Secondary metabolites from the leaves of Terminalia myriocarpa and their α -glucosidase inhibitory potential

The objective of this study is to investigate the chemical composition of the leaves of Terminalia myriocarpa and to evaluate their α -glucosidase inhibitory activity aiming to be used as a safe antidiabetic. Consequently, 10 compounds were isolated using column and preparative thin-layer chromatographic techniques and identified as alphitolic acid, isovitexin, flavogallonic acid, nigaichigoside F1, quercetin

Diabetes mellitus is a widespread metabolic disorder, which affects about 422 million people globally [6].The most prevalent form of diabetes, type 2 (noninsulindependent diabetes mellitus, or T2DM), is characterized by a relative insulin shortage caused by the concomitant presence of insufficient insulin production, tissue insulin resistance, and insufficient compensatory mechanisms [7].Ineffective glycemic control in T2DM patients can lead to serious retinal, renal, and cardiovascular problems, as well as a sharp decline in life expectancy [8].Limiting postprandial hyperglycemia by inhibiting polysaccharide-digesting enzymes in the proximal small intestine, including α-glucosidase, and thus reducing glucose absorption from the gut, is a treatment approach for followed by ethyl acetate (EtOAc) (2l × 5).The solvent in each fraction was separately evaporated under vacuum to dryness.

Identification of the isolated compounds
The structures of compounds 1-10 (Fig. 1) were established, based on their spectral data, as follows.
As a part of our continuous interest in exploring bioactive phytoconstituents, we investigated T. myriocarpa leaf extract aiming at identifying its α-glucosidase inhibitory constituents.

Plant material
The leaves of T. myriocarpa Van Heurck & Müll.Arg. were collected from the Zoo Garden, Giza, Egypt, in March 2020, and identified by Ms. Therese Labib, the taxonomical consultant at Al-Orman and Al-Qubba Botanical Gardens.A voucher specimen, with the identifier M165, was deposited in the herbarium of the National Research Centre, Giza, Egypt.

Extraction and isolation of the leaf constituents
Fresh T. myriocarpa leaves (10 kg) were air-dried and powdered to yield 2.2 kg of leaf powder (22%).The obtained dry powder was thoroughly extracted thrice, at room temperature, by maceration with 100% methanol (MeOH), followed by 70% aqueous MeOH twice.A portion (200 g) of the brownishgreen residue (258 g), resulting from solvent evaporation of the combined leaf extract, was suspended in distilled water (2 l) and then partitioned with dichloromethane (CH 2 Cl 2 ) (2l × 5) s, H-6'').

General method of acid hydrolysis
Each isolated triterpene glycoside (10 mg) dissolved in a 10 ml 2 N hydrochloric acid-methanol mixture (1:1, v/v) was heated under reflux for 2 hours.The reaction mixture was left to cool and then vacuum-evaporated to dryness.The residue suspended in 5 ml of distilled water was extracted with EtOAc (5 ml × 3).The residual acidity of the aqueous layer was eliminated by repeated addition of methanol and evaporation.TLC analysis (Isopropanol-H 2 O, 7:1, v/v) of the residues against authentic material revealed the presence of D-glucose in each of the triterpene glycosides 4, 6, and 7 [13].

Evaluation of the α-glucosidase inhibitory activity
In phosphate buffer saline (pH 6.8), α-glucosidase from Saccharomyces cerevisiae (SIGMA G5003-100UN) was prepared at a concentration of 0.2 U/ml.α-Glucosidase (60 µl, 0.2 U/ml) was combined with each sample (10 µl) at different 6.37 (1H, d, J = 2.0 Hz, H-8), and 6.17 (1H, d, J = 1.9 Hz, H-6).exhibited potent activity with an half-maximal inhibitory concentration (IC 50 ) value of 0.49 ± 0.03 µg/ml.The CH 2 Cl 2 and EtOAc fractions were subjected to further phytochemical analysis, which led to the isolation of compounds 1 through 10 due to their separation using variable chromatographic procedures.The structures of the isolates were determined by spectral means (UV, 1 H-, and 13 C-NMR) and acid hydrolysis.Among these, compounds 1 [17,18], 4 [19,20], 6 [21], and 8 [22,23] are recorded herein for the first time from the genus Terminalia.In addition, this report is the first to mention the occurrence of compounds 7 [24,25], 9 [21], and 10 [26] in T. myriocarpa.Compounds 7, 9, and 10 were previously isolated from Terminalia arjuna bark [27], Terminalia catappa leaves [28], and Terminalia chebula fruits [29], respectively.On the other hand, compounds 2 [30,31], 3 [32], and 5 [31,33] were previously reported from the leaves of T. myriocarpa [2].Results of evaluation of the α-glucosidase inhibitory potential of the isolated compounds (Table 2) revealed that quercetin and flavogallonic acid with IC 50 values equal to 7.5 ± 0.09 and 21.0 ± 1.4 µM, respectively, might be responsible for the activity of the EtOAc fraction.This was in agreement with earlier reports on the ability of quercetin to inhibit α-glucosidase, with an IC 50 value of 7 µM, through the formation of hydrogen bond interactions with the active site pocket of the enzyme [34,35].Furthermore, former studies indicated that arjunic acid [11], nigaichigoside F1, rosamultin, and 19α-hydroxyasiatic acid [36] do not significantly inhibit α-glucosidase enzyme, which agreed with our findings.Moreover, isovitexin and asiatic acid were reported to exert α-glucosidase inhibitory effects with IC 50 values of 266.2 µM [37] and 100.2 ± 4.2 µM [38], respectively, which confirmed our results on the weak α-glucosidase inhibitory effects of these compounds.On the other hand, nothing could be traced in the literature concerning the α-glucosidase inhibitory activity of flavogallonic acid, alphitolic acid, and quadranoside IV.Therefore, to predict the mode of binding of flavogallonic acid to human α-glucosidase, a docking study with human C-terminal maltase-glucoamylase ctMGAM (PDB:3TOP), human N-terminal maltase-glucoamylase ntMGAM (PDB:2QMJ), and concentrations (0.3-700 ppm in the final volume).The mixture was then incubated for 20 minutes at 37°C in a 96-well plate.Subsequently, p-nitrophenyl-D-glucopyranoside (p-NPG) (SIGMA N1377) (150 µl, 1.25 mM) was added to each mixture and incubated for 20 minutes at 37°C, then 50 µl of 2 g/l sodium hydroxide (NaOH) was added to terminate the reaction.The amount of bright yellow p-nitrophenol released from the colorless p-NPG was measured spectrophotometrically at 405 nm to evaluate the activity of the α-glucosidase enzyme.Acarbose was utilized as a positive control, and a reaction mixture with 10 μl of buffer solution in place of the test entity was utilized as a negative control.For blank, p-nitrophenyl-α-D-glucopyranoside with buffer solution was added instead of the enzyme [14,15].

Docking study
The chemical structure of the screened compound was sketched using ChemBioDraw Ultra 14.0 software (CambridgeSoft corporation), and then energy was minimized by MMFF94x force field in the gas phase to a gradient of 0.01 kcal/mol.Å and saved in PDBQT format (A modification of the protein data bank format especially developed to hold the information needed by the protein-ligand docking software AutoDock, including the assigned charges).Cocrystal structures for human N-terminal maltase-glucoamylase ntMGAM (PDB:2QMJ), human C-terminal maltaseglucoamylase ctMGAM (PDB:3TOP), and human N-terminal sucrase-isomaltase ntSI (PDB:3LPP) were downloaded from the protein data bank (https://www.rcsb.org).All target receptors were prepared using MGL tools v1.5.7 to perform the deletion of water molecules and other hetatoms, the addition of polar hydrogens, and the addition of Kollman Charges, then saved in PDBQT format.Grid boxes were centered at the co-crystalized ligands with dimensions 30 × 30 × 30 Å to accommodate the whole binding sites of the target receptors.All docking calculations were implemented with the aid of the open-source software AutoDock vina v1.1.2.The docking poses were ranked according to their docking scores, and the best energy pose was selected.The interactions between the screened compound and the target proteins were analyzed using Discovery Studio Visualizer v21.1.0.20298 [16].

RESULTS AND DISCUSSION
The CH 2 Cl 2 and EtOAc fractions derived from T. myriocarpa methanolic leaf extract were found to exhibit α-glucosidase inhibitory activity (Table 1).The EtOAc fraction  isolated from the EtOAc fraction of T. myriocarpa methanolic leaf extract, point to their possible use as lead compounds to develop α-glucosidase inhibitors.This could only be implemented after confirming the obtained findings by assessing their efficacy and toxicity in-vivo.
human N-terminal sucrase-isomaltase ntSI (PDB:3LPP) was pursued.Results of the docking study revealed that flavogallonic acid was docked successfully to the same binding site of the cocrystalized inhibitor and possessed a binding affinity comparable with or even superior to acarbose (positive control) for the three receptors (Table 3).It adapted similar orientations in the catalytic site of the three receptors showing its gallic acid moiety inserted inside the binding cavity while the ellagic acid moiety heading to the outside.Flavogallonic acid-ntMGAM (PDB:2QMJ) complex was stabilized by six hydrogen bond interactions with asp203, asp327, trp406, asp443, met444, and arg526 in addition to two hydrophobic pi-pi interactions with tyr299 and phe575.Flavogallonic acid-ctMGAM (PDB:3TOP) complex demonstrated seven hydrogen bond interactions with asp1279, trp1355, asp1420, ser1425, lys1460, asp1526, and asp1555, a pi-pi interaction with phe1559, and a pi-alkyl interaction with met1421.For flavogallonic acid-ntSI (PDB:3LPP), seven hydrogen bond interactions with asp355, trp435, asp472, met473, lys509, arg555, a pi-pi interaction with trp327, and a pi-alkyl interaction with leu233 were observed (Fig. 2).

CONCLUSION
The in-vitro and in-silico α-glucosidase inhibitory properties of quercetin and flavogallonic acid, which were

Table 1 .
α-Glucosidase inhibitory activity of the CH 2 Cl 2 and EtOAc fractions derived from T. myriocarpa methanolic leaf extract.
50 : Half-maximal inhibitory concentration, CH 2 Cl 2 : Dichloromethane, EtOAc: Ethyl acetate.Results are represented as the mean value of three independent experiments ± standard deviation (SD).

Table 2 .
α-Glucosidase inhibitory activity of the isolated compounds from T. myriocarpa methanolic leaf extract.

Table 3 .
The binding affinities (kcal/mol) of flavogallonic acid with human α-glucosidase active sites, against those of acarbose.