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Pharmacological evaluation of ethanolic extract of Tagetes erecta on eplepsy
 

Pharmacological evaluation of ethanolic extract of Tagetes erecta on eplepsy

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ABSTRACT...

ABSTRACT
Aims: Flowers of Tagetes erecta (Et E) is considered to be effective in the treatment of epilepsy reported in Ayurveda, but the phytoconstituents present in Et E devoid of antiepileptic activity, but found with phytoconstituets which decrease seizure thresholds. Thus the work undertaken to evaluate the effect of ethanolic extract on epilepsy using in vivo models.Methods and Material: Et E was evaluated using the in vivo models such as pentobarbitone induced sleeping time, MES and PTZ induced convulsions , potentiation of PTZ induced convulsion, spontaneous locomotor activity, forced swim test, learned helplessness test and brewers yeast induced pyrexia model.Results: Present study revealed that extract is devoid of sedative, antiepileptic, proconvulsant activity. The Et E shown CNS stimulant activity, antidepressant and antipyretic activity. Conclusions: The findings suggested that ethanolic extract may reduce the seizure threshold in epileptic patients,chances of seizure precipitation is more, thus usage in epilepsy is cautious.
Keywords: Tagetes erecta, epilepsy, antiepileptic, antidepressant, CNS stimulant

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    Pharmacological evaluation of ethanolic extract of Tagetes erecta on eplepsy Pharmacological evaluation of ethanolic extract of Tagetes erecta on eplepsy Document Transcript

    • Lokesh J Shetty et al. / Journal of Pharmacy Research 2009, 2(6),1035-1038Research Article Available online throughISSN: 0974-6943 www.phresearchjournal.info Pharmacological evaluation of ethanolic extract of flowers of Tagetes erecta on epilepsy Lokesh J Shetty * 1, Harikiran H 2, Dr. J Fernandes 3 *1Dept of Pharmacology 1 and Pharmachemistry 2, Shree Devi College of Pharmacy, Kenjar, Mangalore, Karnataka, India. Dept of Pharmachemistry 3 , N G S M Institute of Pharmaceutical Sciences,Paneer- Deralakatte , Mangalore, Karnataka, India. Received on: 0501-2009; Accepted on: 07-03-2009 ABSTRACTAims: Flowers of Tagetes erecta (Et E) is considered to be effective in the treatment of epilepsy reported in Ayurveda, but the phytoconstituentspresent in Et E devoid of antiepileptic activity, but found with phytoconstituets which decrease seizure thresholds. Thus the work undertakento evaluate the effect of ethanolic extract on epilepsy using in vivo models.Methods and Material: Et E was evaluated using the in vivomodels such as pentobarbitone induced sleeping time, MES and PTZ induced convulsions , potentiation of PTZ induced convulsion,spontaneous locomotor activity, forced swim test, learned helplessness test and brewers yeast induced pyrexia model.Results: Presentstudy revealed that extract is devoid of sedative, antiepileptic, proconvulsant activity. The Et E shown CNS stimulant activity, antidepressantand antipyretic activity. Conclusions: The findings suggested that ethanolic extract may reduce the seizure threshold in epileptic patients,chances of seizure precipitation is more, thus usage in epilepsy is cautious.Keywords: Tagetes erecta, epilepsy, antiepileptic, antidepressant, CNS stimulantINTRODUCTION Traditional medicines have been used to alleviate the suffering Preparation of extractof fellow human beings since the down of human civilization, with Coarse powder of flowers was subjected to maceration sepa-centuries of use of these medicines, there is a notion that ‘natural’ is ration. The powdered material was soaked in 90% ethanol for 72 h with‘safe’ has taken a strong hold in the society. Despite their wide spread occasional shaking. The extract obtained was subjected for evapora-usage traditional medicines have not been evaluated scientifically tion under reduced pressure concentrated to dryness (yield 3.2%w/with regard to their safety and efficacy and has many limitations [1]. In w).addition another criteria related to traditional medicine is that some of Animalsprescriptions of folklore or traditional practitioners often recommends Healthy young 8 to 12 weeks old Albino Wistar rats of labo-the remedy to treat pathological conditions but those scientifically ratory strains of either sex weighing 200 -250g or healthy Adult albinoprooved to be precipitating or contraindicated in the same [1,2]. mice of either sex weighing 25±5g of laboratory strains were employed. Present work mainly concentrated in these parameters, flow- Females were nulliparous and non-pregnant. Room temp: 22°C (± 3°C),ers of Tagetes erecta L (Asteraceae) is considered to be effective in Humidity: 50-60%, Lighting: Provided artificial- the sequence being 12the treatment of epilepsy as in Ayurveda, but the phytoconstituents hours light and 12 hours dark, Placement: Individually.The animalspresent in ethanolic extract (Et E) devoid of antiepileptic activity, but were kept in their cages for at least 5 days prior to dosing to allow forfound with phytoconstituets which decrease seizure thresholds[3,4]. acclimatization to the laboratory conditions and fasted ad libitum prierTherefore the present work was undertaken to determine whether Et to dosing Et E / standard drug / vehicle / convulsant[5].E of flower posses the antiepileptic activity or is it contra-indicated in Toxicity studyepileptic patients if used and the pharmacological mechanism behind Acute Oral Toxicity – Up-and-Down Procedure as per OECDit. guidelines for testing chemicals, OECD/OCDE 425[5].Subjects and Methods: Selection of dosesPlant material For the assessment of Pharmcological activity, three dose The flowers of the plant were collected in the summer sea- levels were chosen in such a way that, middle dose was approximatelyson from the local market of Mangalore, India. The drug was identi- one tenth of the maximum dose during acute toxicity studies, and afied by the department of botany, St Agnes College, Mangalore, In- high dose which was twice that of one tenth dose and a low dosedia. which was 50% of one tenth dose[5].*Corresponding author. Test drug and vehicle usedTel.: + 91-09611201558 Test Drug used: - 100 mg/kg body weight (b.w), 200mg/kgTelefax: +91- b.w, 400 mg/kg b.w, Et E was suspended in 0.6%w/v Sodium CarboxyE-mail: surf_the_imidazole@yahoo.co.in Methyl Cellulose (CMC), 2mL/100g body weight per oral (p.o). Vehicle used: - 2mL/100g b.w, p.o, 0.6%w/v Sodium CMC. Journal of Pharmacy Research Vol.2.Issue 6.June 2009 1035
    • Lokesh J Shetty et al. / Journal of Pharmacy Research 2009, 2(6),1035-1038Table I: Effect of Et E on Spontaneous Locomotor Activity. Treatment Mean Locomotor count ± SEM in 5 min at (min) 0 30 60 90 120 Control 149 ±2.72 151.8±2,52 148±2.72 148.8±2.44 151.2 ±2.688 Caffeine, 8mg/kg b.w; i.p 152.8±2,53 256.5±.95** 295.8±1.55** 320±7.57** 312.3 ±5.518** Et E ,100mg/kg b.w; p.o 146.8±2.34 154.8±2.419 156.7±2.80 158.3±3.69 151.7±4.20 Et E ,200mg/kg b.w; p.o 147.8± 2.65 163.7±2.44* 167.8±2.44** 169.2±2.257** 168±2.301 Et E ,400mg/kg b.w; p.o 152.2±3.69 197.5±2.82** 198.2±2.83** 205±3.23** 211±2.78**Values are expressed as Mean ±SEM (n=6).* P<0.05; **P<0.01compared with control.Table II: Effect of Et E on Learned Helplessness Test. Group Treatment % Incidence of escape response I Control 0.00 ±0.00 II Fluoxetine20mg/kg b.w; i.p 100.00±0.00** III Et E ,100 mg/kg b.w; p.o 0.00±0.00 IV Et E, 200 mg/kg b.w; p.o 16.66±16.67 V Et E ,400 mg/kg b.w; p.o 83.33± 16.67**Values are expressed as Mean ± SEM (n=6). **P<0.01compared withcontrol.Table III: Effect of Et E on brewers yeast induced pyrexia model.Treatment Rectal temperature 0C (mean± SEM) at 0h 1h 2h 3hControl 38.42± 0.11 38.98± 0.17 38.92± 0.14 38.87± 0.16Paracetamol,135 mg/kgb.w; i.p 39.11± 0.19 38.98± 0.17 37.96± 0..09** 37.44± 0.18**Et E, 100mg/kg b.w; p.o 39.11 ± 0.15 39.04± 0.18 39.08 6± 0.15 38.99± 0.17Et E, 200mg/kg b.w; p.o 38.94± 0.17 38.81± 0.13 37.93± 0.19** 37.62 ± 0.14**Et E, 400mg/kg b.w; p.o 39.12± 11.00 38.63± 0.12* 37.83± 0.14** 37.59.±0.16**Values are expressed as Mean ± SEM (n=6). **P<0.01compared with control.Effect of extract on Pentobarbitone induced sleeping time [6] to 5 groups comprising of 6 animals each. Group I served as control Overnight fasted animals (albino Wistar rats) were divided in received vehicle p.o one hour prior, Group III, IV and V received the Etto four groups comprising of six animals each. Pentobarbitone was E at different doses ;p.o one hour prior to administration of sub- con-administered (40 mg /Kg b.w, i.p) one hour after oral administration of vulsive dose of PTZ. Group II received the convulsant dose of PTZEt E to animals of group II and IV, sleeping time was recorded as the (80 mg/kg b.w; s.c) and the other groups were treated with sub con-period starting with loss and regaining righting reflex. Group I served vulsive dose PTZ (60 mg/kg b.w; s.c) at the end of one hour andas control received vehicle and Pentobarbitone. individually placed in wire mesh cage. All animals were observed forMaximal Electroshock (MES) induced convulsions [7] % incidence of convulsion up to 30 min after PTZ treatment. Overnight fasted animals (albino Wistar rats) were divided in Spontaneous Locomotor Activity [7]to five groups comprising of six animals each. Group III, IV and V Animals (albino mice) fasted for 3-4 hr were divided in to fivereceived the Et E at different doses orally 60 min prier to induction of groups comprising of six animals each and basal spontaneous motorMES. Group II received the standard drug (Diazepam, 2 mg/Kg b.w, activity was measured (5 min). Group I received the vehicles served asi.p) 30 min before induction of MES, Group I received the vehicles control, group III to V received the Et E at different doses; p.o 60 minserved as control.A supramaximal electrical stimulus of 150 mA was prior, group II received the standard drug (Caffeine, 8mg/Kg b.w; i.p)given to the animals for 0.2 seconds through ear clip electrodes. Ani- 30 min before the first reading. Animals were placed in actophotometermals observed for various phases of MES seizures viz. tonic hind limb and spontaneous locomotor activity was measured for 5 min at 30minflexion, tonic hind limb extensor and tonic- clonic phase. Abolition or interval.decrease in the duration of extensor phase was taken as an index ofForced Swim Test (FST [8]anti epileptic activity. Drug administration: - Animals (albino mice) fasted for 3-4hrPentylene Tetrazole (PTZ) induced convulsions [7] were divided in to five groups comprising of six each, group III to V Overnight fasted animals (albino Wistar rats) were divided in received Et E at different dose; p.o, group I received vehicle one hourto 5 groups comprising of 6 animals each. Group III, IV and V received prior and to the group II standard drug (Imipramine, 20mg/kg b.w; i.p)the Et E; p. o one hour prior to administration of PTZ. Group II re- was administered 30 min prior on the second day before the test ses-ceived the standard drug (Diazepam, 2mg/kg b.w; i. p) 30 min prior to sion.PTZ treatment. Group I served as control received vehicle. Animals in Procedure: - FST was performed based on the method de-each group were treated with PTZ (80mg/kg b.w; s.c) and individually scribed by Porsolt et al.placed in wire mesh cage. All animals were observed for onset of Briefly animals were forced to swim individually for 15 min, in glassconvulsion up to 30 min after PTZ treatment. beaker (11 cm diameter, 15 cm height) containing fresh water up to aSub-Convulsive Dose of Pentylene Tetrazole with Et E Induced Con- height of 6 cm at a temperature 22± 1ºC. This constituted the pretestvulsion [2] session. Twenty four hours later each animals were again forced to Overnight fasted animals (albino Wistar rats) were divided in swim for a period of 6 min in a test session. Each animals made vigor- Journal of Pharmacy Research Vol.2.Issue 6.June 2009 1036
    • Lokesh J Shetty et al. / Journal of Pharmacy Research 2009, 2(6),1035-1038ous attempts to get out of the glass beaker during first couple of to be antidepressant[12].minutes and there after surrendered to experimental conditions and GABA serves as an inhibitory neurotransmitter in brain.became immobile with occasional escape attempts. The total duration GABAergic interneuron’s in the cortex terminate local sustained burstof immobility during the last 4 min of a 6 min test was recorded. firing and through inhibitory surround, limit lateral spread of seizureLearned Helplessness Test [8] activity. Those that mimic or potentiate the effects of GABA are gen- Overnight fasted Animals( albino Wistar rats) were subjected erally sedative are reported to posses good antiepileptic activity[13]. Infor escapable shock treatment (10 sec duration, 0.8 mA current) i.e., the present study sedative activity of the ethanolic extract was stud-animals placed in shock treatment chamber and trained to escape to- ied, but the result was not significant shown that the extract does notwards the safe chamber when the foot shock was applied. Escape posses sedative property.from the shock treatment chamber to the safe chamber when the foot Previous studies reported that compounds which mimic orshock applied was considered as the escape response. Animals shown potentiate the GABA exert there action primarily mediated by the re-positive escape response were selected for inescapable shock treat- ceptor containing a 1, a 2, a 3 and a 5 subunits. In contrast, sedative ac-ment. During this session animals placed in shock treatment chamber tions are mediated primarily by a1 subunit not by a 2, a 3 and a 5 sub-without any escape route and repeated foot shock was applied (10 sec units. Studies shown that TPA023 which selectively modulates a 2 orduration per min, 0.8 mA current) for an hour. Failure to exhibit escapea 3 subunit of GABAA receptor, ELB139 selective for a3 containingresponse by animal was taken as indicative of its depressive state. receptor both exhibited anticonvulsant properties but is non sedat-Animals failed to exhibit escape response were taken for study. Ani- ing[13]. Therefore if the compound is devoid of sedative property thatmals were divided into five groups comprising of six each, group III- cannot be considered as devoid of antiepileptic property. ThereforeV fed with Et E at different dose; p.o and group I served as control the antiepileptic activity was confirmed with the help of PTZ- inducedreceived vehicle one hour prier, for group II test drug ( Fluoxetine,20 convulsions. But the results of present study of MES and PTZ in-mg/kg b.w, i.p) was administered 30 min prior to study. Animals sub- duced convulsions revealed that the Et E is devoid of antiepilepticjected to escapable shock treatment and incidence of escape response activity on garndmal epilepsy and petitmal epilepsy.was recorded. Studies reported that CNS stimulants, antidepressants areAssessment of antipyretic activity using Brewers Yeast induced Pyr- contraindicated in the epileptic patients; reported to decrease the sei-exia Model[10] zure threshold [12]. Results of increase in spontaneous locomotor ac- Animals with approximately constant rectal temperature tivity was significant thus the Et E of Tagetes erecta may have CNS(37.5OC – 38.50C) were selected for study. After recording basal tem- stimulant property; may due to the presence of indole as one of theperature, fever was induced by injecting 10ml/kg of 15% suspension major phytoconstituents [4]. Results of reduction in duration of immo-of brewer’s yeast in 0.9% Saline s.c in the back of hope of neck. The bility learned helplessness test revealed that Et E posses antidepres-Site was massaged to spread the suspension beneath the skin. Ani- sant property; mechanism of action may via serotonergic pathway[8].mals were kept in standard laboratory conditions in individual cages; Present study also revealed that Et E does not posses proconvulsantfood was withdrawn immediately after the injection. Eighteen hours property.later rise in rectal temperatures was recorded at zero h. Animals were Due to the CNS stimulant and antidepressant property of the Et E;divided in to 5 groups comprising of 6 animals each. Group I served as may decrease the seizure threshold, it may cautious if used in epilepticcontrol received vehicle p.o, group III to V received the Et E at differ- patients. Chances of incidence of seizure development may be more.ent dose; p.o one hour prior and group II received the standard drug Results of decrease in brewers yeast induced pyrexia was significant;(Paracetamol, 135 mg/kg, i.p) 30 min prior to next rectal temperature may be used in benign febrile epilepsy in Ayurveda in earlier days,measurement. Rectal temperature was measured at 1, 2, and 3 hr after where the relief from the convulsions may due to decrease in elevatedoral administration of drug. body temperature [12]. Thus the above findings suggested that Et E of flowers of Tagetes erecta does not have antiepileptic activity; mayStatistical analysis[11] contraindicated in epileptic patients if used. The data was presented as mean ± SEM. The data was ana- References:lyzed by one-way analysis of variance (ANOV followed by Dunnett’s A) 1. Gupta SK, Rajan M. Safety monitoring of traditional medicines: why and how? The Pharm Review 2004; 5: 21-33.test using Graph Pad Prism software, version 4.01. 2. Rumana S, Divya V, Vohara SB. Proconvulsant Effects of CalcinedResults: Arsenic Preparation Used In Unani Medicine. Ind J Pharmacol 1999;The Tables found statistically significant are given in Table I to III. 31: 150-52. 3. Nadakarni KM. The Indian Materia Medica. Bombay: PopularDiscussion: prakashan1954:1191 4. Emari E, Ali. Revised phytochemical study of Tagetes erecta. Et E of flowers of the Tagetes erecta is considered to be Fitoterapia.1983;54(1):9-12 5. New OECD 425 Guidelines. OECD Guidelines for testing of Animals.effective in the treatment of epilepsy as in Ayurveda, but the 2001: 1-26.phytoconstituents present in Et E devoid of antiepileptic activity, but 6. Manna s, Battaacharya D, Mandel TK, Dey S. Neuropharmacologicalfound with phytoconstituets which decrease seizure thresholds such effects of Alfa – Cypermethrin in rats. Indian J Pharmacol 2005: 37: 18-20.as menthol, indole alkaloids and verbenone[2,4]. Menthol is already 7. Girish SA, Sudhir G, Wadodkar. Evaluation of sedative and anticonvulsantproved to be proconvulsant in nature, indole alkaloids are CNS stimu- activities of Unmadnashak Ghrita . J of Ethnopharmacol; 2004; 94:lants thus thus may reduce seizure threshold and verbenone is found 77-83. Journal of Pharmacy Research Vol.2.Issue 6.June 2009 1037
    • Lokesh J Shetty et al. / Journal of Pharmacy Research 2009, 2(6),1035-10388. Porsolt RD, Bertin A, Jalfre M. Behavioral despair in mice. A primary sissoo leaves Indian J Pharmacol 2000; 32 (6): 357-60 screening test for antidepressants. Arch int Arch int. Pharmacodyn 11. Graph Pad Prism. Version 4.01for windows. Graph Pad Software Inc: 1977; 229: 327-36. San Diego, California, USA 20049. Vogel GH, Vogel WH (Eds). Drug Discovery & Evaluation of Phar- 12. James EF, Kathleen P, Anne VP, Sean CS. Martindale: The extra macological Assay. New York: Springer Varlag Berlin Heidelburg Pharmacopoeia. London Royal Pharmaceutical Society 1996; 21: 2002: 422-23,561-62. 363-36610. Hajare SW, Suresh C. Analgesic and antipyretic activities of Dalbergia 13. Meldrum B, Michael AR. Molecular targets for antiepileptic drug de- velopment. Neurotherapeutics 2007 4(1):18-61 Source of support: Nil, Conflict of interest: None Declared Journal of Pharmacy Research Vol.2.Issue 6.June 2009 1038