Anxiolytic and Anticonvulsant Activity of Methanol Extract of Leaves of Alternanthera Brasiliana (L.) Kuntze (Amaranthaceae) in Laboratory Animals

Indian Journal of Experimental Biology Vol. 51, June 2013, pp. 450-457

Anxiolytic and anticonvulsant activity of methanol extract of leaves of Alternanthera brasiliana (L.) Kuntze (Amaranthaceae) in laboratory animals

Chandana C Barua1*, Shameem A Begum2, Acheenta G Barua3, Rumi S Borah4 & Mangala Lahkar5 Department of 1Veterinary Pharmacology and Toxicology, 2Veterinary Pathology, 3Veterinary Public Health & Hygiene and 4Animal Production and Management, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati 781 022, India and 5Department of Pharmacology, GMCH, Guwahati 781 006, India

Received 9 October 2012; revised 13 March 2013

Anxiety related disorders are the most common mental illnesses and major cause of disability in man. Anxiolytic activity of methanol extract of leaves of A. brasiliana (L.) Kuntze (MEAB) was evaluated using hole board (HB), open field (OF), elevated plus maze (EPM) and light/dark exploration test (LDE) in mice. Its locomotor activity was studied using actophotometer and anticonvulsant effect was studied using maximal electroshock-induced seizures and pentylenetetrazole- induced seizures in mice. Single oral administration of MEAB at different doses (100, 300 and 600 mg/kg, ip) significantly increased the number and duration of head poking in the HB test; rearing, assisted rearing and number of square traveled in the OF test; entries and time spent in open arm in the EPM test; time spent in lighted box, and numbers of crossings and transfer latency time in the LDE test. There was significant reduction in the time spent in close arm in the EPM test and time spent in dark box in LDE test. In the actophotometer, the activity count was reduced in MEAB and diazepam treated group than control group. All the three doses of the extract significantly reduced the duration of seizures induced by pentylenetetrazole (chemoshock convulsion). However, the extract did not show any appreciable effect in electroshock convulsion model. The results of the present study suggest promising anxiolytic and anticonvulsant activity of MEAB which might be accredited to different phytoconstituents like alkaloids, steroids and triterpenes present in the methanol extract of A. brasiliana.

Keywords: Alternanthera brasiliana, Anticonvulsant activity, Elevated plus maze, Hole board, Light/dark exploration, Locomotor activity, Open field test, Seizures

Anxiety related disorders such as panic, phobias, treatment of psychotic disorders especially for anxiety obsessive-compulsion and post traumatic stress are in traditional medicinal practice, most of which the most common mental illnesses and major cause directly or indirectly affect the central nervous of disability in the world1. It affects about 1/8th of system, noradrenaline, serotonin, GABA and BDZ the total population worldwide and have become neurotransmitters activities3-7. a very important area of research interest in Alternanthera brasiliana (L.) Kuntze, 2 psychopharmacology . (Amaranthaceae) is an evergreen, perennial herb, Benzodiazepines (BZDs), barbiturates and tricyclic native to tropical and sub-tropical regions of Australia antidepressants (TCA’s) have been used for long and South America. It is a Brazilian plant commonly time to treat anxiety disorders. The serious side known as “penicilina” or terramicina in Brazil and effects associated with these drugs, namely rebound widely used by rural communities as medicinal agent insomnia, sedation, muscle relaxation, anterograde to cure diseases like inflammation, and dolorous or amnesia, withdrawal and tolerance (BZD’s, infection processes, wound healing, as analgesic, barbiturates and alcohol), sexual dysfunction, for antitumor activity, as immunomodulator and for anticholinergic, antihistaminic effects (TCA’s) have lymphocyte proliferation. Aqueous or ethanol extracts limited their use in patients. Many of the plant of A. brasiliana were able to block human mitogen secondary metabolites are being used for the induced lymphocyte proliferation without any toxic —————— effect8. Different extracts of A. brasiliana exhibited *Correspondent author antinociceptive9, antimicrobial10 and also anti-herpes Telephone: 91-361-2361485 simplex virus activity11. The plant showed Cell: 098640-13231 12 Fax: 91-361-2337700 antinociceptive activity . Methanol extract of E-mail: [email protected] A. brasiliana also exhibited wound healing activity BARUA et al.: ANXIOLYTIC & ANTICONVULSANT ACTIVITY OF ALTERNANTHERA LEAVES EXTRACT 451

in both in vivo (excision and incision wound model) as administered orally with MEAB at 100, 300 well as in vitro (chorioallantoic membrane) models13 and 600 mg/kg, respectively and group V mice and in immunocompromised wound model in rats14. received the standard drug diazepam (1 mg/kg), The present study has been undertaken to elucidate intraperitoneally. The study was conducted after anxiolytic and anticonvulsant activity of the extract of obtaining approval of Institutional Animal Ethics its leaves in laboratory animals. Committee (No: 770/03/ac/CPCSEA/FVSc, AAU/ IAEC/06/21). Materials and Methods Determination of LD acute toxicity and gross Plant material—The leaves of the plant, collected 50, effect— LD dose was determined following up-and- from the medicinal garden of the Department 50 down stair case method in mice as per OECD TG-425 of Pharmacology, College of Veterinary Science, guidelines. Doses were adjusted by a constant Khanapara during the month of February to June, multiplication factor (viz., 4) for this experiment. The 2010 were identified by Dr. I. C. Baruah, Principal dose for each successive animal was adjusted Scientist, AAU, Jorhat, and a voucher specimen depending on the previous outcome. The acute (AAU/CVSC/PHT/02) was deposited. toxicity and gross effect of crude methanolic extract Preparation of methanol leaf extract of of A. brasiliana were studied by using 1/2 LD dose. A. brasiliana—The leaves of the plant were cleaned, 50 Six numbers of male albino mice were selected for shade dried, powdered mechanically, weighed and each experiment. Animals were observed at hourly stored in air tight container. About 250 g powder interval for 6 h and again after 24 h. The parameters was soaked in 1000 mL methanol for 72 h in beaker for motor activity and gross effect were determined and mixture was stirred every 18 h using a sterile after administration of A. brasiliana orally at glass rod. Filtrate was obtained after passing thrice 2000 mg/kg body weight. through Whatman filter paper no 1 and concentrated in Rotary evaporator at 50-60 °C under reduced Anxiolytic activity pressure. A dark brown residue was obtained and Hole board test16—The hole board apparatus stored in air tight container at 4 °C till further use. consists of a wooden chamber (40 × 40 × 25 cm3) Recovery of methanol leaf extract of A. brasiliana with 16 holes (each of 3 cm diameter) evenly (MEAB) was 6.12% (w/w). distributed on the floor. It was elevated to a height of Phytochemical screening—The phytochemical 25 cm from the ground so that the mice could peep screening was carried out for the presence of different 15 through the holes. The mice were treated with MEAB, phytoconstituents qualitatively . standard drug or distilled water 30 min prior to test Drugs and chemicals—Diazepam (Ranbaxy and placed in the apparatus. The number of head Laboratories Ltd, Mumbai, India) was used as pokes and the duration of head poking were recorded standard drug and Pentylenetetrazole (PTZ; Sigma during 5 min observation period by observers blind to Chemical Co.), was used for inducing chemoshock the treatment conditions. convulsion. Methanol was procured from Merck Open field test17—The apparatus consists of a (Mumbai, India). × × 3 Experimental animals—Healthy adult male Swiss wooden box (60 60 30 cm ). The floor of the box × albino mice, approximately of 6-8 weeks of age, was divided into 16 squares (15 15 cm). It was weighing 20-30 g procured from the departmental illuminated with a 40-W lamp suspended 100 cm animal house, were used. They were housed under above. The mice were treated with test plant extract controlled conditions of temperature (25±3 °C), (MEAB), standard drug or distilled water and placed humidity (50±5%) and 10/14 h of light-dark in the apparatus. After 30 min, they were placed cycles with free access to food and water. Animals individually in one of the corner squares. The number were housed individually in polypropylene cages of rearing, assisted rearing (forepaws touching the containing sterile paddy husk bedding. They were wall of the apparatus) and the squares traveled were fasted overnight to achieve better drug absorption counted for 5 min. 18 through gastrointestinal tract. Elevated plus maze test —The plus maze The animals were randomly allocated into 5 groups apparatus consists of two open arms (35 × 5 cm2) of 6 animals each. Group I was assigned as vehicle crossed with two closed arms (35 × 5 × 20 cm3). control (distilled water), groups II, III and IV were The arms were connected together with a central 452 INDIAN J EXP BIOL, JUNE 2013

square (5 × 5 cm2). The apparatus was elevated to mice32 and different phase of convulsions were noted the height of 25 cm in a dimly illuminated room. The and reduction/increase in time or abolition of tonic mice were treated with test plant extract (MEAB), extensor phase in test groups was recorded and standard drug or distilled water. After 30 min they compared with control and standard group using were placed individually in the center of the Electroconvulsiometer (Rolex), 30 min after the oral apparatus, facing the closed arm. The time spent administration of vehicle, test drug and standard drug in both the open and closed arms was recorded for (diazepam, 1 mg/kg ip). 5 min. The numbers of entries into open and closed Statistical analysis—Results were expressed as arms were also counted during the test. An entry was mean ± SE and analyzed by one-way ANOVA using defined as having all four paws within the arm. the SPSS software (version 11.5). Light/dark exploration test19—The apparatus consists of two boxes (25 × 25 × 25 cm3) joined Results Phytochemical screening of MEAB showed together. One box was made dark by covering its top 15 with plywood, whereas a 40-W lamp illuminated the presence of alkaloids steroids and triterpenes . other box. The light source was placed 25 cm above MEAB was found to be safe up to 2000 mg/kg the open box. The mice were treated with test plant body weight, po. In acute toxicity study, there was extract (MEAB), standard drug or distilled water no change in motor activity and gross behaviour 30 min before being placed individually in the center at 2000 mg/kg body weight po during 24 h of of the lit box and four behavioural events (number of observation. Hence, the plant extract is relatively crossings, time spent in light and dark box, transfer safe for consumption and did not affect any of the latency) were recorded for the next 5 min. parameters measured. Locomotor activity20—The locomotor activity can In the hole board test, the numbers of head poking be easily studied with the help of actophotometer after administration of MEAB at 300 and 600 mg/kg (Rolex), which operates on photoelectric cells and is was comparable with the standard drug diazepam connected into a circuit with a counter. When the and the values were significantly higher (P<0.05) beam of light falling on the photocell is cut off by the compared to the control group. There was gradual animal, a count is recorded. The mice were treated increase in duration of head poking after single with test plant extract (MEAB), standard drug or oral administration of MEAB from 100–600 mg/kg. distilled water. After 30 min of pre-treatment with the The duration of head poking also increased dose test compound, the mice were placed individually in dependently; MEAB at 300 and 600 mg/kg doses the activity cage for 10 min and activity score of each showed significant (P<0.05) increase in duration of animal was recorded. Percent reduction/increase in head poking as compared to the standard drug treated motor activity in test groups was calculated and group (Table 1). compared with the control and the standard group. In the open field test, there was significant (P<0.05) increase in rearing, assisted rearing and Anticonvulsant activity number of squares traveled in MEAB (100–600 Chemoshock convulsion21—After 1 h of mg/kg) and standard drug treated group, compared to administration of vehicle and MEAB, the control group. The numbers of rearing in the Pentylenetetrazole (PTZ, 80 mg/kg) were Table 1—Effect of A. brasiliana leaf extract (MEAB) on number administered intraperitoneally to all the animals. and duration of head poking in hole board test in mice Each animal was placed individually and latency [Values are mean ± SE from 6 animals in each group] (onset of clonus), onset of tonic convulsions and Treatment Drugs Dose Head poking in percentage protection was recorded initially for groups (mg/kg, po) hole board test 30 min and at the end of 24 h. Number Duration (sec) 22 Maximal electroshock induced convulsion — Gr. I Control - 32±2.13ad 15.12±1.07e Effect of MEAB was tested against electrically Gr. II MEAB 100 34.83±1.49abd 21.83±1.20b induced convulsions. A 60Amp current was delivered Gr. III MEAB 300 37.83±1.87abcd 32.83±1.45a transauricularly for 0.2 sec to mice through small Gr. IV MEAB 600 39.83±1.08bc 40.83±1.40c alligator clips attached to each pinna by Gr. V Diazepam 1 (ip) 42.33±3.01c 27.87±2.42d electroconvulsiometer. This current intensity elicits *Mean in a column bearing same superscript do not differ complete tonic extension of the hind limbs in control significantly (P<0.05). BARUA et al.: ANXIOLYTIC & ANTICONVULSANT ACTIVITY OF ALTERNANTHERA LEAVES EXTRACT 453

groups treated with MEAB at 300 mg/kg was treated groups (Table 3). The numbers of entries into comparable with the standard drug, but at the open arm at 300 and 600 mg/kg of MEAB were 600 mg/kg dose significantly (P<0.05) higher than comparable with the standard group. MEAB also the standard group. The numbers of assisted rearing showed dose dependent increase in the number of at 300 and 600 mg/kg of MEAB treated groups was entries into the close arm. significantly (P<0.05) higher than the standard group. In light/dark exploration test, time spent (sec) in The number of squares traveled also increased lighted box, numbers of crossings and transfer latency significantly (P<0.05) in a dose dependent manner (sec) in MEAB (300 and 600 mg/kg) and standard indicating anxiolytic activity of the plant extract drug treated groups increased significantly (P<0.05) (Table 2). and the time spent (sec) in dark box decreased In elevated plus maze test, there was dose significantly (P<0.05) compared to the control group. dependent increase (P<0.05) in the number of entries At 600 mg/kg of MEAB treated group, the values in the open arm but significant (P<0.05) decrease in were comparable with the standard drug treated group the number of entries in the close arm in MEAB (Table 4). Table 2—Effect of A. brasiliana leaf extract (MEAB) on rearing, assisted rearing and squares traveled in open field test in mice [Values are mean ± SE from 6 animals in each group] Treatment Drugs Dose Open field test groups (mg/kg, po) No of rearing No of assisted rearing No of square traveled Gr. I Control - 2.5±0.67ac 11.17±0.70c 71.83±3.01b Gr. II MEAB 100 3.00±0.58ac 14.33±0.72a 79.67±1.73a Gr. III MEAB 300 7.17±0.60b 17.17±0.70b 84.33±1.71a Gr. IV MEAB 600 12.17±0.70d 19.83±0.48d 92.33±1.69c Gr. V Diazepam 1 (ip) 5.50±0.56b 14.83±1.01a 99.5±2.59d *Mean in a column bearing same superscript do not differ significantly (P<0.05).

Table 3—Effect of A. brasiliana leaf extract (MEAB) on time spent and number of entries in open and closed arm in elevated plus maze test in mice [Values are mean ± SE from 6 animals in each group] Treatment Drugs Dose Elevated plus maze test Groups (mg/kg, po) Time spent in open Time spent in No of entries into No of entries into arm (sec) close arm (sec) open arm close arm Gr. I Control - 21.36±1.02e 270.60±1.89e 3.17±0.6b 11.83±1.17ad Gr. II MEAB 100 59.52±1.10b 230.48±1.30b 3.83±0.6b 5.33±0.67b Gr. III MEAB 300 80.66±3.66a 209.57±3.78a 5.83±0.6a 9.83±0.76a Gr. IV MEAB 600 98.34±5.17d 192.55±4.83d 6.50±0.75a 13.50±1.18cd Gr. V Diazepam 1 (ip) 111.18±2.56c 178.15±3.18c 7.17±0.6ac 11.5±0.92acd Mean in a column bearing same superscript do not differ significantly (P<0.05).

Table 4—Effect of A. brasiliana leaf extract (MEAB) on time spent in light and dark box, number of crossing and transfer latency time in light/dark exploration test in mice [Values are mean ± SE from 6 animals in each group] Treatment Drugs Dose Light/dark exploration test Groups (mg/kg, po) Time spent in light Time spent in No of Transfer latency box (sec) dark box (sec) crossing time (sec) Gr. I Control - 57.33±3.94b 236.46±4.29b 4.33±0.67d 6.21±0.66b Gr. II MEAB 100 61.33±3.32b 230.43±6.35b 10.50±1.34ab 8.24±0.68b Gr. III MEAB 300 82.58±3.43a 200.88±6.02a 13.50±2.17a 16.54±1.80a Gr. IV MEAB 600 131.76±4.41c 147.81±3.54c 24.83±2.65c 20.43±1.98ac Gr. V Diazepam 1 (ip) 141.5±4.81c 137.2±6.18c 23.17±2.02c 21.30±1.50c Mean in a column bearing same superscript do not differ significantly (P<0.05). 454 INDIAN J EXP BIOL, JUNE 2013

In actophotometer reading, locomotor activity duration of 26.53 ± 0.20 sec. MEAB (600 mg/kg, po) of the plant extract treated was reduced dose significantly reduced the latency, but did not alter dependently (Table 5). the incidence of seizures elicited by maximal Pentylenetetrazole produced tonic seizures in all electroshock to any significant extent. The standard the animals used. A dose of 100 mg/kg of MEAB antiepileptic drug, diazepam protected 100% of mice protected 16.66% of animals against seizures and did against seizures (Table 7). not affect the onset of seizures to any significant extent. MEAB, in doses of 300 and 600 mg/kg, respectively, Discussion protected 50 and 66.66% of mice against seizures, MEAB showed the presence of steroids, and significantly delayed the latency of the seizures. triterpenes8 and flavonoids11. Triterpenic steroids The standard antiepileptic drugs, diazepam completely and triterpenoidal saponins possess anticonvulsant protected the animals from seizures (Table 6). activity in some experimental seizure models such Maximal electroshock produced hind limb tonic as MES and PTZ23. extension (HLTE) in all the animals. The vehicle- Hole board test indicates that head dipping treated mice showed tonic hind limb extension for behaviour is sensitive to changes in the emotional Table 5—Effect of A. brasiliana leaf extracts (MEAB) on spontaneous motor activity [Values are mean ± SE from 6 animals in each group] Treatment Drugs Dose Locomotor activity (score) in 10 min Reduction Groups (mg/kg, po) Before treatment After treatment (%) Gr. I Control - 154.66±0.49 147.50±0.56 4.62±0.71 Gr. II MEAB 100 176.00±3.24a 89.17±3.89a 49.30±2.13a Gr. III MEAB 300 77.00±3.98b 44.33±1.98b 44.07±3.93a Gr. IV MEAB 600 171.33±6.02a 107.17±4.38c 37.45±3.54b Gr. V Diazepam 1 (ip) 154.00±3.09c 19.33±1.33d 87.46±0.76c Mean in a row bearing same superscript do not differ significantly (P<0.05).

Table 6—Effect of A. brasiliana leaf extract (MEAB) on chemo shock convulsion test in mice [Values are mean ± SE from 6 animals in each group]. Pentylene tetrazole (PTZ) Control A. Brasiliana Diazepam No. of animals Animals Latency of tonic (mg/kg, po) (mg/kg, ip) convulsed/no. used protected (%) convulsion (sec) - - - 6/6 0 26.53±0.20 80mg/kg, ip 100 - 5/6 16.66 24.70±0.29a 300 - 3/6 50.00 21.25±0.37a 600 - 2/6 66.66 18.40±0.21b - 1 0/6 100 0.0±0.0c Mean in a row bearing same superscript do not differ significantly (P<0.05).

Table 7—Effect of A. brasliana leaf extract (MEAB) on electro convulsion test in mice [Values are mean ± SE from 6 animals of each group] Electro shock (ES) Control A. Brasiliana Diazepam No. of animals Animals Duration of (mg/kg, po) (mg/kg, ip) convulsed/no. used protected (%) HLTE (sec) - - - 6/6 0 17.48± 0.47 60 amp for 20 100 - 6/6 0 4.65±0.26a sec 300 - 6/6 0 2.95±0.23b 600 - 6/6 0 1.80±0.28c - 1 0/6 100 0.0±0.0d *Mean in a column bearing same superscript do not differ significantly (P<0.05). BARUA et al.: ANXIOLYTIC & ANTICONVULSANT ACTIVITY OF ALTERNANTHERA LEAVES EXTRACT 455

state of the animal and suggests that the expression of the level of excitability of the CNS and decrease may an anxiolytic state in animals may be reflected by an be related to sedation resulting from depression increase in head poking behaviour19. of CNS34. MEAB showed reduction in spontaneous The anxiolytic activity of novel substances motor activity which might be closely related to including herbal remedies is generally assessed by sedation resulting from CNS depressant activity. using elevated plus maze test. The test is based on a In anti-convulsant study, the standard antiepileptic premise where the exposure to an open arm of EPM drugs, diazepam significantly protected the evokes an approach-avoidance conflict that is animals from convulsions in both MES induced considerably stronger than evoked by the exposure to electroconvulsion and PTZ induced electroconvulsion. 24 an enclosed arm . The decrease in aversion to the In MES induced convulsion, none of the doses of open arm is the result of an anxiolytic effect, MEAB (100, 300 and 600 mg/kg) alter the incidence expressed by an increase in time spent and entries into of seizures significantly, however, MEAB at the dose the open arm. The primary index is spatiotemporal in 600 mg/kg shortened the latency of seizures in mice. nature; it is reduced by anxiolytic drugs and can be 25 On the other hand, in PTZ induced convulsion, increased by anxiogenic compounds . This parameter MEAB protected animals from seizures at all doses as is accepted as reliable indicator of anxiety and 26 well as significantly reduced the latency of seizures. fearfulness . The results of the present study indicate that Animal models based on spontaneous behaviour or methanol leaf extract of A. brasiliana possesses ethologically based models27 like the light/dark test anticonvulsant activity in mice in Pentylenetetrazole may be more sensitive to the behavioural responses induced seizures which may elicit seizures by than conditioned paradigms28. In light/dark inhibiting GABAergic mechanisms23. GABA is the exploration test, animals always try to spend more major inhibitory neurotransmitter, while glutamic time in dark box compared to light box because of acid is an excitatory neurotransmitter in the brain. fear about new environment. Transitions have been The inhibition of GABA neurotransmitter and the reported to be an index of activity exploration because enhancement of the action of glutamic acid have of habituation over time and the duration of time spent in each compartment to be a reflection of been shown to be the underlying factors in epilepsy. aversion29. The standard antiepileptic drug, diazepam is believed to produce their effects by enhancing GABA Anxiolytics are known to exert pharmacological 23 action by causing an increase in GABA content in the mediated inhibition in the brain . It is, therefore, cerebral hemisphere30. GABA receptors are involved possible that the anticonvulsant effects shown in in anxiety and their direct activation would have an this study by MEAB against seizures produced by anxiolytic effect31. Alkaloids are the most important PTZ may be due to the activation of GABA secondary metabolites in many plants that are held neurotransmission. The results show that the MEAB responsible for their sedative and anxiolytic actions32. protected significant number of animals against MEAB contains alkaloids and triterpenes as seizures induced by pentylenetetrazole and also phytoconstituents8, which might be responsible for its delayed the latency of the seizures. anxiolytic action. There are various ways of Maximal electro-shock (MES) convulsion test is explaining the mechanisms of action of anxiolytic considered to be a predictor of likely therapeutic agents because of involvement of many CNS efficacy against generalized tonic-clonic seizures. chemical mediators. The anxiolytic effect of MEAB The MES induced convulsion in animals represents may be due to interaction of the extract with the grandmal type of epilepsy35. Antiepileptic drugs that neural substrates or chemical mediators like nor- block MES-induced tonic extension are known to adrenaline, serotonin, GABA, BZD, hormones act by blocking seizure spread23. Diazepam does not (testosterones) and magnesium or natural endogenous suppress the focal activity but prevents it from mediators of the body33 which are implicated to be spreading23. MEAB (100, 300 and 600 mg/kg) was responsible for aggressive and anxiety-like condition. able to reduce the duration of tonic phase and the The locomotor activity is an index of alertness and reaction time in a dose dependent manner to recover a decrease indicates sedative effect. Decrease in from electrically induced convulsions indicating locomotion reveals depressant effect of CNS drugs. anticonvulsant effect of the plant extract. Triterpenes Increase in motor activity gives an indication of and steroids are reported to possess anticonvulsant 456 INDIAN J EXP BIOL, JUNE 2013

activity in some experimental seizure models such effects on lymphocyte proliferation in vitro, J Brazil Chem as MES and PTZ36. Isolation of the bioactive Soc, 14 (2003) 451. 12 Barua C C, Talukdar A, Begum S A, Sarma D K, Pathak D C principles, which are responsible for these activities & Borah P, Antinociceptive activity of methanolic extract of is in process. The present findings justify the use leaves of Alternanthera brasiliana Kuntz. in animal models of this plant in the control and/or treatment of of nociception, Pharmacologyonline, 3 (2009) 55. 13 Barua C C, Talukdar A, Begum S A, Sarma D K, Pathak D convulsions and epilepsy. C, Barua A G & Bora R S, Wound healing activity of methanolic extract of leaves of Alternanthera brasiliana Acknowledgement Kuntz using in vivo and in vitro model, Indian J Exp Biol, 47 Authors are grateful to Defence Research (2009) 1005. 14 Barua C C, Begum S A, Talukdar A, Datta Roy J, Development Organization, GOI, New Delhi for Buragohain B, Pathak D C, Sarma D K, Saikia Bora financial help and the Director of Research (Vety), R & Gupta A, Influence of Alternanthera brasiliana (L.) CVSc, AAU, Khanapara for facilities. Kuntze on Altered Antioxidant Enzyme Profile during Cutaneous Wound Healing in immunocompromised Rats, ISRN Pharmacology (2012) doi:10.5402/2012/948792. References 15 Harborne J B, Phytochemical methods, in Guide to modern 1 Ernst E, Herbal remedies for anxiety—A systematic review techniques of plant analysis, 2nd edition, (Chapman and Hall, of controlled clinical trials, Phytomed, 4 (2006) 208. India) 1991, 653. 2 Rabbani M, Sajjadi S & Ezarei H R, Anxiolytic effects of 16 Sonovane G S, Sarveiya V P, Kasture V S & Kasture S B, Stachys lavandulifollia on the elevated plus maze model of Anxiogenic activity of Myristica fragrans seeds, Pharmacol anxiety in mice, J Ethnopharmacol, 89 (2003) 276. Biochem Behav, 71 (2002) 244. 3 Wolfman C, Viola H, Paladini A, Dajas F & Medina 17 Bhattacharyya S K & Satyan K S, Experimental methods for J H, Possible anxiolytic effects of chrysin, a central evaluation of psychotropic agents in rodents: Anti-anxiety benzodiazepine receptor ligand isolated from Passiflora agents, Indian J Exp Biol, 35 (1997) 565. coerulea, Pharmacol Biochem Behav, 47 (2005) 4. 18 Jain N N, Ohal C C, Shroff R H, Somani R S, Kasture V S & 4 Viola H, Wasowski C, Levi de Stein M, Wolfman C, Kasture S B, Clitoria ternatea and the CNS, Pharmacol Silveira R, Dajas F, Medina J H & Paladini A C, Apigenin, Biochem Behav, 75 (2003) 536. a component of Matricaria recutita flowers, is a central 19 Takeda H, Tsuji M & Matsumiya T, Changes in head- benzodiazepine receptor-ligand with anxiolytic effects, dipping behavior in the hole-board test reflect the anxiogenic Planta Medica, 61 (1995) 216. and/or anxiolytic state in mice, Euro J Pharmacol, 350 5 Salgueiro J B, Ardenghi P, Dias M, Ferreira M B, Izquierdo I (1998) 29. & Medina J H, Anxiolytic natural and synthetic flavonoid 20 Finney DJ, Probit analysis, (Cambridge University Press, ligands of the central benzodiazepine receptor have no Cambridge), 1952. effect on memory tasks in rats, Pharmacol Biochem Behav, 21 Khosla P & Pandhi P, Anticonvulsant effect of nimodipine 58 (1997) 891. alone and in combination with diazepam on PTZ induced 6 Paladini A C, Marder M, Viola H, Wolfman C, Wasowski C convulsions, Indian J Pharmacol, 33 (2001) 211. & Medina J H, Flavonoids and the central nervous system: 22 Swinyard E A, Brown W C & Goodman L S, Comparative from forgotten factors to potent anxiolytic compounds, assays of antiepileptic drugs in mice and rats, J Pharmacol J Pharmacy Pharmacol, 51 (1999) 526. Exp Ther, 106 (1952) 330. 7 Dhawan K, Dhawan S & Chhabra S, Attenuation of 23 Hegde K, Thakker S P, Joshi A B, Sashtri C S & benzodiazepine dependence in mice by a tri-substituted Chandrashekhar K S, Anticonvulsant activity of Carissa benzoflavone moiety of Passiflora incarnata Linneaus: A carandas Linn. root extract in experimental mice, Trop J non-habit forming anxiolytic, J Pharmacol Pharmaceut Sci, Pharm Res, 8 (2009) 125. 6 (2003) 222. 24 Rang H P, Dale M M, Ritter J M & Moore P K, Pharmacology, 8 Macedo A F, Barbosa N C, Esquibel M A, Souza M M & 5th edition (Churchill Livingstone, London), 2003, 15. Cechinel-Filho V, Pharmacological and phytochemical 25 Gupta M, Mazumder U K, Kumar R S, Sivakumar T & studies of callus culture extracts from Alternanthera Vamsi M L, Anti-tumour activity and antioxidant status of brasiliana, Pharmazie, 54 (1999) 777. Caesalpinia bonducella against Ehrlich ascites carcinoma in 9 Bivatti M W, Bellever M H, Valpato L, Costa C & Bellaver Swiss albino mice, J Pharmacol Sci, 94 (2004) 184. C, Preliminary studies of alternative feed additives for 26 Saeed M A & Sabir A W, Antibacterial activity of broilers: Alternanthera brasiliana extract, propolis extract Caesalpinia bonducella seed, Fitoterpia, 72 (2001) 809. and linseed oil, Revista Brasileira de Ciencia Avicola 27 Oderpalm B, Hjorth S & Engel J A, Effects of 5-HT1A campinas, 5 (2003) 1535. receptor agonists and L-5-HTP in Montgomery's conflict 10 Lagrota M H C, Wigg M D, Santos M M G, Miranda test, Pharmacol Biochem Behav, 32 (1989) 265. M M F S, Camara F P, Couceiro J N S S & Costa S S, 28 Griebel G, Lanfumey L, Blanchard D C, Rettori M C, Inhibitory activity of extracts of Alternanthera brasiliana Guaardiola L B & Hamon M, Preclinical profile of the mixed (Amaranthaceae) against the herpes simplex virus, Phytother 5-HT1A/5-HT2A receptor antagonist S21357, Pharmacol Res, 8 (1999) 361. Biochem Behav, 54 (1996) 516. 11 Brachadoa C O, Almeidaa A P, Barretoa B P, Costaa L P, 29 Belzung C, Misslin R, Vogel E, Dodd R H & Chapounthier Ribeiroa L S, Pereira R L C, Koatz V L G & G, Anxiogenic effects of methyl-carboline-carboxylate Costa S S, Flavonol Robinobiosides and Rutinosides in a light-dark choice situation, Pharmacol Biochem Behav, from Alternanthera brasiliana (Amaranthaceae) and their 28 (1987) 33. BARUA et al.: ANXIOLYTIC & ANTICONVULSANT ACTIVITY OF ALTERNANTHERA LEAVES EXTRACT 457

30 Makota T, Tsutomu S, Miwa M & Hiroshi N, Involvement 34 Barua C C, Talukdar A, Begum S A, Borah P & of the opioid system in the anxiolytic effect of diazepam Lahkar M, Anxiolytic activity of methanol leaf in mice, Eur J Pharmacol, 307 (1996) 14. extract of Achyranthes aspera. Linn in mice using 31 Vogel H G, Drug discovery and evaluation, in experimental models of anxiety, Indian J Pharmacol, Pharmacological assay, (Springer-Verlag Berlin Heidelberg, 44 (2012) 67. New York) 2002, 385. 35 Viajayalakshmi A, Ravichandran V, Anbu J, Veraj M & 32 Elisabetsky E & Costa-Campos L, The alkaloid alstonine: a Jayakumari S, Anticonvulsant and neurotoxicity profile review of its pharmacological properties, e CAM, 3 (2006) of the rhizome of Smilax china Linn in mice, Indian J 348. 33 Shih J C, Ridd M J, Chen K, Meehan W P, Kung M, Pharmacol, 43 (2011) 30. Self I & Demaeyer E, Ketanserin and tetrabenazine abolish 36 Kasture V S, Kasture S B & Chopde C T, Anticonvulsive aggression in mice lacking mono amino oxidase A, Brain activity of Butea monosperma flowers in laboratory Res, 835 (1999) 112. animals, Pharmacol Biochem Behav,72 (2002) 965.