August 30, 2016
Archives • 2016 • vol.2 • 10-16
ACUTE TOXICITY AND NEUROPHARMACOLOGICAL STUDIES OF MICROCOS PANICULATA & RICHARDIA SCABRA
Aziz, M.A.1*; Sarkar, K.K.1; Akter, M.I.2; Kabir, A.K.L.3 1Department of Pharmacy,
Jessore University of Science & Technology, Bangladesh, Bangladesh, 3Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Dhaka, Bangladesh. 2Department of Pharmacy, Stamford University
*
[email protected]
Abstract The present study was designed to evaluate the safety of the methanolic extract of Microcos paniculata roots (RME), as well as neuropharmacological activity of the methanolic extracts of M. paniculata fruits (FME), RME and Richardia scabra (whole plant) (MRS) by using OECD guidelines, Y-maze and Elevated plusmaze test respectively. Mortality, sign of any toxicity or behavioral changes were not observed up to the dose as high as 4000mg/kg. In Y-maze test, anti-depressive and depressive activities of FME 200 mg/kg and 400 mg/kg, RME 200 mg/kg and 400 mg/kg, MRS 200 mg/kg and 400 mg/kg were noticed. Again, the Elevated plus maze test revealed that every extract including both 200 and 400 mg/kg doses demonstrated anxiolytic and depressive activities. The results obtained in the present study point out that FME, RME and MRS can be the possible sources of CNS depressant, anti- depressant and anxiolytic agents. But further investigation is required for the confirmation of their activities. Key Words: Acute toxicity, neuropharmacological studies, Microcos paniculata, Richardia scabra.
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Introduction Microcos paniculata L. of Tiliaceae family is locally known as ‘Kathgua’ or ‘Fattashi’ in Bangladesh. It has the growth form of a shrub or small tree, grows wildly and is cultivated throughout Bangladesh. Traditionally the plant is used to treat fever, diarrhea, dyspepsia, heat stroke, colds, hepatitis, wounds, for its activity in the digestive system and to kill insects. A review of the literature showed that M. paniculata has been found to have a wide range of activities, including neuropharmacological, larvicidal, insecticidal, free radical scavenging, antimicrobial, brine shrimp lethality, antidiarrheal, analgesic, anti-inflammatory, antipyretic, αglucosidase inhibition, cytotoxic and nicotinic receptor antagonistic activities, as well as preventative effects for coronary heart disease and angina pectoris. Moreover, acute toxicity study of the methanolic extract of M. paniculata fruits were conducted also [1, 2]. Richardia scabra also called Florida Pusley of Rubiaceae family is locally known as ‘Riim-raaz’ in Bangladesh. It is a branched plant that possesses distinctive characteristics because of its hairy stems and leaves. It can grow annually up to 80 cm but is frequently prostrate. As a forage plant, green manure and soil covering it is grown in Southern North America. The whole plant is used as tonic and emetic, along with its activity against asthma and dermatitis. The root of this plant possesses diaphoretic property. Analysis of the literature showed that acute toxicity study, antiinflammatory and CNS depressant activities of Richardia scabra were performed. However, some surveys were carried out locally and internationally on few medicinal plants which disclosed several valuable information of Richardia scabra [3]. Therefore, the present study was designed to evaluate the neuropharmacological activity of the methanolic extracts of M. paniculata fruits (FME) and roots (RME), along with Richardia scabra (whole plant) (MRS).
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Extraction Methanol extraction was carried out on 200 g of powdered fruits and roots of M. paniculata, along with R. scabra (whole plant). Plant parts and whole plant were rinsed 3–4 times successively with running water and once with sterile distilled water that were then dried in the shade for a period of 7 d. The dried plant parts and plant were then ground by using a laboratory grinding mill (Model 2000 LAB Eriez®) and passed through a 40-mesh sieve to get fine powders. Powdered fruits and roots of M. paniculata and whole plant of R. scabra (200 g) were extracted individually in 2 L of methanol, using a soxhlet apparatus and a hot extraction procedure. Whatman No.1 filter papers were used to filter the liquid extracts. The filtrates were then dried in a hot air oven at 400C. The extraction yield of fruits and roots of M. paniculata and whole plant of R. scabra were 11.08% (w/w), 1.56% (w/w) and 1.79% (w/w) respectively. Extracts were stored at 40C for additional studies. Experimental Animals Ninety Swiss albino mice of either sex, 6–7 weeks old, weighting 25–30 g were collected from the Department of Pharmacy, Jahangirnagar University, Savar, Dhaka, Bangladesh. These animals were kept under standard environmental conditions, having relative humidity 55%–65%, 12 h light/12 h dark cycle and (27.00±1.00) 0C temperature. Proper supply of foods and water ad libitum were ensured. Before the experiment, animals were adapted to the laboratory conditions for 1 week. The Institutional Animal Ethical Committee of Jahangirnagar University, Savar, Dhaka, Bangladesh approved all the protocols used in the experiments conducted with these animals.
Acute Oral Toxicity Study Adverse effects that result either from a single exposure or from multiple exposures over a short time (normally less than 24 h) are known as acute Materials and Methods toxicity. To find the half lethal dose (LD50) of the Collection and Identification of the Plant experimental samples, the acute toxicity study was Fruits and roots of M. paniculata and R. scabra carried out following the Organization of Economic (whole plant) were collected from the Cooperation and Development (OECD) guidelines [2]. Jahangirnagar University campus, Savar, Dhaka, Ten mice were divided into two groups: control Bangladesh in November, 2012. Species group and test group (RME), with five animals per identification was verified by Sarder Nasir Uddin, group. The experimental sample (RME) was Principal Scientific Officer at the Bangladesh administered orally at different concentrations (100, National Herbarium. Dried specimens were 250, 500, 1 000, 2 000, 3 000 and 4 000 mg/kg body deposited in the herbarium for future references. weight). After that the animals were observed every 1 h for next 5–6 h for mortality, behavioral pattern changes such as weakness, aggressiveness, food or _______________________________________ http://pharmacologyonline.silae.it ISSN: 1827-8620
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water refusal, diarrhea, salivation, discharge from eyes and ears, noisy breathing, changes in locomotor activity, convulsion, coma, injury, pain or any sign of toxicity in each group of animals. A final evaluation at the end of a 2-week observation period was also conducted [2]. Neuropharmacological Study Y-maze Test Y-maze test was completed according to Mandal et al., 2001; Rushton et al., 1961 and Ma et al., 2007 [4,5,6]. Fourty mice were divided into control group (distilled water, 10 mL/kg, p.o.), positive control or standard group (Diazepam, 1mg/kg, p.o.) and test groups (FME, RME and MRS at 200 and 400 mg/kg body weight, p.o.), containing five mice in each group. Three wooden arms with an angle of 120° between each of the two arms made the Y-maze apparatus, where dimensions of the arms were 30 cm x 8 cm x 15 cm (length x width x height). Each mouse was placed in the centre of a Y – shaped runway. The number of entry of the test animals into the closed and open arms was counted at 0, 30, 60, 120 and 180 min after respective treatment and the every counting was continued for 3 min. Arm entry was defined as the entry of all four paws into one arm Elevated Plus-maze Test (EPM) Elevated plus-maze test was performed following the method of Lister [7]. Mice grouping and administration were performed as mentioned before. The apparatus was made of two opposing closed arms (50 x 10 x 30 cm) (length x width x height) and two opposing open arms (50 x 10 cm) (length x width) that was placed at 70 cm high from the floor level. Each mouse was placed in the centre of elevated plus-maze apparatus. The number of entry of the test animals into the closed and open arms was counted at 0, 30, 60, 120 and 180 min after respective treatment and the every counting was continued for 3 min. Arm entry was defined as the entry of all four paws into one arm. Statistical Analysis All the results were expressed as mean ± S.E. (Standard Error). Statistical analyses for neuropharmacological studies were performed by one-way ANOVA following Dunnet’s test through the SPSS software (version 20; IBM Corporation, New York, USA). (P < 0.05, vs.control) was considered statistically significant.
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Results Acute Oral Toxicity Study After acute toxicity study, no mortality was observed up to the dose as high as 4000 mg/kg for RME or control group. Sign of any toxicity or behavioral changes were not observed up to the dose as high as 4000 mg/kg for RME (test group) or control group, before or after their administration in any animal, which lived up to 14 days. This apparently indicated that the test group does not show acute oral toxicity. Neuropharmacological Study Y-maze Test The result of the table-1 showed that standard drug diazepam revealed depressive activity with time. Moreover, FME 200 mg/kg exhibited significant (p
