ANTI-STRESS POTENTIAL OF COCOS NUCIFERA

ABSTRACT

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
medicine.

Keywords: Anacyclus pyrethrum, anti-anxiety, elevated plus
maze test.

 

INTRODUCTION

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
animals.

MATERIALS
AND METHODS

Plant
material

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.

Fresh endocarp
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.

Preliminary
phytochemical screening

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.

Experimental
animals

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).

Determination
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.

Evaluation
of antistress activity of Cocos nucifera

Experimental design

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
mg/kg, p.o.)

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
mg/kg, p.o.

Immobilization
stress

The treatment
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.

Hematological and
biochemical estimations

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
neurotransmitters

Norepinephrine
and serotonin levels of brain of all rats exposed to immobilization stress were
estimated after their isolation using high-performance liquid chromatographic
(HPLC) technique12.

Statistical
analysis

The
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.

Results

Evaluation
of antistress activity of Cocos nucifera

Immobilization stress model

Effect of EECNE on hematological parameters

The
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

The
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.

Effect
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<0.01) and 500 mg/kg (p<0.001) doses were significantly reversed the altered weight of organs. Effect of EECNE on body weight The rats subjected to immobilization stress shown slow body weight accumulation as compared to normal control animals, where normal body weight accumulation observed. It was observed that the test extract at different doses significantly (250 mg/kg (p<0.01) and 500 mg/kg (p<0.001)) slow down the stress mediated loss of body weight in experimental animals.  The results are displayed in table.No.5. Effect of EECNE on brain neurotransmitters The results are tabulated in table 6. HPLC method of determination of brain neurotransmitters indicates that, rats exposed to stress shown a significant reduction of NA and 5-HT levels compared to normal control. Pre-treatment of animals with EECNE demonstrated significant restoration of these altered neurotransmitters level except the lowest dose i.e. 125 mg/kg. Discussion The literature survey reveals that, the immobilization stress is considered as the most severe type of stress and one of the most widely used experimental animal models. The results of the present study indicate that, the stress has altered the haematological parameters and animals pre-treated with test extract for the period of 10 days, significantly ameliorated the adverse effect of stress on these haematological changes. This might be due to anti-oxidant or anti-lipid peroxidation effect of EECNE. In our research it was observed that, immobilization stress caused a significant rise in serum glucose, cholesterol and BUN. Literature reports reveal that, stress causes significant elevation of serum glucose level in humans and animals. Adrenal cortex secretes cortisol in human and corticosterone in rats to maintain internal homeostasis by regulating gluconeogenesis and lipogenesis. Immobilization stress leads to hyper-activation of adrenal cortex, which in turn releases excessive amount of cortisol, which influences mobilization of carbohydrate reserves and stored fat that causes hyperglycemia and increased triglyceride levels13. In the present research work we found that, the title plant has significantly reduced stress induced hyperglycemia. This effect may be due to reduction in the cortisol level probably by prevention of hyper activation of adrenal cortex6.    The stress causes over-activation of hypothalamo-hypophyseal axis (HPA) which in turn releases excess catecholamines and corticosteroids, this could lead to rise in blood cholesterol level since adrenaline is known for mobilization of lipids from adipose tissues. This release of excess catecholamines also leads to rise in glucose and blood urea nitrogen levels14. From our research it was found that the extract significantly restored all these levels probably due to inhibition of stress induced catecholamines release by regulating hyperactive hypothalamo-hypophyseal axis. Exposure of animals to stress resulted in significant reduction of weight of spleen with concomitant gain in liver and adrenal glands weight. The previous reports suggest that rise in weight of liver is attributed to increased cortisol which in turn enhances mRNA levels in liver. Whereas adrenal gland weight is increased because stress stimulates adreno–medullary response leads to release of adrenaline brings about excessive release of ACTH, which in turn stimulates the adrenal medulla and cortex results in rise of adrenal gland weight. Stress reduces the weight of spleen; this is due to constriction of spleen to release more RBCs under stressful conditions15-16. However, the animals pretreated with extract significantly reverted the altered weight of organs and this may be due to normalization of cortisol release (maintains liver weight), regulation of adreno–medullary response (maintains adrenal gland weight) and reduced constriction of spleen (maintains spleen weight). In our study, it was observed that the normal control animals showed normal weight gain, whereas the stressed animals exhibited decrease in the body weight accumulation. The decrease in body weight in stressed rats may be due to reduced food intake or increase in metabolic demands, reduced digestion and increased adrenal steroid secretion. The animals pre-treated with EECNE have exhibited prevention in the weight loss induced by immobilization stress. This observation is probably may be due to increase in food intake, decrease in metabolic demands, increased digestion process or decreased adrenal steroid secretion17. Reports suggest that, the neurotransmitters such as nor-adrenaline (NA) and serotonin (5-HT) plays a vital role in tackling of stress18. Under severe stress conditions these neurotransmitters levels are significantly decreased. In our study also, brain levels of NA and 5-HT were found to be reduced significantly in immobilization stress model. Animals pretreated with extract demonstrated significant amelioration of levels of these neurotransmitters indicates the adaptogenic potential of EECNE.   Conclusion: Cocos nucifera endocarp extract has shown significant improvement in stress-induced alterations of hematological and biochemical parameters, body weight, organs weight, stomach ulcer index and brain neurotransmitters levels, indicating its protective effect against stress. However, further experiments are needed to establish the mechanism behind the adaptogenic potential of plant.     References 1.      Desai SK., Desai SM., Navdeep S, Arya P and Pooja T. “Antistress activity of Boerhaavia diffusaroot extract and a polyherbal formulation containing Boerhaavia diffusa using cold restraint stress model,” International Journal of Pharmacy and Pharmaceutical Sciences, vol. 3, no. 1, pp. 130–132, 2011. 2.      Khan MN, Suresh J, Yadav KSH., and Ahuja J. “Formulation and evaluation of antistress polyherbal capsule,” Der Pharmacia Sinica, vol. 3, pp. 177–184, 2012. 3.      Joshi T, Sah SP and Singh A. “Antistress activity of ethanolic of Asparagus racemosus Willd roots in mice,” Indian Journal of Experimental Biology, vol. 50, pp. 419–424, 2012. 4.      Solomon Charles Ugochukwu, Arukwe Uche I, Onuoha Ifeanyi. Preliminary phytochemical screening of different solvent extracts of stem bark and roots of Dennetia tripetala G. Baker. 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