2.1 Plant Material Collection and Identification Fruits and shoots of Brassica oleracea L. var. italica were collected from Savar area district of Dhaka and were identified by Mohammad Omar Faruk, department of Botany, University of Chittagong and preserved in the herbarium (Acc. No: CU/DP/PS/2015600321) department of pharmacy, University of Chittagong.
2.2 Trial Registration For an experimental study on animal trial, registration and permission was issued from the departmental clinical ethical review committee, department of pharmacy, University of Chittagong. The trail registration reference number is ERC/DP/CU/2015/0014.
2.3 Extraction of Plant Material Dried, ground fruits and shoots of Brassica oleracea L. var. italic (900 g) was taken in a clean flat bottomed glass container and soaked in 2L of 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 clean, white cotton then followed by a filtration through Whatman filter paper. The filtrate was allowed to keep for 7 days to evaporate the solvent followed by vacuum desiccation. Finally a blackish crude extract was obtained with yield of 5.5%.
2.4 Phytochemical Screening All of the crude extracts were qualitatively analyzed for the presence of different chemical groups, such as alkaloids, glycosides, tannins, flavonoids and saponins.
2.5 Antioxidant Activity: Antioxidant activity of the methanol extract was determined by DPPH free radical scavenging activity on the basis of the modified method of Gupta[19]. Stock solutions (10 mg/ml) of the plant extracts were prepared in ethanol from which serial dilutions were carried out to obtain concentrations of 1, 5, 10, 50, 100 and 500 g/ml. Diluted solutions (2 ml) were added to 2 ml of a 0.004% ethanol solution of DPPH, mixed and allowed to stand for 30 min to react. The absorbance was determined at 517 nm using a double beam UV-visible spectrophotometer and from these values corresponding percentage of inhibitions were calculated. Then % inhibitions were plotted against log concentration and from the graph, the half maximal inhibitory concentration (IC50) was calculated. The experiment was performed in triplicate and average absorption was noted for each concentration. Ascorbic acid was used as positive control. Free radical scavenging activity was expressed as the inhibition percentage (I%) and calculated as following equation:
Inhibitin percentage (I%) = [(Blank absorbance - Sample absorbance)/Blank absorbance]× 100
2.6 Antibacterial Activity: Antibacterial activity of the methanol extract was assessed by the disc diffusion method according to the previously described method. Bacteria used as test organisms for the antibacterial activity test is listed in Table 5 (vide infra).
2.7 Experimental Animals Young Swiss-albino mice aged 4-5 weeks old and average weight 20-25 g was employed for the experiment. The mice were purchased from the Animal Research Branch of the International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR, B). They were kept in standard environmental condition (RH 55% to 60%, room temperature 25± 2°C and 12 h light/ dark cycle) for one week for adaptation and fed ICDDRB formulated rodent food and water ad libitum. The experimental study was performed under the guidelines of the Institutional Animal Ethics Committee.
2.8 Chemicals and Drugs The standard drug, metformin hydrochloride was a generous gift from Beximco Pharmaceuticals Ltd of Bangladesh. Alloxan monohydrate was purchased from Loba Chemie, India. Carrageenan was purchased from Otto Chemika, India. Blood samples were analyzed for blood glucose by using the OK meter Match glucose test meter (Hsinchu, Taiwan). Acetic acid was prepared from the laboratory of Bangladesh University. The standard drug diclofenac-Na was purchased from Square Pharmaceuticals Limited of Bangladesh.
2.9 Experimental Induction of Diabetes Mellitus Experimental induction of diabetes mellitus in mice, freshly prepared solution of alloxan monohydrate in normal saline solution at a dose of 120 mg/kg body weight injected to mice intraperitoneally. Alloxan can produce initially fatal hypoglycemia as a result of massive pancreatic insulin release mice were treated with 20% glucose solution (5 - 10 ml) orally after 6 h. The mice were then kept for the next 24 h on 5% glucose solution bottles in their cages to prevent hypoglycemia. After 1 week, mice with moderate diabetes mellitus that exhibited glycosuria and hyperglycemia (i.e. blood glucose concentration >200 mg/dL) were taken for the experiment.
2.10 Experimental Design for Antidiabetic Activity Study Fifteen mice were divided in to five groups as Group I: normal rats received only distilled water during the experimental period, Group II: diabetic control rats received only distilled water during the experimental period, Group III: Diabetic mice administered 500 mg/kg sample, Group IV: diabetic mice administered 250 mg/kg sample, Group V: diabetic mice administered 0.25 mg/kg glibenclamide. Treatment was continued for a period of 6 hours following oral administration to the experimental animals by gastric intubation, using a force-feeding needle. Blood samples were collected from tail vein prior to dosing (0 hour) and then after 1st hour, 3rd hour and 5th hour respectively from all groups of mice after treatment. Blood glucose was estimated on withdrawing blood samples. Fixed amount of rat chow and fluid was given to each rat and replenished the next.
2.11 Acetic Acid-induced Writhing Test for Analgesic Activity The analgesic activity of the samples was also studied using acetic acid-induced writhing model in mice. Test samples and vehicle were administered orally 30 munities before intraperitoneal administration of 1% acetic acid but diclofenac-Na was administered intraperitoneally before 15 mins, the mice were observed for specific contraction of body referred to as “writhing” for the next 10 munities.
2.12 Statistical Analysis Results were expressed as mean ± SEM or mean ± SD. One-way ANOVA was used for analysis of data followed by Dunnet’s multiple comparisons. Differences were considered significant at P ≤0.05.