Evaluation Of anti-epileptic activity of Psidium Gujava Extract


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Evaluation Of anti-epileptic activity of Psidium Gujava Extract

  2. 2.  EPILEPSY: Epilepsy is a collective term for a group of chronic seizure disorder having in common ,sudden and transient episodes (seizure) of loss or disturbance of consciousness , usually but not always with a characteristic body movements (convulsions) and sometimes with autonomic hyperactivity. SEIZURES : Seizures are discrete, time limited alterations in brain function including changes in motor activity, autonomic function, consciousness or sensation that result from an abnormal and excessive electrical discharge of a group of neurons with in the brain. CONVULSIONS: Convulsions are sudden, violent, involuntary contractions of the muscles of the body, often accompanied by loss of resconsciousness. mssist-cop
  3. 3.  Psidium guajava (Linn.) belongs to the family-Myrtaceae has many pharmaceutical applications. ROOTS - anti-emetic, and are useful in hemorrhages, diarrhoea and dysentery especially in children, ulcers, gingivitis, proctoptosis and vomiting. LEAVES - wounds, ulcers, cholera, diarrhoea, vomiting, nephritis, cachexia and epilepsy. FLOWERS - bronchitis, ophthalmodynia, colic andulemorrhagia. FRUITS - aphrodisiac, laxative, tonic and also useful in diarrhoea, dysentery.9 mssist-cop
  4. 4. AIM : An attempt is proposed to evaluate the effect of Psidium guajava leaves extract on electro shock induced convulsions and Pentylenetetrazole induced convulsions in rats or mice. The whole study is divided into two phases:1. Phase -1 : Identification and authentication of the plant Psidium guajava(Linn.)2. Phase -2 :To evaluate antiepileptic activity of hydro alcoholic extract of Psidium guajava (Linn.) Leaves in various validated animal models such as: - PTZ (Pentylenetetrazole) Induced convulsive model - Maximal Electro Shock (MES) Induced convulsive model mssist-cop
  5. 5. METHODOLOGY:The hydro alcoholic extract of the leaves was collected and the following preliminary tests were performed:Nature : StickyColor : Greenish%Yield : 39.68(% w/w) in g. mssist-cop
  6. 6.  PRELIMINARY PHYTOCHEMICAL TESTS:EXPERIMENT OBSERVATION INFERENCEBenedicts test Formation of orange red presence of reducing precipitate sugar.Fehling’s Test red precipitate presence of reducing sugarBiuret test purplish violet color presence of proteins.Millions test brick red precipitate Presence of proteinsNinhydrin test purple or bluish color appears. Presence of amino acidsTyrosine test dark red color Presence of amino acidsMayer’s Test yellow cream precipitate presence of alkaloidsDragendroff’s red precipitate presence of alkaloidsTestBorntrager’s test Ammonical layer turns pink or red. Presence of glycosidesLead acetate test Yellow colored precipitate Presence of flavonoids mssist-cop
  7. 7. EXPERIMENT OBSERVATION INFERENCESalkowski’s Test lower layer turns red Sterol are present. lower layer turns golden triterpenes are presents yellowStain Test oily stain on filter paper presence of fixed oilLead acetate test White precipitate Presence of tannins and phenolsAcetic acid test red color solution. Presence of tannins and phenols mssist-cop
  8. 8. Experimental animal : Albino mice of either sex weighing between 20-30g were procured.Principle of the test : Animals are dosed, one at a time, at 24 hour intervals. The first animal receives a dose at the level of the best estimate of the LD50. Depending on the outcome for the previous animal, the dose for the next animal is adjusted up or down. If an animal survives, the dose for the next animal is increased; if it dies, the dose for the next animal is decreased. After reaching the reversal of the initial outcome, i.e. the point where an increasing (or decreasing) dose pattern is reversed by giving a smaller (or a higher) dose, four additional animals are dosed following the same UDP. The LD50 is calculated using the method of maximum likelihood. mssist-cop
  9. 9. Procedure: The systemic acute oral toxicity (LD50) profile of the extract was evaluated in female wistar albino rats according to OECD 425 guidelines. In brief, this method was carried out in three steps, the initial investigation in which nine animals were used, three animals per treatment group. The animals used were fasted overnight, note down the fasted body weights and calculate the doses, the dose volume should not be exceeded 1ml/100gm . The different doses selected were 500, 1000, 2000 mg/kg of the extract per body weight. Animals are observed individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter for a total of 14 days. However, the duration of the observation period should not be fixed rigidly. It should be determined by the toxic reactions, time of onset and length of recovery period, and may thus be extended when considered necessary. mssist-cop
  10. 10. DETERMINATION OF ANTICONVULSANT ACTIVITY :PTZ INDUCED CONVULSIONS: Albino mice of either sex weighing between 22-25g were randomly selected and segregated in to five groups, each group consisting of six animals. Group A - Normal control (2%w/v Gum acacia p.o.) Group B - Standard (Diazepam 5mg/kg p.o) Group C - Leaf extract of Psidium guajava (Linn.) (100mg/kg p.o) Group D - Leaf extract of Psidium guajava (Linn.) (200 mg/kg p.o) Group E - Leaf extract of Psidium guajava (Linn.) (400 mg/kg p.o) mssist-cop
  11. 11. Experimental procedure(for anti convulsant activity) Group A served as normal control and was administered with 2%w/v Gum acacia suspension orally, Group B with diazepam (5mg/kg p.o.) and served as standard, Groups C, D and E with three different doses of leaf extracts (low, medium and high respectively) hydroalcoholic of Psidium guajava(Linn.) for seven consecutive days. On the eighth day one hour after the oral administration of either acacia suspension/standard drug/extracts respectively to different groups, PTZ 60 mg/kg was administered subcutaneously. Each animal was then placed into individual plastic cages and were observed initially for 30min and later up to 24 hrs.The following parameters were recorded during test session of initial 30min and up to 24 hrs respectively: ► Latency (onset of clonus) ► Onset of tonic-clonic convulsions ► Status of animal after 1hr ► Status of animal after 24 hrs ► Percent protection mssist-cop
  12. 12. MAXIMAL ELECTRO SHOCK (MES) INDUCED CONVULSIONS Albino mice of either sex weighing between 22-25g were divided into five groups each group was consisting of six animals. Group A - Normal control (2% gum acacia p.o) Group B - Standard (Diazepam 5mg/kg p.o) Group C - Leaf extract of Psidium guajava (Linn.) (100mg/kg p.o) Group D - Leaf extract of Psidium guajava (Linn.) (200 mg/kg p.o) Group E - Leaf extract of Psidium guajava (Linn.) (400 mg/kg p.o) mssist-cop
  13. 13. Experimental procedure(For MES induced convulsions) : Group A served as control and was administered with 2% gum acacia suspension, Group B with phenytoin (25mg/kg p.o.) and served as standard. Group C, D and E with three different doses of hydroalcoholic extracts of Psidium guajava(Linn.) (low, medium and high respectively) for seven consecutive days. On the eighth day one hour after oral administration of acacia suspension/standard drug/different extracts to respective groups, MES seizures were induced by electroconvulsometer. A 60 mA current was delivered transauricularly for 0.2sec in mice.. In the pilot study various phases of convulsions, viz., tonic flexion, extension, clonus, stupor and mortality due to convulsions were selected as the parameters. Phenytoin (25mg/kg p.o.) used as standard drug.The following parameters were recorded during 1hr test session. Tonic flexion Tonic extension Clonus convulsions Percent protection mssist-cop
  14. 14. RESULTS. Assessment of anti-convulsant activity of hydro alcoholic extract of psidium guajava leaves :PTZ (Pentylenetetrazole) induced convulsions: Lower dose(100mg/kg)produced significant anticonvulsant activity when compared to control Medium and high doses (200 & 400mg/kg respectively) exhibited a very significant anti convulsant effect by increasing onset time of seizures and reducing the duration of tonic-clonic seizures. The lower, medium and higher doses of leaves offered a protective effect of 66.66%, 83.33% and 83.33% up to 1hr interval respectively. The standard drug diazepam (5mg/kg) exhibited a significant anti- convulsant activity and offered 100% protection mssist-cop
  15. 15. ONSET OF CONVULSIONS (seconds) STATUS OFTREATEME ANIMAL AFTER MEAN ± SEM NT No. of animals in the group 1hr Death/Recovery 1 2 3 4 5 6 Control (2% Gum 654 648 699 687 643 692 670.5±10.135 6/0acacia p.o.) Diazepam 885 925 880 870 923 970 908.8±15.396** 0/6(5mg/kg p.o.) PGE (100mg/kg 735 789 690 733 690 709 724.3±15.235* 2/4 p.o.) PGE (200mg/kg 790 755 820 805 782 795 806.1±15.844** 1/5 p.o.) PGE (400mg/kg 800 830 832 805 852 820 823.1±7.816** 1/5 p.o.) mssist-cop
  16. 16. Effect of hydro alcoholic extract of psidium guajava(linn.) leaveson MES induced convulsions (Duration of tonic extensor seizure) in mice. mssist-cop
  17. 17. DURATION OF TONIC EXTENSOR SEIZURES (seconds) MEAN ± SEM No. of animals in the groupTREATEMENT 1 2 3 4 5 6 Control (2% Gum 21 24 23 22 25 23 23 ±0.577 acacia p.o.) Diazepam 7 5 6 4 8 5 5.83 ±0.600, ** (5mg/kg p.o.) PGE 22.16 21 24 23 20 22 23(100mg/kg p.o.) ±0.600 PGE 13 16 14 15 12 16 14.33 ±0.666, **(200mg/kg p.o.) PGE 10 14 11 13 12 11 12.16 ±0.600, **(400mg/kg p.o.) mssist-cop
  18. 18. ) Effect of hydro alcoholic extract of psidium guajava (linn.) leaveson MES induced convulsions (Onset of clonic seizure) in mice mssist-cop
  19. 19. DURATION OF ONSET OF TONIC CLONUS EXTENSOR % AVG.WT. AVG.VOL.OF (Sec)S.NO TREATEMENT (Sec) PROTECTION (g) DOSE (ml) (1hr) Mean ± SEM Mean ± SEM Control 1 (2% Gum acacia 24.6 0.25 25±0.57 23 ±0.577 33.33 p.o.) Diazepam (5mg/kg 2 23.8 0.23 6.8±0.60** 5.83 ±0.600** 100 p.o.) PGE 3 23 0.23 24.1±0.60 22.16 ±0.600 33.33 (100mg/kg p.o.) PGE 4 22 0.22 16.3±0.66** 14.33 ±0.666 ** 66.66 (200mg/kg p.o.) PGE 83.33 5 24 0.24 13.8±0.60** 12.16 ±0.600 ** (400mg/kg p.o.) mssist-cop
  20. 20. Effect of hydro alcoholic extract of psidium guajava (linn.) leaveson MES induced convulsions in mice mssist-cop
  21. 21.  The present study was carried out to find out, evaluation of anti-epileptic activity on Psidium Guajava (Linn.) leaves extract in mice. From the results obtained, we conclude that the Psidium Guajava (Linn.) Leaves extract at higher and medium doses produces highly significant and sustained increase in the delay of onset of convulsions and decrease in the no of convulsions. This activity may be due to the presence of different phytoconstituents viz, flavanoids and saponins in the extract. However, long term studies in different animals and epileptic subjects may further substantial our study result mssist-cop
  22. 22.  Namara JO. Drug effective in the therapy of the Epilepsies. Goodman and Gilman’s The Pharmacological basis of Therapeutics. 10th Ed. New York, Mc Graw Hill; 2001: 521. Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for revised clinical and electroencephalographic classification of epileptic seizures. Epilepsia, 1981, 22: 489-501 HL Sharma, KK Sharma, Principles of pharmacology, Paras medical publisher, Delhi, 2007; 1:531. http://en.wikipedia.org/wiki/Convulsion. Access date 25/12/2008 Walczak TS, Leppik IE, DAmelio M, Rarick J, Ahman P, Ruggles K, et al. Incidence and risk factors in sudden unexpected death in epilepsy: a prospective cohort study. Neurology 2001; 56: 519-525. Lathers C, Schraeder P. Epilepsy and Sudden Death. Dekker; 1990 NY. Hitiris N, Mohanraj R, Norrie J, Brodie M J. Mortality in epilepsy. Epilepsy Behaviour; 2007, 363-376. mssist-cop
  23. 23. THANK YOU mssist-cop