Plant materials collection A. paniculata (Kalmegh) samples (stem along with leaves) were garnered from Owshudi Gram, Natore, Bangladesh, during September 2018. Having properly identified by an expert botanist, a voucher specimen (DACB Accession Number: 35939) of the plant sample was then deposited to Bangladesh National Herbarium, Mirpur, Dhaka. After collection, cleansing and chopping of the sample into small pieces, in the next step dried samples were grained into powder with the help of a cyclone grinder machine. The powdered samples were eventually used for different experiments.
Chemicals, reagents and equipment 1, 1-Diphenyl-2-picryl hydrazyl (DPPH), aluminum chloride (AlCl3), disodium hydrogen phosphate (Na2HPO4), FeCl2.4H2O, FeCl3, Ferrozine, Folin–Ciocalteu reagent, L-Ascorbic acid, Sodium carbonate (Na2CO3), KH2PO4, K3Fe(CN)6, Sodium nitroprusside, Griess reagent (2% H3PO4, 0.1% naphthalene diamine dihydrochloride, 1% sulphanilamide), phosphate buffer and Trichloroacetic acid were taken from Sigma Chemical Co. (USA). The methanol for the mobile phase was HPLC grade (Merck, Germany). Deionized water was obtained from a water purification system. All other chemicals and reagents were of analytical quality. Soxhlet apparatus (Fisher Scientific, USA), Rotary Vacuum Evaporator (Labfirst Scientific and Industrial, China), UV VIS spectrophotometer SPECORD 205 (Analytik Jena, Germany) were used in this study.
Preparation and extraction of extracts The powdered samples of A. paniculata were then subjected to extract preparation. For extract preparation, powdered samples were extracted with solvents using a Soxhlet apparatus. The extraction solvents used for the extraction were methanol, acetone, and water. Then the extracted solvents were evaporated and concentrated using a rotary evaporator. Finally, it was desiccated in a vacuum drier and stored for further analysis (WHO, 2011).
DPPH radical scavenging activity The stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was assessed concurring with the modified method explicated (Chang et al., 2001; Gupta et al., 2004; Uddin et al., 2020). Concisely, 2 ml volume of 0.1 mM DPPH solution having purple color and another 2 ml of plant sample of varying concentrations (5, 10, 20, 40, 60, 80, 100, 250 and 500 µg/m) were first brought together and stirred vigorously for 15 s. To get a uniform mixture followed by keeping in the dark at room temperature for a time period of half an hour. After the specified interval, a UV/Visible spectrophotometer was used to read absorbance against a condign blank at 517 nm (Model 205, Germany). The antioxidant viability was estimated from the ability of the extract to eradicate the purple color that appeared in DPPH solution and the percentage of DPPH radical-scavenging activity was computed as:
DPPH radical-scavenging activity (%I), = A0 - A / Ao x 100
Where A0 is the absorbance of the control solution; A is the absorbance of the DPPH solution containing plant extract. IC50 values for the plant extract and standard were obtained by analysis of the respective percentage scavenging of DPPH radical.
Determination of reducing power assay It was performed with faint modifications, based on the method expounded by Oyaizu (1986). First of all, a mixture of 0.75 ml of plant extract at varying concentrations with 1% potassium hexacyanoferrate (0.75 ml) and 0.2 M potassium dihydrogen phosphate (0.75 ml, pH 6.6) was conducted with subsequent incubation in a water bath at 50°C for 20 min. The mixture was then subjected to centrifugation for 10 min at 800 rpm after the addition of trichloroacetic acid (10%). After that, the obtained upper layer was blended with 0.1% ferric chloride (w/v, 0.1 ml) and distilled water (1.5 ml) for ten minutes. The optical density (OD) against a blank was ascertained at 700 nm. Increased OD value of the mixture of reaction indicates increased reducing power.
Total antioxidant capacity determination The total antioxidant capacity was ascertained following the phosphomolybdenum assay method (Prieto et al., 1999) which operates via the reduction of Mo (VI) to Mo (V) by extracting and subsequently forming a green phosphate-Mo (V) complex in an acidic state. The extract was permitted to amalgamate with 3.0 ml of reagent solution (0.6 M H2SO4, 28 mM Na3PO4, 4 mM ammonium molybdate), and afterward, the reaction mixture was incubated at 95 0C for 90 min. Upon cooling at room temperature, a antioxidant activity was elicited as the number of ascorbic acids UV-Visible spectrophotometer was used to measure the solution’s absorbance against an opposite blank at a wavelength of 695 nm.