Chemicals and reagents
1,1-diphenyl-2-picrylhydrazyl radical (DPPH), Folin-Ciocalteu reagent were obtained from Sigma-Aldrich (St. Louis, USA). Methanol was bought from SIGMA® (Sigma- Aldrich®, St Louis, USA). Chloroform, dichloromentane, carbon tetrachloride, petroleum ether, gallic acid, quercetin, sodium carbonate (Na2CO3), ferric chloride (FeCl3), potassium ferricyanide [K3 Fe(CN)6], trichloroacetic acid (TCA), buffer and ascorbic acid were purchased from Merck (Darmstadt, Germany). All chemicals used were of analytical grade.
Collection of plant material
Fresh leaves of the plants were collected in May, 2012 and identified by Botany Department, Rajshahi University, Bangladesh. A voucher specimen with accession no.3798 has been deposited in Bangladesh National Herbarium, Dhaka, Bangladesh.
Preparation of plant extracts
The leaves were left to dry under shade, grinded and extracted with methanol by cold maceration for 7 days at room temperature. The extract was then filtered off through a cotton plug and finally through filter paper. The filtrate was concentrated using vacuum rotary evaporator at 50°C. The concentrated methanol extract was fractionated by the modified Kupchan partitioning method [11] into petroleum ether, dichloromethane, carbon tetrachloride, and ethyl acetate fractions. After that, all the extracts were stored in a refrigerator for further use.
DPPH radical scavenging activity
The free radical scavenging activity of the extracts and ascorbic acid as positive control was measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH by the method described by Susanti et al. [12] with slight modifications. 2mL of each extract and control at various concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.625, and 0.812 μg/mL) were added to 3 ml of freshly prepared DPPH solution (0.004%) in methanol. The reaction was allowed for 30 min and absorbance was measured at 515 nm using a spectrophotometer (HACH 4000 DU UV – visible spectrophotometer). All experiments were repeated three times independently. The degree of decolorization of DPPH from purple to yellow indicated the scavenging efficiency of the extract. The percentage inhibition of DPPH free radical scavenging activity was calculated using the following equation:
Where:
ADPPH = Absorbance of DPPH
Asample = Absorbance of sample (extract/ascorbic acid)
The % inhibition data was then plotted against log concentration fitted in a graph and IC50 (half-maximal inhibitory concentration) value was calculated by linear regression analysis.
Reducing power capacity
Reducing power of the extract was evaluated by Oyaizu method [13]. Different concentrations of leaves extract and ascorbic acid as standard (6.25, 12.5, 25, 50, and 100 μg/mL) in 0.25ml methanol were mixed with phosphate buffer (0.625 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe (CN)6] (0.625 ml, 1% w/v). The mixture was vortex and incubated at 50°C for 20 min. After incubation, 0.625ml of 10% trichlorocacetic acid solution was added to each tube and the mixture was centrifuged at 3000 rpm for 10 minutes. 1.8 ml of the upper layer solution was mixed with equal volume of distilled water and 0.36 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. Phosphate buffer (PH 6.6) was used as blank solution.
Phosphomolybdate assay (total antioxidant capacity)
Total antioxidant activity of the fractions was evaluated by the phosphomolybdate method using AA as a standard [14]. The assay is based on the reduction of Mo (VI)-Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acidic pH. An aliquot of 0.3 mL extract was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of the solution was measured at 695 nm against blank using a spectrophotometer. Methanol (0.3 mL) in the place of extract is used as the blank. Ascorbic acid equivalents were calculated using standard graph of AA. The experiment was conducted in triplicates and values are expressed as equivalent of ascorbic acid in mg per g of extract.
Estimation of total phenolic content
Total phenolic content of all the extracts was evaluated with Folin-Ciocalteu method [15]. Samples containing polyphenols are reduced by the Folin-Ciocalteu reagent there by producing blue colored complex. The phenolic concentration of extracts was evaluated from a gallic acid calibration curve. To prepare a calibration curve, 0.5mL aliquots of 12.5, 25, 50, 100, 200, and 400 μg/mL methanolic gallic acid solutions were mixed with 2.5 mL Folin–Ciocalteu reagent (diluted ten-fold) and 2.5 mL (75 g/L) sodium carbonate. After incubation at 25°C for 30 min, the quantative phenolic estimation was performed at 765 nm against reagent blank by UV Spectrophotometer 1650 Shimadzu, Japan. The calibration curve was constructed by putting the value of absorbance vs. concentration. A similar procedure was adopted for the extracts as above described in the preparation of calibration curve. All determinations were performed in triplicate. Total phenolic content was expressed as milligrams of gallic acid equivalent (GAE) per g of extract.
Phytochemical screening of APM
Phytochemical screening was done as described by Dohou et al. [16]. Qualitative screening of APM and various fractions of A. procera leaves was performed for the identification of phytochemicals like saponins, tannins, glycosides, flavonoids, steroids and alkaloids.
Statistical analysis
All analyses were carried out in triplicates. Data were presented as mean ± SD. Free R-software version 2.15.1 (http://www.r-project.org/) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical and graphical evaluations.
