Chemicals Donezepil, galantamine, acetylthiocholine and S-butyrylthiocholine, DPPH (2,2’-diphenyl-1-picrylhydrazyl), ammonium molybdate, Folin-Ciocalteu reagent, thiobarbituric acid (TBA), tricholoroacetic acid (TCA), 2-deoxy-D-ribose, 5,5’-dithio-bis-(2-nitro) benzoic acid (DTNB), triton X-100, aluminum chloride, potassium ferricyanide, and Tris-HCl were procured from Sigma-Aldrich, Germany. Catechin, ascorbic acid and gallic acid were obtained from Wako Pure Chemical Company Ltd., Japan. Methanol, ethylacetate, chloroform and petroleum ether were purchased from Active Fine Chemicals Limited, Dhaka, Bangladesh. All other chemicals, unless specified, were of analytical grade.
Animals and collection of brain
Mice were used only for collection of brain as a source of crude acetylcholinesterase enzyme. Swiss Albino Mice having 5-6 weeks were purchased from the Animal House, Jahangirnagar University, Savar, Dhaka and were caged in the experimental room. A standard diet and water ad libitum were given to the mice. The study was carried out in compliance with the ARRIVE guidelines. The international ethical guidelines were followed to deal with the laboratory animals. The procedures were approved by the by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) of the University of Rajshahi, Bangladesh (Ethical clearance number: 102).
To collect brain from mice, mice were anesthetized with sodium pentobarbital (30 mg/kg; intraperitoneal injection; Taj Pharmaceuticals Ltd., India) and then sacrificed by cervical dislocation. Cervical dislocation was performed by a trained person. Brains were taken quickly; washed in ice cold saline and homogenized in 50 mM Tris buffer, PH 7.4 to yield the crude acetylcholinesterase enzyme [17].
Collection and extraction of plant materials
The whole plant was collected from the district of Natore, Bangladesh in March 2016 and authenticated by Professor Dr. A.H.M. Mahbubur Rahman, Department of Botany, Rajshahi University, where a voucher specimen (accession no. 370) have been deposited.
Crude methnol extract (CME, 18.5 g) was prepared from plant powder (500 g) of W. chinensis by hot extraction with methanol in a soxhlet apparatus. The extract (10 g) was then concentrated in vacuo with a rotary evaporator and suspended in water. It was then sequentially partitioned with solvents of increasing polarity such as petroleum ether, chloroform, ethylacetate and water by the method as described earlier [17] to yield the corresponding petroleum ether (PEF, 3.2 g), chloroform (CLF, 2.5 g), ethylacetate (EAF, 1.4 g) and aqueous (AQF, 2.9 g) fractions. All the fractions were preserved in a refrigerator at 4°C until further use.
Phytochemical analysis
Phytochemical screening of the plant extract
Qualitative tests were performed to identify the classes of phytochemicals such as flavonoids, glycosides, alkaloids, tannins, saponins, and steroids in the different fractions by the methods as described earlier [18].
Quantitation of total phenolic content (TPC)
Folin-Ciocalteu method was used to determine the the total phenolic content of the extractives of W. chinensis as described [19]. To a mixure of 2.5 ml of 10% Folin-Ciocalteu reagent and 2.5 ml of 7.5% sodium carbonate solution, 0.5 ml sample was added and left in the dark for 20 minutes at 250C. The absorbance of the reaction mixure was recorded by a spectrophotometer at 760 nm. A standard curve was obtained for gallic acid and the phenolic content was determined from extrapolation of this curve.
Quantitation of total flavonoid content (TFC)
Aluminum chloride colorimetric method was used to measure the total flavonoid content of the extracts of W. chinensis as described earlier [20]. To a mixure of methanol (3.0 ml), 10% AlCl3 (0.2 ml), 1M potassium acetate (0.2 ml) and 5.6 ml of distilled water, plant extract (1.0 ml) was added and left at room temperature for 30 minutes. The absorbance of the reaction mixture was recorded by a spectrophotometer at 420 nm. A standard curve was obtained for catechin and the flavonoid content was determined from extrapolation of this curve.
Antioxidant activity
Reducing power assay
The reducing ability of the extracts of W. chinensis was determined by the method of Oyazu et al. (1986) [21]. To a mixure of 0.2 M potassium buffer (2.5 ml) and 1% potassium ferricyanide (2.5 ml), plant extract (1 ml) was added and incubated at 50°C for 20 minutes. Then 10% TCA solution (2.5 ml) was added to the reaction mixture and centrifuged (3000 rpm) for 10 minutes. Finally, 2.5 ml of solution was mixed with 2.5 ml of ultrapure water and 0.5 ml of 0.1% ferric chloride solution. The absorbance of the reaction mixture was recorded at 700 nm. A reference standard catechin was used for comparison.
Total antioxidant capacity assay
The antioxidant capacity of the extracts of W. chinensis was assessed by the method as described earlier [18]. To a mixture of sulphuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4 mM), plant extract was added and heated in a water bath at 950C for 90 min. After cooling to room temperature, the absorbance of the mixture was recorded at 695 nm against blank. A reference compound catechin was used for comparison.
DPPH radical scavenging assay
The ability of the extracts of W. chinensis to scavenge DPPH radical was measured by the modified method of Choi et al. (2000) [22]. A reference compound catechin was used for comparison. Methanolic solution of plant extract or reference compound was mixed with 0.135 mM of methanolic DPPH and left in dark for 30 minutes. The absorbance of the reaction mixture was recorded at 517 nm. The percent scavenging was calculated using the equation:
[(A control – A sample) / A control] × 100
Where, A control is the absorbance of control and A sample is the absorbance of extract or reference compound. The percentage inhibition was plotted against the compound concentration in order to calculate the IC50 values.
Determination of hydroxyl radical scavenging activity
The capacity of the extracts of W. chinensis to scavenge hydroxyl radical was assessed by the modified method of Elizabeth et al. (1990) [23]. A reference compound catechin was used for comparison. Plant extract or reference compound was added to a 1 ml reaction mixture containing 2.8 mM 2-deoxy-2-ribose, 20 mM phosphate buffer (pH 7.4), 100 μM FeCl3, 100 μM EDTA, 1 mM H2O2 and 100 μM ascorbic acid and then incubated at 37°C for 60 minutes. 0.5 ml of the reaction mixture was mixed with 1 ml of TCA (2.8%) and 1 ml of TBA (1%) and heated in a water bath at 90°C for 15 minutes. After cooling to room tempaerature, the absorbance of the mixture was recorded at 532 nm in a spectrophotometer against an appropriate blank solution. The percent scavenging of hydroxyl radical was calculated as in DPPH radical scavenging assay.
Determination of lipid peroxidation inhibition activity
The ability of the extracts of W. chinensis to inhibit the peroxidation of lipid was assesses by the method as described [18]. Brain homogenate was employed for in vitro lipid peroxidation assay. Mice brain homogenates were prepared by homogenizing brain in 50 mM phosphate buffer (pH 7.4) containing 0.15 M KCl using a homogenizer and centrifuged at 10000 g at 4oC for 20 min. To a mixture of brain homogenates (0.5 ml), 0.15 M KCl (1 ml) and 10 µM hydrogen peroxide (100 µl), plant extract was added and incubated at 37oC for 30 minutes. A solution of 2 ml of HCl (0.25 N) containging TCA (15%), TBA (0.38%), and BHT (5%) was added to the reaction mixture and heated in a water bath at 80oC for 60 minutes. After cooling to room temperature, the mixture was centrifuged to separate the supernatant and then the absorbance was measured at 532 nm by spectrophotometer. The percent inhibition of lipid peroxidation was determined as in DPPH radical scavenging assay. A reference standard catechin was used for comparison.
Cholinesterase inhibitory activities
The assessment of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibiting activities were performed by the colorimetric method of Ellman et al. (1961) [24]. Crude AChE enzyme was prepared from mice brain as mentioned above and BChE enzyme was prepared from human blood according to the method as described earlier [18]. The acetylthiocholine iodide and butyrylthiochoilne iodide were used as substrates for investigation of AChE and BChE assays, respectively. The hydrolysis of acetylthiocholine and butyrylthiocholine were determined spectrophotometrically. Plant extract or reference compound was added in an enzyme solution and incubated at 37°C for 15 min for interaction. This was followed by the addition of a 50 mM sodium phosphate buffer (pH 8.0) containing 0.5 mM acetylthiocholine and 1 mM DTNB and immediately the absorbance of the solution was recorded against a blank solution. All the experiments were taken in triplicate. For comparison, a reference compound donepezil was used for AChE activity and galantamine was used for BChE activity. The percent inhibition of cholinesterase activity was computed using the equation:
[(A control – A sample) / A control] × 100
Where, A control is the absorbance of control and A sample is the absorbance of extract or reference compound. IC50 value could be calculated from the dose response curve obtained by plotting the percent inhibition values against test concentrations of each extract or compound.
Isolation and characterization of an active compound from the bioactive extract
The EAF (5.6 g) of W. chinensis was subjected to column chromatography using silica gel 60 (Merck, Germany) as a stationery phase in an open column and sequentially eluted with n-hexane, dichloromethane and methanol stepwise gradient to yield five major subfractions (F1 to F5). Fraction F2 with potent AChE and BChE inhibitory activity was purified on silica gel GF254 by preparative thin layer chromatography with n-hexane-acetone (6:4) as the mobile phase to obtain the pure compound 1 (18 mg).
1H- and 13 C-NMR spectra of the compound 1 was recorded in DMSO-d6 on a Jeol-Ex 400 MHz and FT-NMR 100 MHz spectrometers. The chemical structure of the compound 1 was confirmed by comparing its spectral data with the reported values in the literature [27].
Statistical Analysis
All experiments were carried out in triplicate. The results were expressed as mean ± SD. Graph Pad Prism (version 8.0.1) and Microsoft Excel 2010 were used for the statistical and graphical evaluations. T-test was employed to estimate the statistical significance (P-value < 0.05) between the average values. IC50 values of different fractions/extractives were calculated using non-linear regression (Dose-Response -- Inhibition equation; log10 (inhibitor) vs. normalized response -- variable slope) in Graph Pad Prism - 8.0.1. Correlation study was performed using Pearson correlation test.