2.1 Chemicals and ReagentsAcetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI),5’dithio-bis-(2-nitro)benzoic acid (DTNB), acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), eserine, galantamine, gallicacid, catechin and Streptokinase were purchased from Sigma-Aldrich (Japan). Tris.HCl buffer, sodium chloride, sodium carbonate, sodium acetate, sodium hydroxide, magnesium chloride, Triton X-100, folin-ciocalteau reagent (FCR), aluminium chloride, ammonium sulphate were collected from Wako Pure Chemical Company Ltd. (Japan). Analytical grade chemicals and solvents were used in this study. 2.2 Preparation of Fruit SampleFresh fruits of C. lemon, C. aurantifollia, C. bergamia, C. maxima, C. sinensis and C. macroptera at the mature commercial stage were harvested from several commercial orchards in the month of October-November, from Sylhet, Hobiganj, Mymensingh, Dhaka and Rajshahi, Bangladesh. Only healthy fruits were selected randomly for their uniformity in colorand shape. Fruits were then washed thoroughly with distilled water and then dried in air. Then fruits were chopped into thin slices and dried under a shadow. Dried fruit slices were then grounded into finer powder using a powerful grinder. The ground sample was sieved to get uniform particle size and kept it in an air-tight container to prevent it from any photolytic degradation.2.3 Extraction Powdered fruits (500g) were placed into an amber coated bottle and soaked into 1000 ml of methanol and contents were sealed into the bottle for ten days with occasionally stirred and shaken. After ten days, the whole mixtures were filtered by Whitman No. 1 filter papers, and the filtrated solutions were concentrated under reduced pressure, heating below 50°C. Finally, near about 20g of crude methanolic extracts (CMEs) of fruits were obtained. 2.3.1 Determination of total phenolics: The total content of phenolics in fruits was measured by using substrate FCR, where gallic acid used as a standard. In a reaction mixture 0.5 mL CME of fruits, 2.5 ml of FCR and 2 ml of sodium carbonate (7.5%) were added. The tubes were mixed and let to stand for 2 hours. At 760 nm absorbance was measured. 2.3.2 Estimation of total flavonoids: The total content of flavonoids was measured according to the method of Zhishen et al. Fruit extract was added with 0.5 mL in 0.15 ml of 5% sodium nitrite and well mixed. After 5 min of incubation, 0.3 mL of 10% aluminium chloride solution was added. After 6 min of the interval, one mL of 1M sodium hydroxide was added to the mixture, and the volume was made up to 10 mL with distilled water. The absorbance was taken at 510 nm with UV–vis spectrophotometer. The total content of flavonoids was calculated from a Catechin standard curve and expressed as mg Catechin equivalents/gm (mg CE/gm). 2.3.3 Determination of total flavonols: [38]Total amount flavonol was determined by using aluminium chloride as a substrate and standard Gallic acid as a standard. 300μl/mL CME was placed in a 10mL test tube & methanol was added up to 1 mL. Then, one of aluminum chloride solution (2%) is added to it. Finally, 1.5 mL of 5% w/v sodium acetate was added to the test tube which is then incubated at room temperature for two and half hours. Absorbances were taken at 440 nm. Total Flavonol amounts were expressed as Gallic acid equivalents/g (mg GAE/gm) dry matter. All samples were analyzed thrice and resulted averaged.
3.4 Determination of AChE inhibitory activity: Modified Ellman's colourimetric method was applied to run In-vitro ACh Einhibitory assay, and ATCI used as a substrate. AChE hydrolysis rate was monitored spectrophotometrically. Each fruit extract or standard (various concentrations) was mixed with 200 μL of enzyme solution (5.21 x 10-3U) and incubated at 37°C for 30 min. After that, Ellman’s reaction mixture (400 μL of 0.35 mM ATCI, 200 μLof 0.7 mM DTNB) was placed in an extraction buffer saline (50 mM Tris.HCl buffer, 50 mM MgCl2, 50mM NaCl, 1% Triton X-100, pH 8.0) to adjust it 3 ml of final volume. Absorbance at 412 nm was taken after 30 min incubated this mixture at 37°C. The blank reaction was measured by substituting buffer saline for the enzyme. Eserine was used as a standard drug. Percentage of inhibition of AChE enzymes were determined by comparison of reaction rates of samples related to blank using the formula of (E-S)/E x 100, where E is the activity of enzyme without test sample, and S is the activity of the enzyme with the test sample. 2.3.5Determination of BuChE inhibitory activity: [39]BuChEinhibitory assay was performed by modified Ellman's colourimetric method, where BTCI acts as a substrate. BuChE hydrolysis rate was spectrophotometrically examined to run this test. Each fruit extract or standard (various concentrations) was mixed with 50μL enzyme solution (4.16 x 10-3U) and incubated at 37°C for 30 min. After adding Ellman’s reaction mixture (400 μLof 0.35 mM BTCI, 200 μLof 0.7 mM DTNB) in a buffered saline (50 mM of Tris.HCl buffer, 50 mM of MgCl2, 50 mM of NaCl and 1% Triton X-100, pH 8.0) to the above reaction mixture, to adjust final volume of 3 mL. To verify the result, all reading was repeated three times. The blank reaction was measured by substituting buffer saline for the enzyme. Galantamine was used as a reference standard. The percentage of inhibition of BuChE enzymes was determined by comparison of reaction rates of samples related to blank using the formula of (E-S)/E x 100, where E is the activity of enzyme without test sample, and S is the activity of the enzyme with the test sample.2.3.6 Thrombolytic activity test: For thrombolytic activity test for the fruits, human blood was used. Blood was withdrawn from healthy human volunteers (n=10) having no history of blood-related disorder, oral contraceptive pills administration or ongoing anticoagulant therapy. 1.0 ml of venous blood from each volunteer was transferred to the sterilized eppendorf tubes (volume 1.5 ml) and incubated for 45 min at 37°C and was allowed to form a clot. Fruits extracts (100 mg) were suspended in 10 ml of distilled water. After clot formation, the serum was completely removed from eppendorf tube. The blood clot was again weighed to determine the weight of clot. For each Eppendorf tube with the pre-weighed clot, 100 μLaqueous solution of the crude extract was added separately. 100 μLof SK (30,000 IU) was added to the positive control and 100 μLdistilled water were added to negative control tubes, respectively. All tubes were then again incubated for 90 min at 37°C to observe clot lysis. Then, the released fluid was removed, and tubes were again weighed. The difference obtained in weight taken before and after clot lysis by the extract, positive control and negative control, was expressed as a percentage of clot lysis and the equation is shown below:% of Clot lysis = (Weight of clot after release of fluid/Weight of clot before releasing of fluid) x 100%2.4 Statistical AnalysisValues in this experiment are expressed as the mean of triplicate determination ± Standard Deviation. All data used are subjected to one-way analysis of variance (ANOVA) and the significant difference between means was determined by Dancan’s Multiple Test (P<0.05) using Statistical Package for the social science version 13.0 (SPSS Inc., Chicago, IL, USA).