Plant For the investigation, A. salvifolium wang root was collected from Rajshahi, Bangladesh between January and June, 2013 and identified by an expert of the Bangladesh National Herbarium, Dhaka, where a voucher specimen number was retained with an accession no. 40214. The collected plant part was cleaned, dried for one week and pulverized into a coarse powder using a suitable grinder. Powdered material was stored in an airtight container and kept in a cool, dark, and dry place until further analysis was taken.
Extract preparation Approximately 500 g of the powdered root was placed separately in four clean and flat-bottomed glass containers and soaked in ethanol, dichloromethane, chloroform and distilled water. All the containers with their contents were sealed and kept for 7 days. Then extraction was carried out using an ultrasonic sound bath accompanied by sonication (40 min). The entire mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then the extract was filtered through Whatman filter paper and concentrated by using a rotatory evaporator at reduced pressure. The gummy extracts were then dried by using an electric oven, and finally obtained EASR (12.25 g), DASR (9.5 g), CASR (7.5 g) and AASR (14.17 g). The dried extracts were separately stored in air tight containers until completion of the analysis.
Drugs and chemicals Enzymes including acetylcholinesterase (AChE) electric eel (typeVI-S), butyrylcholinesterase (BChE) equine serum lyophilized, substrates acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BuTCI), chromogen 5, 5-dithio-bis (2-nitrobenzoic) acid (DTNB) and serine were purchased from Sigma-Aldrich, USA. 1,1-Diphenyl-2-picryhydrazyl (DPPH), dimethyl sulfoxide (DMSO), trichloroacetic acid (TCA), quercetin, ascorbic acid, gallic acid, ferric chloride, and glacial acetic acid were purchased from Merck, Germay. All other reagents and solvents used for the study were of the highest purity grade and commercially available.
Phytochemical screening: Phytochemical screening of the extracts was done by applying some previously established methods. Alkaloids, saponins, terpenoids and steroids were detected by applying Harborne (Harborne, 1973) method. Flavonoids and tannins were examined by applying methods of Sofowara (Sofowara, 1993). Reducing sugar and resins were evaluated by following methods of Dipali (Dipali et al., 2013). Coumarins, anthraquinones, cardiac glycosides and phlobatannins were detected by applying the methods of Trease and Evans (Trease and Evans., 1989).
Determination of total phenolic content (TPC) TPC of the extracts was determined by using the Folin-Ciocalteau method with slight modification (Gao et al., 2000). Briefly, the extracts and standard gallic acid solution (1 ml) were mixed with 2.58 ml of Folin-Ciocalteu’s phenol reagent. After 3 min, 0.3 ml of saturated sodium carbonate solution was added to the mixture and incubated at room temperature (25°C) for 20 min. Then, absorbance of each sample was measured at 760 nm with a spectrophotometer. TPC of the extracts was calculated from the regression equation (r2 = 0.958) of the standard gallic acid and the results were expressed as milligram per gram of gallic acid equivalent of the dried extracts.
Determination of total flavonoid content (TFC) 1 ml extract in methanol (200 mg/ml) was mixed with 1 ml aluminium trichloride in ethanol (20 mg/ml, and a drop of acetic acid), and then the mixture was diluted by the addition of ethanol up to its 25 ml volume. Blank samples were prepared by adding all the reagents with equal volume used in the sample, except the extract. The absorbance of the solution was read at 415 nm after 40 min of incubation at room temperature. Using the same procedure for absorbance of quercetin, standard compound of flavonoid was read and TFC of the extracts was calculated from the standard curve (r2 = 0.902) of the quercetin (12.5 to 200 mg/ml). Total flavonoid content was expressed as mg/g of quercetin equivalent (Kumaran and Karunakaran, 2007).
Estimation of total flavonol content (TFlC) TFlC was determined by applying a method previously described by Mbaebie et al. with slight modification (Mbaebie et al., 2012). According to the method, 1 ml of the extracts (200 µg/ml) was taken separately in different test tubes. 2 ml ethanol solution of AlCl3 and 3 ml of (50 g/l) sodium acetate solution were added in the test tubes. After gently mixing, all the test tubes were allowed to stand for 2.5 h at 20°C temperature. Then, absorbance was determined by using a spectrophotometer at a wavelength of 440 nm. Quercetin was used as standard flavonol compound. Following the aforementioned procedure, absorbance of the quercetin was taken at various concentrations (25 to 400 μg/ml) of series dilution. TFlC of the extracts was calculated from regression equation (r2 = 0.951) of the standard quercetin and the results were expressed as milligram per gram of quercetin equivalent of the dried extracts.
Determination of total antioxidant content (TAC) TAC of the extracts was evaluated by phosphomolybdenum complex method with slight modification, which was described by Prieto et al. (1999). Briefly, a reagent solution was prepared having 0.6M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate in distilled water. 1 ml of each extract was combined with the reagent solution in separate test tubes. After shaking gently, the test tubes were incubated for 90 min at 95°C temperature. Then after cooling at room temperature, absorbance was measured at 695 nm wavelength using a spectrophotometer. Similarly, ascorbic acid, a standard antioxidant, was run through the process at different concentration gradients (25 to 400 μg/ml). Using this absorbance value, a standard calibration curve and a regression equation (r2 = 0.964) was derived, from which TAC of each of the extracts was determined and expressed as mg/g of the ascorbic acid equivalent of the dried extracts.