A. H. M. Zulfiker*
Division of Molecular and Gene Therapies, School of Medical Science and Griffith Health Institute, Griffith University, Gold Coast Campus, QLD., 4222, Australia.
P. P. Roy
Department of Pharmacy, School of Science & Engineering, Southeast University, Banani, Dhaka-1213, Bangladesh.
M. A. M. Momin
Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.
M. S. Khan
Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.
I. J. Bulbul
Department of Pharmacy, School of Science & Engineering, Southeast University, Banani, Dhaka-1213, Bangladesh.
T. Ahmed
Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.
M. S. Rana
Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.
Antioxidant; Antimicrobial; Chloroform; Petroleum ether; Medicinal plant.
Resource Development and Management
Medicinal Plants
2.1 Collection and Identification of Plant Material The plants namely Manilkara zapota L., Polyalthia longifolia, Abroma augusta L., Ficus hispida L., Vitex negundo L. were collected from Village: Jabra Thana: Dumuria, District: Khulna, Bangladesh in July 2010 and was identified at the Bangladesh National Herbarium, Mirpur, Dhaka where the Voucher specimen no: 32767 has been deposited for future reference. 2.2 Chemicals and Drugs DPPH (1, 1-diphenyl-2-picrylhydrazyl), Ascorbic acid & sodium nitroprusside, sodium phosphate, sulphanilamide, phosphoric acid, naphthyl ethelene diamine were obtained from SD Fine Chem. Ltd. India, ammonium molybdate from Merck, Germany, ferric chloride & neocuproine were obtained from Sigma Chemical Co. USA. Kanamycin and nystatin were collected from Square Pharmaceuticals Ltd., Bangladesh.
2.3 Drying and Pulverization The fresh leaves of the plants were first washed with water to remove adhering dirt and then cut into small pieces, sun-dried for 4 days. After complete drying, the entire portions were pulverized into a coarse powder with the help of a grinding machine and were stored in an airtight container for further use. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced. 2.4 Cold Extraction About 50 gm of Manilkara zapota, 50 gm of Polyalthia longifolia, 75 gm of Abroma augusta Linn, 100 gm of Vitex negundo Linn, 50 gm of Ficus hispida Linn powered material was taken in a clean, flat bottomed 5 glass container and soaked in 500 ml of 95% methanol. The container with its contents was sealed and kept for a period of 7 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then it was filtered through Whatman filter paper (Bibby RE200, Sterilin Ltd., UK). 2.5 Extraction with Chloroform The concentrated methanol extract was made slurry with water. The slurry was taken in a separating funnel and few ml Chloroform (50 ml) was added to the aqueous solution and the mass was shaken vigorously in a separating funnel. Then the funnel was allowed to stand for few minutes for the complete separation of the layers. The organic (lower layer) layer was collected. The process was repeated two times. 2.6 Extraction with Petroleum Ether After chloroform extraction petroleum ether (50ml) was added to the Methanolic aqueous solution and the mass was shaken vigorously in a separating funnel. Then the funnel was allowed to stand for few minutes for the complete separation of the layers. The organic (upper layer) layer was collected. The process was repeated two times. The filtrates (chloroform and petroleum ether extract) obtained were concentrated at 50ºC under reduced pressure using vacuum pump rotary evaporator (STUART RF3022C, UK). It rendered a gummy concentrate of reddish black color. The extract was transferred to a closed container for further use and protection. 2.7 Determination of Antioxidant Potentials 2.7.1 DPPH radical scavenging activity The free radical scavenging capacity of the extracts was determined using DPPH. The absorbance was read at 515 nm using a spectrophotometer. Ascorbic acid was used as a standard. The inhibition curve was plotted and IC50 values were calculated. 2.7.2 Nitric oxide scavenging assay: Nitric Oxide Scavenging assay was carried out according to the procedure described earlier. The method is based on the generation of NO from sodium nitroprusside and subsequent estimation of nitrite ions using Griess reagent produced by the reaction of NO with oxygen in an aqueous solution at physiological pH (7.2). In a test tube, 4 ml of plant extract or standard of different concentrations were mixed with 1.0 ml of Sodium nitroprusside (5mM) solution. Then the test tube was incubated for 120 minutes at 30ºC. After incubation 2 ml of solution was withdrawn from the mixture and mixed with 1.2 ml of Griess reagent (1% Sulfanilamide in 5% H3PO4 and 0.1% Naphthylethylene diamine dihydrochloride). The absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with Naphthylethylenediamine dihydrochloride was measured at 550 nm using a spectrophotometer against a blank. The percent (%) inhibition of nitrite formation was calculated from the following equation.
{(A0 – A1)/A0} X 100
Where C is the total content of phenolic compounds in mg/g plant extract; c is the concentration of Gallic acid established from the calibration curve in mg/ml; V is the volume of extract in ml and m is the weight of Chloroform or Pet. ether extract in g. The value of the total content of phenolic compounds is expressed as GAE (Gallic Acid Equivalent) in mg/g extract.
British Journal of Medicine & Medical Research 3(4): 1418-1436, 2013
Journal