Aim: Cocos nucifera endocarp was screeden for
anti-stress effect using Immobilization stress model. Method: Control and extract treated animals were subjected for
immobilization stress for 10 days. Results
and discussion: Ethanolic extract of Cocos
nucifera at 250mg and 500mg/kg have shown significant anti-stress activity further
it was supported by restoration of all the altered values. Conclusion: Present findings confirm its use in Aurvedic system of
Keywords: Anacyclus pyrethrum, anti-anxiety, elevated plus
Stress is a common phenomenon in
the present scenario of life. It has become an integral part of life. Extreme stressful conditions results in
modulation of homeostasis which may lead to pathogenesis of many diseases like
diabetes, hypertension, anxiety, peptic ulcer etc1. To
overcome this stress many drugs like diazepam, amphetamine, anabolic steroids
are extensively used. But these are misused as these are causing drug
dependence and their over-use is associated with toxicity.
Since ancient times, herbal plants
are used as remedy to combat stress. Many plants like Withania somnifera2, Asparagus
racemosus3 etc were reported to having antistress effect.
Cocos nucifera is known to possess
adaptogenic activity in Ayurvedic system of medicine. Hence, the present
research aimed to explore the antistress potential of Cocos nucifera in immobilization stress model in experimental
The Cocos nucifera endocarp was collected from local market after
identification and authentication by Dr. M. B.Mulimani, Professor of Botany,
S.B Arts and K.C.P. Science College, Bijapur, Karnataka. A voucher specimen
(CN03) has been deposited at the herbarium of Dept. of Pharmacology, HSK
College of Pharmacy, Bagalkot.
was air dried, pulverized to a coarse powder by using grinder and passed
through a 40-mesh sieve. Then the powdered material was packed into Soxhlet
column and extracted ethanol. After this, the extract was concentrated using
rotary flash evaporator.
Test extract was
subjected to preliminary phytochemical screening for the detection of various
phytoconstituents. Tests for the presence of phytoconstituents were performed
by following the standard procedures described in the literature4.
In the present
study, albino mice (20 – 30 g) of either sex were used. These animals were
procured from BLDEA’s Sri B.M.Patil Medical College, Hospital and Research
Center, Bijapur. Before initiation of experiment, the rats were acclimatized
for a period of 10 days under standard environmental conditions such as
temperature (26 + 20C), relative humidity (45-55%) and 12 hr
dark/light cycles. All the animals were fed with rodent pellet diet (VRK
Nutritional industries, Pune, India) and water was allowed ad-libitum under strict hygienic conditions. Ethical clearance for
performing the experiments on animals was obtained from Institutional Animal
Ethics Committee (IAEC).
of Acute toxicity (LD50)
The acute toxicity of Cocos nucifera endocarp extracts was determined in female albino
mice, this is because literature surveys of conventional LD50 tests show that,
females are generally slightly more sensitive33. In this study, the animals were fasted for 4
hr before the experiment. After dosing, food but not water was withheld for
further 1 hr. Mortality and general behavior of the animals observed
individually after dosing at least once during the first 30 minutes,
periodically during the first 24 hr, with special attention given during the
first 4 hr, and daily thereafter, for a total of 14 days. Fixed dose method of
OECD Guideline No. 423; (Annexure-2d: Starting dose is: 2000 mg/kg bw) was
followed for toxicity study.
Based on the results of the study, 1/5th,
1/10th and 1/20th of LD50 cut off value were
selected as screening doses for investigation5.
of antistress activity of Cocos nucifera
Adult albino rats of either sex weighing 150 – 200 g
were selected and divided into six groups of six animals each.
Group I – Normal control, untreated
Group II – Stress control, received vehicle only
Group III –
Standard (Withania somnifera 100
Group IV – EECNE
(Ethanolic extract of Cocos nucifera endocarp) 125 mg/kg, p.o.
Group V – EECNE 250 mg/kg, p.o.
Group VI – EECNE 500
was made as stated above for 10 days 1hr. before the exposure of stress. Stress
was induced by immobilizing rats with head down, supine position by fixing the
forelimbs and hind limbs to a wooden board inclined at an angle of 600,
daily 2 hrs. for a period of ten days, except normal control rats6.
At the end of 10th
day one hour after drug treatment the blood was collected from retro orbital
plexus in sodium citrated tubes for estimation of hemoglobin (Hb), RBC, WBC,
differential leucocytes count (DLC) and platelets carried out using digital
cell counter and for the estimation of biochemical parameters such as, serum
glucose (GOD-POD method), cholesterol (CHOD-PAP method), triglycerides
(GPO-Trinder method), BUN (Blood Urea Nitrogen, GLDH-UREASE method) were
measured using semi auto analyzer7-10.
The rats then
scarified and their organs such brain, liver, spleen and adrenal gland were
removed. The weight of liver, spleen and adrenal gland were recorded after
washing with alcohol per 100 g body weight of animal11.
Estimation of brain
and serotonin levels of brain of all rats exposed to immobilization stress were
estimated after their isolation using high-performance liquid chromatographic
data obtained from the above findings were subjected to statistical analysis
following one-way ANOVA followed by Tukey’s Kramer Multiple Comparison Test to
assess the statistical significance of the results using GraphPad Prism-5
software. p-values less than 0.05 were
considered as statistically significant.
of antistress activity of Cocos nucifera
Immobilization stress model
Effect of EECNE on hematological parameters
effect of EECNE pretreatment on hematological parameters is shown in table
no.1. In the present study, a decreased Hb level, percentage of lymphocytes and
eosinophils were observed in stress control rats, however, elevated RBC, WBC,
platelets count and percentage increase in neutrophils
and monocytes monitored over normal control. Animals pretreated with
test extract (at different doses (250 and 500 mg/kg)) significantly restored these altered
hematological parameters compared to stress control group. The effect of test
extract was found to be dose dependent at higher doses i.e. at 250 mg/kg and
500 mg/kg. However, the effect of test extract at lower dose (125 mg/kg) found
to be statistically not-significant. Effect of higher dose, 500 mg/kg extract
on hematological data was found to be almost similar to that of Withania somnifera.
Effect of EECNE on biochemical parameters
results are tabulated in table 2. It was observed that the exposure of animals
to immobilization stress caused significant elevation of serum
triglycerides, glucose, total cholesterol and BUN levels over normal control
rats. Animals pre-treatment with graded doses (250 mg/kg and 500 mg/kg) of EECNE demonstrated significant attenuation of
these altered biochemical parameters, however the effect of 125 mg/kg of
extract was found to be statistically not-significant.
of EECNE on organs weight
The results are tabulated in table
3. The immobilization stress induced a marked change in weight of organs i.e.
significant gain in liver and adrenal glands weight and reduction in weight
spleen when compared to normal control. Pre-treatment of animals with EECNE at
250 mg/kg (p