Materials
The following materials were purchased from Frutarom (UK): β-carotene, porcine pancreatic amylase enzyme solution, starch, dinitrosalicylic acid (DNSA), methanol, trolox, gallic acid, Na2CO3, and acarbose.
All the tested compounds were synthesized previously by our team. The compounds are classified into three different groups: benzodioxole aryl acetate (3a–3 g), benzodioxole aryl acetic acid (4a–4 h) [20], benzodioxole-carboxamide (6a & 6b) [21], see Table 1.
α-Amylase inhibitory activity method
The α-amylase inhibitory assessment was based on the Wickramaratne et al. protocol [30] with some slight alterations in some steps. The experimental part was carried out by following the 3,5-dinitrosalicylic acid (DNSA) procedure. Solutions of 20 mm sodium phosphate monobasic and sodium phosphate dibasic buffer involving 6.7 mm sodium chloride (NaH2PO4 and Na2HPO4, both including 6.7 mm NaCl, pH 6.9) were constructed by partially filling the beaker with NaH2PO4 and NaCl solution; the mixture was subjected to a magnetic stirrer. In contrast, the pH was adjusted by inserting a calibrated pH electrode in the solution. Then, the Na2HPO4 and NaCl solution was gradually added until the pH reached 6.9. A weight of 5.36 g of 20 mm Na2HPO4. 7H2O and 0.39 g of 6.7 mm NaCl were dispersed in distilled water to make 1 L and a weight of 2.76 g of NaH2PO4. H2O and 0.39 g of NaCl dissolved in distilled water to make 1 L. The stock solution for the synthesized molecules had a concentration of 1 mg/ml and was put in a minimum amount of 10% DMSO (1:100 dilution) and was then dispersed in a buffer of Na2HPO4/NaH2PO4 (0.02 M) and NaCl (0.006 M) at adjusted pH 6.9. Working solutions with concentrations of 1, 10, 50, 100 and 500 µg/ml were obtained by mixing 0.01, 0.1, 0.5, 1, and 5 ml of our synthesized molecules, respectively, and then diluting them with a buffer of Na2HPO4/NaH2PO4 (0.02 M) and NaCl (0.006 M) at pH 6.9 and then brined up to 10 ml using VF (10 ml). The acarbose was considered a reference and was established following the same previous steps used for the synthesized molecules.
A solution of α-amylase was (2 units/ml) was produced by dissolving 12.5 mg of amylase in a minimum amount of DMSO10%, which was then brined up to 100 ml with the previous phosphate buffer in a volumetric flask (V.F) (100 ml). A starch solution with a concentration of 1% (w/v) was prepared by suspending 1000 mg of starch in 100 ml of distilled water using V.F (100 ml), and then it was kept in a water bath at 37 °C until use, with occasional mixing to prevent starch precipitation. DNSA was used as a reactive reagent to react with reducing sugars to produce 3-amino-5- nitro salicylic acid that is highly absorbent of light at about 540 nm. It was prepared by dissolving 12 g of sodium potassium tartrate tetrahydrate in 8.0 ml of 2 M NaOH (8 g in 100 ml distill. water) then further dissolved in 20 ml of 96 mm of 3.5-dinitrosalicylic acid solution.
Then, 200 μl of the amylase solution (2 units/ml) was gently shaken with 200 μl of each of the VOs established working solutions, and then this was incubated at 37 °C for 10 min. Then 200 μl of the starch solution was added to each test tube, and there was further incubation for 3 min at 37 °C. The addition of 200 μl DNSA terminated the reaction and then boiling for 10 min at 85 to 90 °C. The mixture was then cooled to room temperature and diluted with 5 ml of distilled water, and the absorbance was recorded at 540 nm using a UV–Visible spectrophotometer. Replacement of the synthesized compounds with 200 μl of buffer was established to obtain the blank sample. In this protocol, acarbose was the positive control sample. The enzyme inhibitory activity was expressed as percent inhibition, and the following equation was used to determine the IC50 value for the tested compounds [31].
$$\alpha-{\text{amylase inhibitory }}\left( \% \right){\text{ }} = {\text{ }}\left[ {{\text{ABSblank }}{-}{\text{ ABStest}}} \right]/\left[ {{\text{ ABSblank}}} \right]){\text{ }}*{\text{1}}00\%$$
Anti-lipase activity
A solution of 1 mg/ml of the synthesized compounds was mixed with 10% dimethyl sulfoxide (DMSO) and then diluted with 10% DMSO to produce five dilutions (40, 100, 200, 300, and 400 µg/ml). Orlistat was considered a reference in this inhibition protocol for pancreatic lipase and was then tested following the same steps used previously.
A freshly prepared stock solution of pancreatic lipase enzyme was established by suspending this enzyme in 10% DMSO to form 1 mg/ml. Firstly, 25 mg of lipase was suspended in a small amount of 10% DMSO, bringing the volume up to 25 ml in V.F (25 ml), this was then put in a water bath sonicator at 37 °C for 15 min. The stock solution of PNPB was constructed depending on the manufacturing structure (20.9 mg was obtained from PNPB and dispersed in 2 ml of acetonitrile) by dissolving 104.5 mg of PNPB in acetonitrile brined up to the volume of 10 ml in V.F (10 ml). The pancreatic lipase inhibition assay was conducted by adopting the procedure in the references with slight modifications [32,33,34]. From each working solution of the synthesized compounds prepared above, 200 µl of the synthesized compounds were taken and put in a separate test tube, then 100 µl of porcine pancreatic lipase (1 mg/ml) was added to it. The resulting mixture was then adjusted to 1000 µl after addition to the 700 µl of Tris–HCl solution, and then it was incubated in a water bath at 37 °C for 15 min. After the incubation, 100 µl of PNPB (p-nitrophenyl butyrate) solution was added to each test tube. This mixture was then incubated again in a water bath at 37 °C for 30 min. A solution characterized as a negative control was constructed without the synthesized compounds, using 100 µl of porcine pancreatic lipase (1 mg/ml) solution mixed with the Tris–HCl solution up to 1 ml after the addition of 900 µl. The same procedure was adopted for orlistat, which was a positive control. A Tris–HCl buffer was used to zero UV–Vis spectrophotometer at 405 nm. The pancreatic lipase effect was reported by measuring the hydrolysis of p-nitrophenolate to p-nitrophenol at 405 nm using a UV–Vis spectrophotometer device. The lipase inhibition activity of the synthesized compounds, or orlistat as a reference, was identified by measuring the effect on the enzyme reaction rate after the addition of the synthesized compounds and then comparing it with the control. The % inhibition of the synthesized compounds was calculated by using the following equation:
$${\text{Inhibitory lipase }}\left( \% \right){\text{ }} = {\text{ }}\left[ {\left( {{\text{A}}_{{\text{b}}} - {\text{ A}}_{{\text{s}}} } \right)/{\text{A}}_{{\text{b}}} } \right]{\text{ }}*{\text{1}}00$$
Chemo-informatics properties of the tested compounds
The Chemo-informatics properties of the synthesized compounds were evaluated based on chemo-informatics properties and the Lipinski rule of five (RO5). Multiple online servers such as Molinspiration (http://www.molinspiration.com/), and Molsoft (http://www.molsoft.com/) were employed to predict the molecular properties designed compounds [35].
Molecular docking
Protein Data Bank structure of human pancreatic α-amylase (PDB code: 4w93) was downloaded from the RCSB repository. The protein structures were analyzed with PyMOL v1.8. The native ligand, non-proteins atoms and crystallographic waters were removed. Polar hydrogen atoms were added to the previous structure, and side-chain amides and imidazoles were protonated assuming a physiological pH using an H++ server. Docking studies were performed using the open-source program rDOCK (rdock.sourceforge.net); which is a development of RiboDock [36]. The cavity was defined using the native ligand of crystal structure 4w93 (a flavonol glycoside called Monotrobtin A) within 6 Å around it. The standard docking protocol in rDOCK was used, including 3 stages of Genetic Algorithm search (GA1, GA2, GA3), followed by low-temperature Monte Carlo (MC) and Simplex minimization (MIN) stages (rDock Reference Guide, August 2015). Three representatives of the synthesized inhibitors (3a, 4f, and 6b) were selected to be docked in the binding pocket of the prepared crystal structure using the empirical score function of rDOCK keeping 20 docking solutions for each inhibitor to be sorted by their binding scores and later visually analyzed for the interactions between the pocket’s residues and the inhibitors.