ABSTRACTAim: Cocos nucifera endocarp was screeden foranti-stress effect using Immobilization stress model. Method: Control and extract treated animals were subjected forimmobilization stress for 10 days. Resultsand discussion: Ethanolic extract of Cocosnucifera at 250mg and 500mg/kg have shown significant anti-stress activity furtherit was supported by restoration of all the altered values. Conclusion: Present findings confirm its use in Aurvedic system ofmedicine.
Keywords: Anacyclus pyrethrum, anti-anxiety, elevated plusmaze test. INTRODUCTIONStress is a common phenomenon inthe present scenario of life. It has become an integral part of life.
Extreme stressful conditions results inmodulation of homeostasis which may lead to pathogenesis of many diseases likediabetes, hypertension, anxiety, peptic ulcer etc1. Toovercome this stress many drugs like diazepam, amphetamine, anabolic steroidsare extensively used. But these are misused as these are causing drugdependence and their over-use is associated with toxicity. Since ancient times, herbal plantsare used as remedy to combat stress.
Many plants like Withania somnifera2, Asparagusracemosus3 etc were reported to having antistress effect.Cocos nucifera is known to possessadaptogenic activity in Ayurvedic system of medicine. Hence, the presentresearch aimed to explore the antistress potential of Cocos nucifera in immobilization stress model in experimentalanimals. MATERIALSAND METHODS PlantmaterialThe Cocos nucifera endocarp was collected from local market afteridentification 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, HSKCollege of Pharmacy, Bagalkot.
Fresh endocarpwas air dried, pulverized to a coarse powder by using grinder and passedthrough a 40-mesh sieve. Then the powdered material was packed into Soxhletcolumn and extracted ethanol. After this, the extract was concentrated usingrotary flash evaporator.
Preliminaryphytochemical screening Test extract wassubjected to preliminary phytochemical screening for the detection of variousphytoconstituents. Tests for the presence of phytoconstituents were performedby following the standard procedures described in the literature4.ExperimentalanimalsIn the presentstudy, albino mice (20 – 30 g) of either sex were used. These animals wereprocured from BLDEA’s Sri B.M.
Patil Medical College, Hospital and ResearchCenter, Bijapur. Before initiation of experiment, the rats were acclimatizedfor a period of 10 days under standard environmental conditions such astemperature (26 + 20C), relative humidity (45-55%) and 12 hrdark/light cycles. All the animals were fed with rodent pellet diet (VRKNutritional industries, Pune, India) and water was allowed ad-libitum under strict hygienic conditions. Ethical clearance forperforming the experiments on animals was obtained from Institutional AnimalEthics Committee (IAEC).
Determinationof Acute toxicity (LD50)The acute toxicity of Cocos nucifera endocarp extracts was determined in female albinomice, 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 4hr before the experiment. After dosing, food but not water was withheld forfurther 1 hr.
Mortality and general behavior of the animals observedindividually after dosing at least once during the first 30 minutes,periodically during the first 24 hr, with special attention given during thefirst 4 hr, and daily thereafter, for a total of 14 days. Fixed dose method ofOECD Guideline No. 423; (Annexure-2d: Starting dose is: 2000 mg/kg bw) wasfollowed for toxicity study. Based on the results of the study, 1/5th,1/10th and 1/20th of LD50 cut off value wereselected as screening doses for investigation5.Evaluationof antistress activity of Cocos nuciferaExperimental designAdult albino rats of either sex weighing 150 – 200 gwere selected and divided into six groups of six animals each.Group I – Normal control, untreatedGroup II – Stress control, received vehicle onlyGroup III -Standard (Withania somnifera 100mg/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 500mg/kg, p.o.
ImmobilizationstressThe treatmentwas made as stated above for 10 days 1hr. before the exposure of stress. Stresswas induced by immobilizing rats with head down, supine position by fixing theforelimbs 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 andbiochemical estimationsAt the end of 10thday one hour after drug treatment the blood was collected from retro orbitalplexus in sodium citrated tubes for estimation of hemoglobin (Hb), RBC, WBC,differential leucocytes count (DLC) and platelets carried out using digitalcell counter and for the estimation of biochemical parameters such as, serumglucose (GOD-POD method), cholesterol (CHOD-PAP method), triglycerides(GPO-Trinder method), BUN (Blood Urea Nitrogen, GLDH-UREASE method) weremeasured using semi auto analyzer7-10. The rats thenscarified and their organs such brain, liver, spleen and adrenal gland wereremoved. The weight of liver, spleen and adrenal gland were recorded afterwashing with alcohol per 100 g body weight of animal11.
Estimation of brainneurotransmittersNorepinephrineand serotonin levels of brain of all rats exposed to immobilization stress wereestimated after their isolation using high-performance liquid chromatographic(HPLC) technique12.StatisticalanalysisThedata obtained from the above findings were subjected to statistical analysisfollowing one-way ANOVA followed by Tukey’s Kramer Multiple Comparison Test toassess the statistical significance of the results using GraphPad Prism-5software. p-values less than 0.05 wereconsidered as statistically significant.ResultsEvaluationof antistress activity of Cocos nuciferaImmobilization stress modelEffect of EECNE on hematological parametersTheeffect of EECNE pretreatment on hematological parameters is shown in tableno.1.
In the present study, a decreased Hb level, percentage of lymphocytes andeosinophils were observed in stress control rats, however, elevated RBC, WBC,platelets count and percentage increase in neutrophilsand monocytes monitored over normal control. Animals pretreated withtest extract (at different doses (250 and 500 mg/kg)) significantly restored these alteredhematological parameters compared to stress control group. The effect of testextract was found to be dose dependent at higher doses i.e. at 250 mg/kg and500 mg/kg. However, the effect of test extract at lower dose (125 mg/kg) foundto be statistically not-significant.
Effect of higher dose, 500 mg/kg extracton hematological data was found to be almost similar to that of Withania somnifera. Effect of EECNE on biochemical parametersTheresults are tabulated in table 2. It was observed that the exposure of animalsto immobilization stress caused significant elevation of serumtriglycerides, glucose, total cholesterol and BUN levels over normal controlrats. Animals pre-treatment with graded doses (250 mg/kg and 500 mg/kg) of EECNE demonstrated significant attenuation ofthese altered biochemical parameters, however the effect of 125 mg/kg ofextract was found to be statistically not-significant.
Effectof EECNE on organs weightThe results are tabulated in table3. The immobilization stress induced a marked change in weight of organs i.e.significant gain in liver and adrenal glands weight and reduction in weightspleen when compared to normal control. Pre-treatment of animals with EECNE at250 mg/kg (p<0.01) and 500 mg/kg (p<0.001) doses were significantlyreversed the altered weight of organs. Effectof EECNE on body weight The rats subjected to immobilizationstress shown slow body weight accumulation as compared to normal controlanimals, where normal body weight accumulation observed.
It was observed thatthe test extract at different doses significantly (250 mg/kg (p<0.01) and500 mg/kg (p<0.001)) slow down the stress mediated loss of body weight inexperimental animals. The results aredisplayed in table.No.5.
Effectof EECNE on brain neurotransmittersThe results are tabulated in table6. HPLC method of determination of brain neurotransmitters indicates that, ratsexposed to stress shown a significant reduction of NA and 5-HT levels comparedto normal control. Pre-treatment of animals with EECNE demonstrated significantrestoration of these altered neurotransmitters level except the lowest dose i.e.125 mg/kg.DiscussionThe literature survey reveals that, theimmobilization stress is considered as the most severe type of stress and oneof the most widely used experimental animal models. The results of the presentstudy indicate that, the stress has altered the haematological parameters andanimals pre-treated with test extract for the period of 10 days, significantlyameliorated the adverse effect of stress on these haematological changes. Thismight be due to anti-oxidant or anti-lipid peroxidation effect of EECNE.
Inour research it was observed that, immobilization stress caused a significantrise in serum glucose, cholesterol and BUN. Literaturereports reveal that, stress causes significant elevation of serum glucose levelin humans and animals. Adrenal cortex secretes cortisol in human andcorticosterone in rats to maintain internal homeostasis by regulatinggluconeogenesis and lipogenesis. Immobilization stress leads tohyper-activation of adrenal cortex, which in turn releases excessive amount ofcortisol, which influences mobilization of carbohydrate reserves and stored fatthat causes hyperglycemia and increased triglyceride levels13. Inthe present research work we found that, the title plant has significantlyreduced stress induced hyperglycemia. This effect may be due to reduction inthe cortisol level probably by prevention of hyper activation of adrenal cortex6. The stress causesover-activation of hypothalamo-hypophyseal axis (HPA) which in turn releasesexcess catecholamines and corticosteroids, this could lead to rise in bloodcholesterol level since adrenaline is known for mobilization of lipids fromadipose tissues. This release of excess catecholamines also leads to rise inglucose and blood urea nitrogen levels14.
From our researchit was found that the extract significantly restored all these levels probablydue to inhibition of stress induced catecholamines release by regulatinghyperactive hypothalamo-hypophyseal axis.Exposure of animals tostress resulted in significant reduction of weight of spleen with concomitantgain in liver and adrenal glands weight. The previous reports suggest that risein weight of liver is attributed to increased cortisol which in turn enhancesmRNA levels in liver. Whereas adrenal gland weight is increased because stressstimulates adreno–medullary response leads to release of adrenaline bringsabout excessive release of ACTH, which in turn stimulates the adrenal medullaand cortex results in rise of adrenal gland weight. Stress reduces the weight ofspleen; this is due to constriction of spleen to release more RBCs understressful conditions15-16.
However, the animalspretreated with extract significantly reverted the altered weight of organs andthis may be due to normalization of cortisol release (maintains liver weight),regulation of adreno–medullary response (maintains adrenal gland weight) andreduced constriction of spleen (maintains spleen weight). In our study, it was observed that thenormal control animals showed normal weight gain, whereas the stressed animalsexhibited decrease in the body weight accumulation. The decrease in body weightin stressed rats may be due to reduced food intake or increase in metabolicdemands, reduced digestion and increased adrenal steroid secretion. The animalspre-treated with EECNE have exhibited prevention in the weight loss induced byimmobilization stress. This observation is probably may be due to increase infood intake, decrease in metabolic demands, increased digestion process ordecreased adrenal steroid secretion17. Reports suggest that, theneurotransmitters such as nor-adrenaline (NA) and serotonin (5-HT) plays avital role in tackling of stress18. Under severe stress conditionsthese neurotransmitters levels are significantly decreased. In our study also,brain levels of NA and 5-HT were found to be reduced significantly inimmobilization stress model.
Animals pretreated with extract demonstratedsignificant amelioration of levels of these neurotransmitters indicates theadaptogenic potential of EECNE. Conclusion: Cocos nucifera endocarp extracthas shown significant improvement in stress-induced alterations of hematological and biochemical parameters, bodyweight, organs weight, stomach ulcer index and brainneurotransmitters levels, indicating its protective effect against stress.However, further experiments are needed to establish the mechanism behindthe adaptogenic potentialof plant.
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