Plant material: A. macrorrhizos rhizomes were collected from a local vegetable market in Chittagong, Bangladesh, in the month of August, and were authenticated by Dr. Shaikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong, Chittagong, Bangladesh.
Preparation of extract: The rhizomes were sun-dried and ground. The ground rhizomes (250 g) were soaked in a sufficient amount of methanol for 1 week at room temperature, with occasional shaking and stirring, and were then filtered through a cotton plug followed by Whitman filter paper No. 1. The solvent was evaporated under vacuum at room temperature to yield a semisolid. The extract was then preserved in a refrigerator until further use.
Experimental animals: Male Swiss albino mice, about 25-30 g in weight and 4-6 weeks of age, were collected from the International Center for Diarrheal Diseases Research, Bangladesh (ICDDRB), and housed in polypropylene cages under controlled conditions. The animals were exposed to an alternating cycle of 12 h of light and 12 h of dark. The animals were allowed free access to drinking water and pellet diet, obtained from the ICDDRB. The mice were acclimatized for 7 days.
Drugs: Alloxan monohydrate (Sisco Research Laboratories Pvt. Ltd., India), metformin hydrochloride (Square Pharmaceuticals Ltd., Bangladesh), 1,1-diphenyl-2- picrylhydrazyl (DPPH; Sigma Chemical Co., USA), and ascorbic acid (Fine Chem. Ltd., India) were used. All of the other chemicals and reagents used were of analytical grade.
Induction of diabetes and treatments: The alloxan was first weighed individually for each animal according to its weight and then solubilized with 0.2 mL of saline (154 mM NaCl) just prior to injection. Diabetes was induced by injecting the alloxan at a dose of 150 mg/kg body weight intraperitoneally after overnight fasting. After 48h, animals with fasting blood glucose levels from 16 to 19 mmol/L were separated and included in the study. Twenty-five male Swiss albino mice were used, divided randomly into 5 groups containing 5 mice each. Group 1 served as a control, which received a vehicle alone. In groups 2-5, each animal received a single dose of alloxan (150 mg/kg intraperitoneally) after overnight fasting. Group 1 received only dimethyl sulfoxide (DMSO) as the normal control group and group 2 was the diabetic control group, which did not receive either metformin or rhizome extract. Metformin (150 mg/kg body weight) was injected intraperitoneally into group 3, and extract at a dose of 250 mg/kg body weight and 500 mg/kg body weight was injected intraperitoneally into group 4 and group 5, respectively. Metformin and the extract were both dissolved in 0.2% DMSO vehicle. Blood samples were then analyzed for blood glucose content at 0, 2, 4, 8, and 16h, respectively, using a glucometer kit (Accu-Check Active, Roche Diagnostic GmbH, Germany).
Antioxidant activity
DPPH free radical scavenging activity: DPPH scavenging activity was carried out using the method of Braca et al. (2001). Different concentrations (500, 300, 100, 50, and 10 μg/mL) of Alocasia macrorrhizos extract were dissolved in methanol and placed in different test tubes, and 3 mL of a 0.004% w/v methanol solution of DPPH was added to each test tube. Absorbance at 517 nm was determined aft er 30 min against a blank, and the percent inhibition activity was calculated from [(A0–A1)/A0] × 100, where A0 is the absorbance of the control and A1 is the absorbance of the sample. Ascorbic acid was used as a reference standard and dissolved in methanol to make the stock solution with the same concentration. The control sample was prepared containing the same volume without any extract or reference drug. Methanol served as a blank. The inhibition curves were prepared and the half maximal inhibitory concentration (IC50) values were calculated using linear regression analysis. Reducing power capacity The reducing power of the extract was evaluated using the method of Oyaizu (1986). Different concentrations of rhizome extract of A. macrorrhizos (125, 250, 500, and 1000 μg/mL) in 1 mL of distilled water were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 mL, 1% w/v). The mixture was incubated at 50°C for 20 min. After incubation, 2.5 mL of 10% trichloroacetic acid solution was added to each tube and the mixture was centrifuged at 3000 rpm for 10 min. Subsequently, 5 mL of the upper layer solution was mixed with 5 mL of distilled water and 1 mL of ferric chloride solution (0.1% w/v), and the absorbance was measured at 700 nm. The reducing power of the extract was linearly proportional to the concentration of the sample. Ascorbic acid was taken as a reference standard. Phosphate buffer (pH 6.6) was used as a blank solution. Brine shrimp lethality bioassay The cytotoxicity assay was performed on brine shrimp (Artemia salina) nauplii using the Meyer method (Meyer et al.1982). The dried cysts of the brine shrimp were collected from an aquarium shop (Chittagong, Bangladesh) and hatched in artificial seawater (3.8% NaCl solution) with strong aeration for a 48-h light/ dark cycle to mature shrimp called nauplii. The test sample (extract) was prepared by dissolving extract in DMSO (not more than 50 μL in 5 mL of solution) plus sea water (3.8% NaCl in water) to attain concentrations of 10, 50, 100, 150, 200, and 400 μg/mL. A vial containing 50 μL of DMSO diluted to 5 mL was used as a control. Standard vincristine sulfate was used as the positive control. Th e matured shrimp were then applied to each of the experimental vials and the control vial. Aft er 24 h, the vials were inspected using a magnifying glass and the number of surviving nauplii in each vial was counted. From these data, the percent (%) mortality of the brine shrimp nauplii was calculated for each concentration using the following formula:
% mortality = (Nt/N0) × 100,
where Nt = the number of killed nauplii after 24h of incubation and N0 = the number of total nauplii transferred, i.e. 10. The median lethal concentration (LC50) was then determined.
Statistical analysis: The experimental data are presented as the means ± standard error of the mean (SEM). Th eresults were analyzed for statistical significance using one-way ANOVA followed by Dunnett’s test using Graph Pad Prism Version 5.0 for Windows (Graph Pad Soft ware, USA). P<0.05 was considered significant.