Theaflavin (TF), theaflavin monogallate (TFMG) and theaflavin digallate (TFDG),Acetyl cholinesterase (AChE) type VI-S from electric eel was purchased from Sigma-Aldrich (Saint Louis, Missouri, USA). Uric acid and allopurinol powder were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.1. Preparation of Solution
2.1.1. Preparation of tea extract
The leaf dusts of Camellia sinensis (CS) were collected locally and dried in incubator for 2 days at 40ºC. Extraction was done by taking 10mg/ml in distilled water. When the color of the aqueous extract became brown, it was collected for the experiment.
2.1.2. Preparation of Arsenic Solution
For this experiment, we used sodium-meta-arsenite as arsenic source by dissolving it in distilled water maintaining 0.6 ppm concentration. We are taking this dose because it is not lethal for the animal but the exposure of arsenic in animal model for a moderate time period like 21 days can generate toxicity in their body.
2.1.3. Preparation of Uric acid- Purchased uric acid was dissolved in deionized water and filtered for having a crystal free solution and the concentration was 8 mg/dL (Hua et al., 2015).
2.1.4. Preparation of allopurinol-Stock allopurinol powder (> 99% pure; Sigma; cat. no. A-8003) was dissolved in sterile 0.9% NaCl solution, pH 10, on the day of use. Typical volumes for oral dosing are around 0.5–0.6 ml of solution in a 150g rat.
2.2. Treatment and Group Distribution of Animal Model
For this experiment, female albino rats weighing 150± 10 were acclimatized for 10 days at 12 hours light-dark cycle, 32± 2ºC temperature, humidity 50-70%. Standards pellet diet (Hindustan Lever, Mumbai, India) was given to them. Rats were purchased from Government accredited rodent firm house (CPCSEA-Committee for the Purpose of Control and Supervision of Experiments on Animals: Reg. no 1A2A/PO/BT/S/15/CPCSEA. <http://cpcsea.nic.in/Auth/index.aspx>) organization under the Dept of Animal Husbandry and Dairy, Ministry of Agriculture and Farmer’s Welfare, Govt. of India. Animal experiments were performed in the OIST Animal Resource Facilities. Institutional ethical concerns (Oriental Institute of Science and Technology) Review Board; OIST-IRB, reference no. oist/EC/sm/19_3) were maintained throughout the investigation.
Rats were randomly selected for the experiment and they were divided into 5 different groups having four in each group. Control or group-I – animals were fed only drinking water. Arsenic or group-II -0.5 ml of arsenic solution at a concentration of 0.6 ppm was given for 21 days. Arsenic+CS or group-III – 0.5 ml arsenic at a concentration of 0.6 ppm + 0.5 ml lyophilized tea extract for the same time period. Group – IVwas treated with 0.5 ml of arsenic and uric acid 8mg/dL concentration. In group-V,same dose arsenic and 0.5 ml allopurinol from the stock were given.
2.3. Evaluation of Toxicity Level in Brain Sample
By sacrificing the experimental rat, cerebrum and cerebellum were collected. These tissues were homogenized in homogenizer by using ice cold phosphate buffer (pH-4) and cytosols were obtained. Collected cytosols were distributed in two aliquots and were preserved at -20ºC. The cytosol was used for different parameter testing.
2.3.1. Estimation of Non-Protein Thiol Assay (NPSH)
From the prepared cytosol of the brain tissue (cerebrum and cerebellum), NPSH level was measured. This assay was done by a standardized protocol using Ellman’s reagent (DTNB) with a slight modification (Forman et al., 2009). At first the sample of different groups were taken and proteins were precipitated by 5% TCA. In the clear cytosol, Di-thio-bis-nitrobenzoic acid (DTNB) was added as a color reagent with Tris HCL buffer. 10 to 15 minutes incubation was maintained in room temperature (RT). Reading of absorbance was taken at 420 nm and the concentration of NPSH was determined against a standard curve of GSH (reduced glutathione).
2.3.2. Malondialdehyde Assay
At first, we took the samples of different groups and treated it with the same amount of 5% TCAand then it was centrifuged at 8-10k rpm. After discarding pellets, supernatant was collected. Collected supernatant was treated with 3% TBA (Thiobarbituric acid) solution. Then it was incubated at 80ºC in water bath for 30 min and finally cooled at RT for 20 min. For the presence of malondialdehyde which is the end product of lipid peroxidation, a slight pink color appeared. By measuring the color intensity in spectrophotometer MDA concentration was calculated in the different groups with the help of molar extinction coefficient of MDA (1.56 × 105 cm2/mmol) (Maiti and Chatterjee 2001; Maiti et al., 2014).
2.3.3. Acetyl cholinesterase assay
Acetyl cholinesteraseassay was performed by Ellman’s method in which DTNB was used as a chromogen and acetylthiocholine iodide as substrate (Govindappa et al., 1987). In the assay, 1 cm quartz cuvette was gradually filled with 0.5 mL of DTNB (3mM), 375 μL of 50 mM Tris-HCl buffer (pH 8.0) and 100 μL of 0.086 U/mL AChE. The reaction was started by addition of ATCI (25 μL). The reaction mixture was monitored at 405 nm for 10 min. Enzyme activity was calculated using the extinction coefficient, ε = 13600 M−1cm−1.A standard solution of eserine salicylate was used as positive control for AChE inhibitor.
2.3.4. Determination of pure AChE inhibition by pure TFDG, TFMG and simple TF
Acetyl cholinesterase was assayed using the above method except one change. Here100 μL of 0.086 U/mL AChE (from electric eel) was added in the reaction mixture instead of AChE extract from rat cerebellum. A standard solution of eserine salicylate was used as positive control for AChE inhibitor.
The concentrations of TFDG, TFMG and simple TF were used in the range of 1-8 μg/mLthe percentage of AChE inhibitory activity (% IA) was calculated by using the equation:
% IA = [(Ac - At)/Ac] × 100
Where, Ac is the activity of control enzyme (containing all reactants, except the tea components) and Atis the activity of tested compound. All experiments were performed in triplicate.
2.3.5. Estimation of IC50 values
The concentrations of the test samples that inhibited the hydrolysis of substrate (acetylthiocholine iodide) by 50% (IC50) were determined by a linear regression analysis between the inhibition percentages against the log concentrations of inhibitors using the OriginPro 8 software and Microsoft office excel 2007 program.
2.3.6. Assay of Catalase in native polyacrylamide gel
Catalase activity assay was done in 8% native polyacrylamide gel electrophoresis (PAGE). 0.003% H2O2 (30% solution vol/vol) was given as a substrate. 2% ferric chloride (wt/vol) and 2% potassium ferrocyanide (wt/vol) were used for staining purpose. After staining, transparent bands were developed against dark green-blue background where the enzyme was present because the catalase enzyme removes the hydrogen peroxides from the zone of the gel it occupies. Peroxide is oxidized to O2; removal of peroxide does not allow the potassium ferricyanide (a yellow substance) to be reduced to potassium ferrocyanide that reacts with ferric chloride to form a Prussian blue precipitate (Maiti et al., 2014).
2.3.7. Superoxide dismutase (SOD) assay in native polyacrylamide gel
Superoxide dismutase (SOD) enzyme was assessed in 12.5% non-denaturing PAGE gel. After completing the electrophoresis, the gel was kept in NBT (nitro bluetetrazolium)solution in shaking condition for incubation. After incubation, the gel was soaked in SOD solution which contained Potassium phosphate, TEMED and riboflavin for approximately 15 min. Then the gel was illuminated in UV trans-illuminator. Gel became purple except the portion containing superoxidedismutase.In purple background, whitish bands of SOD were observed (Acharyya et al., 2014).
2.3.8. Lactate dehydrogenase (LDH) assay in native polyacrylamide gel
For sample running, 8% native gel was electrophoresed at 4˚ C. After completing gel running, the gel was incubated in staining solution in dark condition for 40-45 min. The component of staining solution was Tris HCL, lithium lactate, Nitro blue tetrazolium, phenazine methyl sulfateand NAD+.Band of LDH wasof prominent blue color against the transparent background (Gilmour et al., 1994).
2.3.9. Matrix metalloproteinase (MMP) in native polyacrylamide gel
Samples were run in SDS-polyacrylamide gel electrophoresis. 8% gel was prepared by mixing sterile distilled water, Tris HCL, 20% SDS (sodium dodecyl sulphate), polyacrylamide and gelatin solution (1%) in a tube. After addition of APS (ammonium per sulfate) and TEMED (tetraethyl ethylenediamine), gel solution was poured into the gel chamber. Gel was run at 110V (Bio-Rad apparatus) using pre-cooled running buffer at 4⁰C.After removing the gel from gel apparatus, the gel was incubated in zymographic development buffer (50mM Tris pH-7.4, 1mM CaCl2,0.02% NaN3, sterile diH2O) at 37⁰C for 42 hours. Then the gel was stained by Coomassie Brilliant Blue R-250 solution at room temperature on a rotary shaker. Staining was followed by using distaining solution (ethanol, acetic acid, dH2O) until the clear transparent bands were observed in bluish background (Frankowski et al.,2012).
2.3.10. DNA Comet assay
The Comet assay was done with minute modifications of Singh and colleagues’ method (Singh et al, 1988). At 37⁰C, 75 ml of low melting point agarose (0.6%) containing Phosphate buffer saline (PBS) was added to 25 ml of cell suspension (105cells). This mixture was then placed onslideswhich were pre-coated with 1% agarose and coverslips were placed. After the solidification of agarose, the coverslip was removed and the slides were immersed in ice cold lysis buffer (2.5 mM NaCl, 1% Triton X-100, 85 mM EDTA, 10 mM Trizma base,10% DMSO and 1% sodium lauryl sarcosinate, pH -10) for 1 hour at 4◦C. After lysis, the slides were washed for three times in Phosphate buffer saline at room temperature. Next, 50 ml of buffer (control) or T4 endo V (Epicentre) (4 U/slide) in buffer was transferred to the slides. Coverslips were kept and the slides were incubated at 37◦C for 45 min. After removing the coverslip, slides were washed in water twice for removing excess salt. Slides were then placed in a gel electrophoresis chamber (Bio-Rad, USA) filled with alkaline electrophoresis buffer (0.3 M NaOH and 1 mM EDTA) for 25 min. Electrophoresis was run for 30 min at 25 V and the current was adjusted to 300 mA. Slides were then neutralized by PBS and stained with a solution of 10 mg/ml ethidium bromide for 5 min. Excess stain was removed by water wash. Slides were observed by using a fluorescence microscope (Nikon, Eclipse LV100 POL), with the help ofVisComet (ImpulsBildanalyze) software. A total of 100 comets /slide were studied for each experiment (Garcia et al., 1993).
2.3.11. Histo-architecture study of rat brain tissues
Intact and fresh brain tissues (cerebrum and cerebellum) were collected just after sacrifice and these tissues were poured in fixative. Those tissues were embedded in paraffin block, sectioned at 5 microns, stained with eosin and Hematoxylin. For the histo-architectural study, stained slides were observed under light microscope (Nikon, Eclipse LV100, magnification 10X).
2.3.12. Bioinformatics study
Protein and ligand structure retrieval
An X-ray crystallographic structure of human acetylcholine esterase was retrieved from Protein Data Bank (PDB ID: 4M0E). Removal of other molecules complexed with 4M0E was performed using BIOVIA Discovery Studio 2017R2 and the edited 4M0E molecule was saved in PDB format for further use. Again, the three-dimensional structures of substrate; acetylcholine, and different inhibiters like catechingallate (CG), gallocatechingallate (GCG), epicatechin (EC), epicatechingallate (ECG), epigallocatechin (EGC), epigallocatechingallate (EGCG), theaflavin (TF), theaflavinmonogallate (TFMG) and theaflavindigallate (TFDG) were retrieved from PubChem [https://pubchem.ncbi.nlm.nih.gov/] database of bioactive compounds in SDF format. The SDF format was converted to PDB through BIOVIA Discovery Studio 2017R2 and saved for further use.
Ligand and protein structures preparation
The PyMol(https://pymol.org/2/) was used as molecular-visualization tool in this study. The ligand molecules were retrieved as .sdf format and structures were saved in a .pdb format.
Surface-Topology Calculation of Proteins
The factors solvent accessibility is definedby two properties of a protein; pocket, where water enters and cavity, where does not. The CASTp: Computed-Atlas-of-Surface-Topography of Protein (http://sts.bioe.uic.edu/castp/index.html?j_5e8c7bec25090) was used to define the pockets and cavities.
Molecular docking study
Molecular docking with acetylcholinesterase and acetylcholine was performed and analyzed using Autodock Tools -1.5.6. Different acetylcholine esterase inhibitors interactions were also analyzed by PatchDock server [https://bioinfo3d.cs.tau.ac.il/PatchDock/].