Antidiabetic, antioxidant, and anti-obesity effects of phenylthio-ethyl benzoate derivatives, and molecular docking study regarding α-amylase enzyme

In addition to their wide therapeutic application, benzoates and benzoic acid derivatives are the most commonly utilized food preservatives. The purpose of this study was to estimate the antioxidant, anti-diabetic, and anti-obesity activities of four 2-(phenylthio)-ethyl benzoate derivatives utilizing standard biomedical assays. The results revealed that the 2a compound has potent antidiabetic activity through the inhibition of α-amylase and α-glycosidase with IC50 doses of 3.57 ± 1.08 and 10.09 ± 0.70 µg/ml, respectively, compared with the positive control acarbose (IC50 = 6.47 and 44.79 µg/ml), respectively. In addition, by utilizing the β-carotene linoleic acid and DPPH methods, the 2a compound showed the highest antioxidant activity compared with positive controls. Moreover, the 2a compound showed potential anti-lipase activity with an IC50 dose of 107.95 ± 1.88 µg/ml compared to orlistat (IC50 = 25.01 ± 0.78 µg/ml). A molecular docking study was used to understand the interactions between four derivatives of (2-(phenylthio)-ethyl benzoate with α-amylase binding pocket. The present study concludes that the 2a compound could be exploited for further antidiabetic, antioxidant, and anti-obesity preclinical and clinical tests and design suitable pharmaceutical forms to treat these global health problems.

Frutarom (UK): β-carotene, DPPH, Na 2 HPO 4 /NaH 2 PO 4 , NaCl, gallic acid, trolox, methanol, starch, porcine pancreatic amylase, lipase, glucosidase, dinitrosalicylic acid (DNSA), Na 2 CO 3 , dimethyl sulfoxide (DMSO), and acarbose. The evaluated compounds 2a-2d were synthesized previously by our team, as outlined in Fig. 2 12 . α-Amylase inhibitory activity. The α-amylase inhibitory evaluation was established following the Wickramaratne et al. procedure 13 with minor changes. Working solutions with concentrations of 10, 50, 100, 500, and 1000 µg/ml were produced from our synthesized compounds, diluting them with a buffer of Na 2 HPO 4 /NaH 2 PO 4 (0.02 M), NaCl (0.006 M) at pH 6.9 and then brined up to 10 ml using a 10 ml volumetric flask. The acarbose was considered a positive control and was established following the same previous steps utilized for the synthesized compounds. As percent inhibition, the enzyme inhibitory potential was expressed, and the following equation was utilized to estimate the IC 50 dose for the samples 14 . www.nature.com/scientificreports/ α-Glucosidase activity. The inhibition of α-glucosidase activity by the synthesized molecules was measured. The α-glucosidase inhibitory activity is expressed as percentages of inhibition, which was calculated using the following formula: where A b and A s are the absorbance values of the blank (containing PBS, α-glucosidase, and PNPG, the colored substrate of glucosidase) and the tested samples containing PBS, samples, α-glycosidase, and PNPG, respectively. The α-glucosidase activity was measured using 10 and 20 mg/ml of the synthesized molecules. Each concentration was recorded by using 1, 3, 6, 9, and 12 mm PNPG. The inhibitory pattern was assessed using a Lineweaver-Burk plot. The constant Ki of enzyme inhibitory effect was determined 15 . Antioxidant activity. DPPH method. Four synthetic compounds were tested for the efficiency of scavenging free radicals matched with trolox and gallic acid as a basic. From the compounds, 1 mg/ml concentration solutions in methanol were prepared, and the solutions were used to prepare concentrations of 5, 10, 20, 30, 40, and 50 μg/ml. The DPPH reagent (0.002% w/v) was dissolved in methanol before mixed with working concentrations in ratios of 1:1:1 (compound:DPPH:methanol). The methanol solution was adopted as a blank. All the solutions were incubated for 30 min at room temperature in the dark. When the antioxidant compound reacts with DPPH, which can donate hydrogen, it is reduced the color changes from deep violet to light. Absorbance values were estimated by using a UV-Vis spectrophotometer at a wavelength of 517 nm. The DPPH inhibition percentage of all tested compound, trolox and gallic acid, was estimated according to the formula: where A b is blank absorbance, and A S is sample absorbance. The IC 50 values for tested synthetic compounds, trolox and gallic acid were determined from the curves 16 .
β-Carotene method. The activity of the synthesized compounds was assessed via performing a modified assay of Gazzani and Miller 17 . The method was based on the coupled oxidation of β-carotene and linoleic acid emulsion. The bleaching mechanism of β-carotene is produced from the hydro-peroxides created from linoleic acid 18 . During the oxidation process, the characteristic orange color of β-carotene and the chromophore will be lost. The presence of antioxidants can hinder the extent of the β-carotene bleaching. Briefly, 1 mg of β-carotene was dissolved in 2 ml of chloroform, 20 mg of linoleic acid, and 200 mg of Tween 20. A rotary evaporator entirely vaporized chloroform at a temperature of less than 30 °C under reduced pressure. Then 200 ml of distilled water saturated with oxygen was added to the flask, which was shaken vigorously for 30 min. A sample (5 ml) of the prepared emulsion was transferred to a series of tubes containing 0.1 ml of the synthesized compounds or tocopherol (2 mg/ml).
The control sample was prepared in the same way but without added antioxidants. Each sample type was prepared in triplicate. The samples were inserted in a water bath at 50 °C for 2 h. The absorbance of the samples was measured using a spectrophotometer at a wavelength of 470 nm, immediately after the preparation of the samples, and at 15-min intervals until the end (t = 120 min) of the experiment.
Anti-lipase activity. The lipase inhibitory activity was conducted by following the method in the references with minor modifications [19][20][21] . The prepared synthesized compounds solutions (1 mg/ml) were diluted with 10% DMSO to produce five different dilutions (0.2, 0.4, 0.6, 0.8, and 1 mg/ml). Orlistat anti-obesity drug was considered a positive control in this protocol and was then tested following the same steps used previously. A Tris-HCl buffer was used to zero UV-Vis spectrophotometer at 405 nm. The % inhibition of the synthesized compounds and orlistat was calculated by using the following equation: where A b is blank absorbance, and A S is sample absorbance. The IC 50 values for tested synthetic compounds and orlistat were determined from the constructed curves.

Molecular docking.
The docking studies on the α-amylase enzyme were based on the human pancreatic amylase (the X-ray crystallographic structure (PDB code 4W93) that was obtained from the RCSB repository. This 3D protein structure was chosen based on its good resolution of crystallography (X-ray diffraction 1.352 A o ). Additionally, this work is considered a continuation of our previous work on the α-amylase enzyme that utilized the ID: 4W93 α-amylase crystal structure 22 .
Preparing this crude obtained crystal structure for docking, using the PyMOL program (v1.8), started with removing the native ligand, non-protein atoms, and the present crystallographic waters. This step was followed by protonating imidazole and amide side chains via an H++ server. Then the polar hydrogen atoms were added and the structure was brought up to a physiological pH. The open-source program rDOCK (rdock.sourceforge. net), a development of RiboDock, was applied to perform the docking studies 23 . Considering the cavity occupied by the native ligand (a flavonol glycoside called montbretin A) of the utilized protein crystal structure (code %a − Amylase Inhibiton = Abs100% control − Abs Sample Abs 100% Control × 100 www.nature.com/scientificreports/ 4W93), the binding site for docking studies was defined within 6A o around it. The default docking procedure was followed that includes 3 stages of Genetic Algorithm search (GA-3, GA-2, and GA-1). This was followed by low-temperature Monte Carlo (MC) and Simplex minimization (MIN) stages 22 .
In the current study, the most potent compound against α-amylase was the 2a compound. Applying the empirical score function of Rdock, the 2a molecule was chosen to be docked in the binding pocket of the prepared crystal structure, keeping twenty docking solutions for each inhibitor to be sorted by their binding scores and later visually investigated for the connections between the inhibitors and the pocket's residues.

ADME-T calculations.
A set of ADME-T related properties were calculated by using the QiKProp module (schrödinger 10.9, LLC, NY) running in normal mode. QikProp generates relevant descriptors and uses them to perform ADMET predictions 24,25 . Statistical analysis. The antidiabetic, antioxidant, and antilipase experimental works were conducted in triplicates and the results were expressed as means ± SD standard deviation while the result was considered significant when the p value was < 0.05.

Results and discussion
Antidiabetic activity. Hyperglycemia can be controlled by several therapeutic protocols, one of which is the inhibition of α-amylase and α-glucosidase metabolic enzymes 6,26 . In fact, α-amylase is a responsible enzyme for the hydrolysis of starch into simple monosaccharides [27][28][29] . While α-glucosidase enzyme can be found on the border of the small intestine and act upon hydrolysis of α- (1,4) bonds between monosaccharide units [30][31][32] .
The antidiabetic potential of the synthesized compounds was estimated by assessing their α-amylase and α-glycosidase inhibitory effects. Acarbose, which is a commercial antidiabetic drug, was used as a positive control. An evaluation of the plot of % α-amylase and α-glycosidase inhibition activities of each compound is shown in Figs. 3 and 4, respectively. The IC50 values were calculated (Table 1). All synthesized compounds show potential activity against the α-amylase enzyme.
Compound 2a showed potent α-amylase inhibitory activity with an IC 50 value of 3.57 ± 1.08 µg/ml in comparison with positive control acarbose (IC 50 = 6.47 ± 1.01 µg/ml), as for compound 2a, it demonstrated potent α-glycosidase inhibitory activity with an IC 50 dose of 10.09 ± 0.70 µg/ml in comparison with acarbose (IC 50 = 44.79 µg/ml). These results are reasonable and justified since the synthesized compounds are modified acid esters and have the same functional groups as phenolic compounds, such as trans-p-coumaric acid.
Antioxidant activity. Antioxidants are valuable organic substances that can neutralize the harmful free radicals in living cells. There are two types of these radicals; natural pathways create the first type in our bodies due to the aging of cells and other factors; the second type is entered into our bodies from external sources like smoking, sun exposure, and other dangerous pollution sources. However, the body needs exogenous sources of synthetic antioxidants or dietary antioxidants from fruits and vegetables to scavenge these harmful radicals 6,33,34 . DPPH inhibitory activity. 1,1-Diphenyl-2-picryl-hydroxyl (DPPH) is one of the most widely used methods for measuring the antioxidant activity of different compounds such as phenols and phenolic acid esters. The decrease in absorbance at 517 nm induced by antioxidants determines the reduction ability of the DPPH. When antioxidants donate hydrogen atoms to the radicals, they lose their purple color. This, in turn, leads to decreased absorption. The decrease in absorption is taken as a measure of the extent of radical scavenging. All the compounds showed free radical scavenging activity near gallic acid at a concentration of 30 µg/ml (Fig. 5).   Table 2). On the other hand, there is not much difference between percent inhibition values for the compounds. This means that the position of functional groups as ortho-, para-or meta-has a slight effect on the antioxidant    β-Carotene linoleic acid activity. The same compounds were tested for their antioxidant activity using the emulsion system of β-carotene linoleic acid, depending on the fact that β-carotene loses its color in the absence of antioxidants. The synthetic compounds 2a and 2b showed higher antioxidant efficiency in concentration 0.2 mg/ml compared with water (control) and α-tocopherol and the other synthetic compounds antioxidant, which gave the lowest antioxidant efficiency. Figure 6 showed the antioxidant activities of the synthetic acid esters and positive reference standard with the coupled oxidation of β-carotene in a concentration of 0.2 mg/ml. The antioxidant activity of all the compounds gradually decreases with the increase of time. Water showed the highest β-carotene bleaching activity, followed by α-tocopherol after 1 h and (2a) compound, which has the best antioxidant activity with the absorbance of 1.04 after 75 min compared with the other synthetic tested compounds. The second one is a 2b compound which showed absorbance 0.84 after 75 min.
Porcine pancreatic lipase enzyme activity. The hydrolysis of p-nitrophenyl butyrate to p-nitrophenyl was used to measure the influence of the synthetic acid esters on the porcine pancreatic lipase enzyme. The assay measures the inhibition percentages of the synthesized compounds and the obtained results compared with orlistat, a pharmaceutical anti-obesity drug, and used in the current study as a positive control. The results of inhibitory percentages for the compounds and orlistat are shown in Fig. 7.
The anti-lipase activity IC 50 values of the synthesized compounds were with the range of 107.25-341.06 µg/ml in comparison with orlistat (IC 50 = 25.01 ± 0.78 µg/ml). The best inhibition activity was observed in compounds 2a and 2d with IC 50 values of 107.95 ± 1.88 and 119.05 ± 2.04 µg/ml, respectively. Compound 2c has the lowest inhibitory activity with an IC 50 value of 341.06 ± 2.40 µg/ml. Docking study. The 2a compound, which exhibited a super anti-diabetic and anti-lipase activity besides super antioxidant activity (considering the β-Carotene method) over the other designed compounds, was docked to the binding pocket of the α-amylase protein structure ID: 4W93). This docking revealed the formation of hydrogen bonds with the residue E233 and charge-π interactions with H201; stabilized by interactions with other surrounding residues like π-π stacking with Y62. Our docking studies suggest the potent inhibition www.nature.com/scientificreports/ of α-amylase is derived from strong interactions with the catalytic residue E233 and the neighbor residue H201, besides other stabilizing interactions with surrounding residues (Fig. 8) as well as the molecular Docking scores and α-amylase inhibtory activities are presented in Table 3.

ADME-T calculations.
All the calculated ADME-T parameters of our new compounds and the ideal recommended values are summarized in Table 4. It was clear that all synthesized compounds are located in the recommended and ideal range and do not exhibit any violations outside the recommended ADMET values. De facto, diabetes mellitus is a growing health problem worldwide without therapeutic agents that can control glucose levels in the bloodstream, leading to many macroangiopathic complications, including neuropathy, glaucoma, carpal tunnel syndrome, diabetic ulcers, atherosclerosis phenomena, and many others. In addition, diabetes also can cause microangiopathies injuries, including neuropathies of the peripheral nervous system, retinopathies, and nephropathies. In addition, it can increase the risk of various infectious diseases and cancer 1,[36][37][38] . Therefore, the search for practical and multi-functional drugs that can treat diabetes, obesity, and oxidative stress at the same time must be one of the main goals of modern scientists worldwide.

Conclusion
The results of the current investigation revealed that synthetic compound 2a exhibited potential anti-diabetic and anti-obesity properties. The 2a compound has strong potential in the inhabitation of metabolic key enzymes, including α-amylase, α-glycosidase, and anti-lipase, compared with the positive controls. Moreover, the 2a molecule demonstrated a potential antioxidant effect against DPPH and β-carotene linoleic acid-free radicals. The discovery of this simple synthetic compound may lead to the emergence of a new generation of antidiabetic, antioxidant, and anti-obesity compounds that can be used to manufacture multi-target medicines based on  www.nature.com/scientificreports/ benzoic acid derivatives. Thus, studying the properties of these bioactive molecules will contribute to combating these dangerous disorders and limiting their repercussions in the world. Moreover, the current study results may encourage other researchers to conduct more studies on these benzoic acid derivatives.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.