In our previous botanical investigation [10], DP leaves were gathered from Al-Abed Palm Garden situated along the Cairo-Alexandria Desert Road in March 2020. Dr. Trease Labib, a plant taxonomy consultant at the Egyptian Ministry of Agriculture, confirmed the plant identity. A specimen (Aun-Phg-0002016) was submitted to the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Assiut University.

The dried powder of DP leaves (5 kg) was extracted with 70% methanol (5 × 20 L) through successive maceration, and the extract was concentrated under vacuum to yield a crude residue of 870 g. The crude extract was suspended in distilled water (500 ml), and partitioned with n-hexane (5 × 1 L), dichloromethane (CH2Cl2) (5 × 1 L), and ethyl acetate (EtOAc) (5 × 1 L). Each phase was concentrated under reduced pressure to give the corresponding fractions: n-hexane fraction (64 g), CH2Cl2 fraction (37 g), EtOAc fraction (30 g), and aqueous fraction (700 g). All fractions were further fractioned and chromatographed to afford compounds 1–13. The methods used for fractionation, isolation, and purification of these compounds were described in our previous phytochemical study [11].

DPPH (2,2-Diphenyl-1-picryl-hydrazine), α-glucosidase enzyme, and acarbose as a reference antidiabetic were obtained from Sigma-Aldrich Chemical Co. (Sigma, St. Louis, Mo., USA). Ascorbic acid as a reference antioxidant was obtained from El-Nasr Pharmaceutical and Chemical Co., Egypt. p-Nitrophenyl-α-D-glucopyranoside (p-NPG), sodium carbonate, sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from Hi-Media Leading BioSciences Company, Mumbai. The absorbance was recorded by UV-visible spectrophotometer (Milton Roy, Spectronic 1201, USA) and a multiplate reader (SunRise, TECAN, Inc., USA).

The DPPH free radical scavenging experiment was conducted as described previously [12]. In brief, the freshly made DPPH radical solution in methanol was created and kept at 10 °C in the dark. After that, a 40 µl aliquot of the test sample’s methanol solution was added to 3 ml of DPPH solution. A UV-visible spectrophotometer (Milton Roy, Spectronic 1201) was used to take instantaneous absorbance readings. Until the absorbance stabilised (16 minutes), the decline in absorbance at 515 nm was monitored constantly, with data being recorded at 1-minute intervals. Both the absorbance of the DPPH radical in the absence of antioxidant (control) and in the presence of the reference substance ascorbic acid were assessed. Three replicates were performed for each determination, and the average was calculated. The following formula was used to determine the percentage inhibition of the DPPH radical:

$$}\,}\,\left[ }_}}}}_}}} \right)}}_}}} \right\}\,}\,}} \right]$$

Where AC = Absorbance of the control at t = 0 min and AT = absorbance of the sample + DPPH at t = 16 min. Using Graphpad Prism software (San Diego, CA. USA), the 50% inhibitory concentration (IC50) was estimated from graphic plots of the dose response curve.

The in vitro antidiabetic activity was evaluated by α-glucosidase inhibitory activity, and performed as described previously [13]. Briefly, in a 96-well plate, the reaction mixture, which included 20 µl of different concentrations of each compound (1000–7.8 g/ml), 50 µl of phosphate buffer (100 mM, pH 6.8), 10 µl of α-glucosidase (1 U/ml), and 15 minutes of preincubation at 37 °C. 20 µl of p-NPG (5 mM) was then added to the reaction mixture as a substrate and incubated for an additional 20 min at 37 °C. By adding 50 µl of Na2CO3 (0.1 M), the reaction was stopped. Using a multiplate reader (SunRise, TECAN, Inc., USA), the absorbance of the produced p-nitrophenol was recorded at 405 nm. As a standard, acarbose was used in a range of concentrations (1000–7.81 g/ml). Each experiment was carried out in triplicates and was set up in parallel without test substance as a control.

The following formula was used to determine the percentage of inhibition:

$$}\,}\,\left( } \right)\,}\,\left( }\, - \,}} \right)\,}\,}$$

Where: As = the absorbance in the presence of test substance; Ac = the absorbance of control. The IC50 was estimated from graphic plots of the dose response curve for each concentration using Graphpad Prism software (San Diego, CA. USA).

All experimental findings are shown as mean ± standard deviation (SD) of three replicates. The 2016 version of Microsoft Excel was used to create the graphs.

Molecular docking was performed using Molecular Operating Environment program (MOE) 2020.01 on May 14, 2023; at Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University. Receptor-ligand docking and scoring is a method used when a three-dimensional structure of the target is available. This method can be divided into two parts: first, docking the ligand structures into the target’s binding site, generating a set of poses for each ligand; and secondly, scoring the poses and ligands based on how well each pose binds into the active site and the quality of interactions formed with the target [14, 15]. In this method, the ligand has conformational flexibility while the receptor remains rigid. The docking program produces a set of poses for each ligand along with a numerical score for each pose. The target ligands for modeling were created using the builder interface of the MOE software package 2020.01, and then subjected to conformational search. Conformers were optimized through energy minimization until RMSD gradient of 0.01 Kcal/mol and RMS distance of 0.1 Å with MMFF94X force-field, and partial charges were automatically calculated. The obtained database was saved as an MDB file to be used in the docking study. The x-ray structure of cytochrome c peroxidase ascorbate bound to the engineered ascorbate binding site (PDB code: 2 × 08) [16] and Co-crystal structure of alpha glucosidase binding site (PDB code: 3wy1) [17] were obtained from Protein Data Bank. We ran docking on the binding site of the co-crystallized ligand, since the crystal structure contains a ligand molecule, the program automatically identifies the binding site, and we docked the tested ligands on it. The following steps were taken for structure preparation: (a) the program checked the connections of the atoms and corrected any broken chains; (b) the program added hydrogen atoms through the Protonate 3D process; and (c) the potential of the enzyme atoms was fixed after selecting the complete enzyme structure. The docking of the target ligand conformations database was performed using the MOE-DOCK software wizard, with the following parameters adjusted: receptor and solvent as receptor, co-crystallized ligand atoms as active site, London dG as the initial scoring function, GBVI/WSA dG as the final scoring function, and MMFF94x force field for calculating energy parameters of the ligand-cleavage complex model. To evaluate the relative binding affinity of the conformers, London dG was employed as the scoring function, with lower values indicating more favorable poses. The docking calculations were run, and the obtained poses were studied. The 2D and 3D ligand interactions for each compound were saved as picture files. Docking was run on the binding site of the co-crystallized ligand. Since the crystal structure contains a ligand molecule, the program automatically identifies the binding site, and we dock the tested ligands on it. For structure preparation, three steps were done: a. Correct: in which the program checks atoms connections and corrects any break in the chains. b. Protonate 3D: in which the program adds the hydrogen atoms. c. Fixing the potential of the enzyme atoms after selection of the whole enzyme structure. Docking of the conformations database of the target ligands was done using MOE-DOCK software wizard. The following parameters were adjusted: Receptor and solvent as receptor, Co-crystalized ligand atoms as active site. Database containing test ligands as ligand, London dG as initial scoring function, GBVI/WSA dG as final scoring function, MMFF94x force field was used for calculating the energy parameters of the ligand – cleavage complex model. To compare between the conformers London dG was used as scoring function, lower values indicate more favorable poses. The dock calculations were run, and the obtained poses were studied. The 2D and 3D ligand interactions for each compound were saved as picture files.