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