urea and creatinine.
The elevated levels of serum urea and creatinine in diabetic
rats are due to catabolism of the protein and nucleic acids
(Wilson et al., 2011). Treatment of diabetic rats with
LMCW showed significant decrease in the concentration
of blood urea and creatinine in serum which could be due
to the prevention of protein and nucleic acid degradation by
LMCW. The results were similar to that of glibenclamide
In addition to this, the present study showed
globulin and A/G ratio in diabetic rats. Hypoalbuminemia
observed in diabetes is generally attributed in the presence
of nephropathy and/ or may be due to increased protein
catabolism (Prakasam, 2004; Sivajothi et al., 2008).
Significant decrease in the concentration of serum protein,
serum albumin and A/G ratio were observed in alloxan
induced diabetic rats when compared to normal control rats.
Treatment of diabetic rats with LMCW and glibenclamide
showed significant increase in the concentration of serum
protein, serum albumin and A/G ratio when compared to
diabetic rats. These findings suggest that LMCW treatment
ameliorates alloxan induced nephrotoxicity.
Serum concentrations of liver function marker
diabetic rats were elevated. This may be due to leaking
out of enzymes from the tissues and migrating into the
circulation by the adverse effect of alloxan and alloxan can
induce the liver injury by free radical mechanism (Kala
et al., 2012). LMCW treatment regulated the activity of
SGPT and SGOT in liver of rats intoxicated with alloxan.
SGPT and SGOT act as indicators of liver function and
restoration of normal levels of these parameters indicate
normal functioning of liver. The measurement of enzymatic
activity of alkaline phosphatase (ALP) is of clinical and
toxicological importance as changes in the activity is
indicative of tissue damage by toxicants. Elevated levels
of ALP in diabetes may be due to extensive damage to
liver in the alloxan induced diabetic rats (Ravikumar et al.,
2010). Treatment with LMCW in alloxan induced diabetic
rats caused a decline in ALP level.
Diabetic rats showed reduced activity of nitric
of nitric oxide. Nitric oxide (NO) represents one of the
signalling molecules involved in the modulation of the
intracellular redox environment. Previous studies reported
that NO acts as a physiological modulator of islet hormone
release (Jimenez-Feltstrom et al., 2004).
hyperglycaemia, and these beneficial actions are associated
with increased NO formation in patients with type 2 diabetes
(Das et al., 1993). There are reports that
rats. In addition to potential action on insulin release,
arginine administration may provide multiple benefits
to ameliorate diabetes-induced endothelial dysfunction
(Pieper 1998). Chemical analysis of LMCW showed that
-arginine (5.85%), ascorbic acid (0.45%),
manganese (0.084%), total proteins (13.6%) etc. Among
-arginine is the major bioactive component which
diabetes (Preetha et al., 2012). LMCW treatment enhanced
the activities of nitric oxide synthase and increased the
-arginine in diabetic rats suggests that
-arginine is a major factor responsible for the beneficial
In diabetic rats, mature coconut water treatment
antinephrotoxicity and antihepatotoxicity. In conclusion,
the present study clearly revealed that the mature coconut
water has beneficial effect against diabetes induced
complications and its effects were comparable to that of
standard drug, glibenclamide.
First author thank Govt. of Kerala, India for
PPP contributed in carrying out the laboratory
interpreting the results scientifically and drafted the
manuscript. VGD involved in drafting the manuscript.
TR designed the study, supervised the laboratory
work and contributed to critical reading and finalizing
the manuscript. All the authors have read the final
manuscript and approved the submission.
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Department of Biochemistry, University of Kerala, Kariavattom
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