Thiocholine

AChE activity was measured using a modified 96-well microplate assay [11] (link) based on Ellman’s method [21] . The enzyme hydrolyses the substrate acetylthiocholine resulting in the product thiocholine which reacts with Ellman’s reagent (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detected at 412 nm. 50 mM Tris–HCl pH 8.0 was used as a buffer throughout the experiment unless otherwise stated. AChE used in the assay was from electric eel (type VI-S lyophilized powder, 518 U/mg solid, 844 U/mg protein). The enzyme stock solution (518 U/ml) was kept at −80°C. The further enzyme-dilution was done in 0.1% BSA in buffer. DTNB was dissolved in the buffer containing 0.1 M NaCl and 0.02 M MgCl₂. ATCI was dissolved in deionized water. In the 96-well plates, 100 µl of 3 mM DTNB, 20 µl of 0.26 U/ml of AChE, and 40 µl of buffer (50 mM tris pH 8.0), 20 µl of each extract in various concentrations (25, 50, 100, 250 and 500 µg/ml) dissolved in buffer containing not more than 10% methanol were added to the wells. After mixing, the plate was incubated for 15 min (25°C) and then the absorbance was measured at 412 nm in Tecan infinite 200 microplate reader and the readings were used as blank. The enzymatic reaction was initiated by the addition of 20 µl of 15 mM ATCI and the hydrolysis of acetylthiocholine was monitored by reading the absorbance every 5 min for 20 min. Physostigmine was used as positive control. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows:
Where; E is the activity of the enzyme without extract and S is the activity of enzyme with the extract. IC₅₀ value could be calculated from the % inhibition values of different concentrations of each plant extract.

The inhibitory activity of these important enzymes was measured thorough the Ellman’s method, as stated previously (Mocan et al., 2017 (link)). Briefly, DTNB was dissolved in buffer Tris-HCl at pH 8.0 containing 0.1 M NaCl and 0.02 M MgCl₂. Then, a filtered sample solution dissolved in deionized water (50 ml, 2 mg ml⁻¹) was mixed with 125 ml of 5-dithio-bis(2-nitrobenzoic) acid (DTNB), acetylcholinesterase (AChE), or butyrylcholinesterase (BChE) solution (25 ml) dissolved in Tris-HCl buffer at pH 8.0 in a 96-well microplate and incubated for 15 min at 25^°C. Initiation of reaction was performed by the addition of acetyl-thiocholine iodide (ATCI) or butyryl-thiocholine chloride (BTCl) (25 ml). In addition, a blank was prepared by adding the solution sample to all reagents without the enzyme(s) (AChE or BChE) solutions. The sample and blank absorbances were then read at 405 nm after 10 min of incubation at 25°C. The absorbance of the sample was subtracted from that of the blank and the cholinesterase inhibitory capacity was expressed as IC₅₀ (µg ml⁻¹, concentration range 0.05 to 25 µg ml⁻¹). Galantamine was used as positive control. All data were collected in triplicate.

The anticholinesterase inhibition of samples was investigated by Ellman’s reaction [4 (link)]. In the in vitro assays, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) catalyze the hydrolysis of acetyl- and butyryl-thiocholine, two alternative analogs of natural substrate acetylcholine. The quantification of free sulfhydryl groups was performed by 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB) and the absorbance was measured at 405 nm for 2 min. The galantamine, a drug used to treat Parkinson’s and Alzheimer’s diseases, was used as positive control. The results were expressed on the basis of the concentration of the sample required to inhibit the activity of the enzyme by 50% (IC₅₀) in μg/mL calculated by nonlinear regression analysis.
In particular, in AChE inhibition in vitro assay, different concentrations of sample (0.10–1000 μg/mL), buffer B (50.00 mM Tris-HCl, pH 8.00 containing 0.10% BSA), 3.00 mM DTNB, and 15.00 mM acetylthiocholine iodide were mixed and the reaction was started by adding 0.18 U/mL of AChE enzyme.
The BChE inhibition in vitro assay was performed in a similar way by using 15 mM butyrylthiocholine iodide as substrate and 0.10 U/mL of BChE enzyme [4 (link)].

The effect of the enriched fraction on the insect’s acetylcholinesterase enzyme (AChE) was studied following Ellman’s method with slight modification (Ellman, 1959 (link)). The AChE enzyme hydrolyses the substrate acetylthiocholine to produce acetate and thiocholine. Thiocholine reacts with Ellman’s reagent (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detected at 412 nm. The enzyme activity was tested against crude enzyme extract of S. oryzae, R. dominica, and T. castaneum in -vitro conditions. The insects (20 adults each) were homogenized using 0.5M Tris-HCl buffer and stored at -20⁰C. For the study, crude enzyme extract was pre-incubated with the enriched fraction and with standard inhibitor (Pyridostigmine bromide) at different doses of 25, 50, 75, and 100 μg/ml of insect’s enzyme extract at 37°C for 30 mins. A microplate reader was used to measure the difference in the absorbance. In a microplate well 200 µl of the reaction mixture, 3 µl of 0.1M acetylthiocholine chloride, 10 µl of insect homogenate, and 87 µl of water were added to make the total volume of 300 µl. The reaction mixture is prepared by adding 10.5 ml of cocktail (13 ml of 1M NaCl, 2 ml 1M MgCl₂, 10 ml of 0.5M Tris-HCl, and 10 ml of 0.2M EDTA), 3 ml of 1mM DTNB and 6.5 ml of water in a reagent bottle. The reaction is initiated either by adding the treated enzyme or substrate and expressed as percentage inhibition.
Inhibition (%) = 100 - Change of sample absorbance/Change of blank absorbance X 100

The AChE/(BuChE) inhibitory test was measured using a spectrophotometric method developed by Elman et al. [31 (link),32 (link)]. A buffer solution containing phosphate (pH 8.0) of volume 140 μL; 20 μL of each AChE/BuChE solution; and 20 μL of the test sample were incubated at room temperature for 15 min. AChE/BuChE 10 μL was used to start the reaction, followed by the addition of DTNB. ATCh or BTCh hydrolyzed the reaction of DTNB with thiocholine for 15 min; unrestricted by AChE and BuChE enzymatic hydrolysis. E − S/E × 100, where E&S represent enzyme activity with and without test samples, were used to compute the percentage (percent) inhibition (30). Each sample’s inhibitory activity was measured in terms of IC₅₀ (g/mL) or μM. For all substances, the IC₅₀ values were derived using a generic graph. The graph was created in Excel and the IC₅₀ values were derived by taking Y = 50 and determining the x value as IC₅₀.