Chemical Profiling and Molecular Docking Study of Agathophora alopecuroides

Natural products continue to provide inspiring chemical moieties that represent a key stone in the drug discovery process. As per our previous research, the halophyte Agathophora alopecuroides was noted as a potential antidiabetic plant. However, the chemical profiling and highlighting the metabolite(s) responsible for the observed antidiabetic activity still need to be investigated. Accordingly, the present study presents the chemical profiling of this species using the LC-HRMS/MS technique followed by a study of the ligand–protein interaction using the molecular docking method. LC-HRMS/MS results detected twenty-seven compounds in A. alopecuroides extract (AAE) belonging to variable chemical classes. Among the detected compounds, alkaloids, flavonoids, lignans, and iridoids were the most prevailing. In order to highlight the bioactive compounds in AAE, the molecular docking technique was adopted. Results suggested that the two alkaloids (Eburnamonine and Isochondrodendrine) as well as the four flavonoids (Narirutin, Pelargonidin 3-O-rutinoside, Sophora isoflavanone A, and Dracorubin) were responsible for the observed antidiabetic activity. It is worth mentioning that this is the first report for the metabolomic profiling of A. alopecuroides as well as the antidiabetic potential of Isochondrodendrine, Sophora isoflavanone A, and Dracorubin that could be a promising target for an antidiabetic drug.


Introduction
Diabetes mellitus is a chronic metabolic disease recognized by an increase in blood glucose levels which develops from a deficiency in insulin secretion, action, or both of them [1]. Type 2 diabetes mellitus (T2DM) is the most common health problem and accounts for about 90% of diabetes cases with 4.9 million mortalities throughout the world [2]. Inhibiting the digestion of dietary carbohydrates is one of the effective procedures for the management of postprandial hyperglycemia in T2DM. One of the essential digestive enzymes is pancreatic α-amylase which converts dietary carbohydrates such as starch into smaller oligosaccharides mixture that are further broken down into glucose by α-glucosidase, another important metabolic enzyme. Upon the absorption of glucose, it enters the bloodstream and causes postprandial elevation in blood glucose levels. Therefore, blocking the enzymes α-amylase and α-glucosidase can inhibit the digestion of carbohydrates, postpone glucose uptake, and subsequently lower blood sugar levels [3]. Recently, medicinal plants proved their great therapeutic potential and negligible side effects in the treatment of T2DM.

Plant Materials
Agathophora alopecuroides var. papillosa was collected from the Qassim region in the northcentral Saudi Arabia during October 2021. The taxonomic identity of the plant was confirmed by Ibrahim Aldakhil, botanical expert, Qassim Area, and avoucher sample number QPP-101 was kept at the College of Pharmacy, Qassim University (Buraydah, Qassim, Saudi Arabia). The aerial parts of the plant were carefully cleaned then dried in the shade for two weeks. The dried powdered plant material (500 g) was extracted by maceration in 80% methanol (3 times × 1000 mL) at room temperature with frequent shaking. The methanolic extract was then concentrated by a vacuum rotary evaporator. The dried extract was then kept in an amber-colored vial at 4 • C till further use.

Metabolites Profiling of the Methanolic Extract of A. alopecuroides
AAE was reconstituted in HPLC grade methanol, filtered through a 0.22 µm PTFE membrane, and then separation was performed adopting Thermo Scientific C 18 column (Ac-claimTM Polar Advantage II, 3 × 150 mm, 3 µm particle size) on an UltiMate 3000 UHPLC system (Dionex). Gradient elution was performed at a flow rate of 0.4 mL/min and a column temperature of 40 • C, using H 2 O + 0.1% Formic Acid (A) and 100% Acetonitrile (B) with a 22 min total run time. The injection volume of the sample was 3 µL. The gradient started at 5% B (0-3 min), 80% B (3-10 min), 80% B (10-15 min), and 5% B (15-22 min). High-resolution mass spectrometry was carried out using a MicroTOF QIII (Bruker Dal- tonic, Bremen, Germany) using an ESI positive ionization and adjusting the following settings: capillary voltage: 4500 V; nebulizer pressure: 2.0 bar; drying gas: 8 L/min at 300 • C. The mass range was 50-1000 m/z. The accurate mass data of the molecular ions, provided by the TOF analyzer, were processed by Compass Data Analysis software (Bruker Daltonik GmbH).

Molecular Docking Study
AutoDock Vina software was used in all molecular docking experiments [13]. All dereplicated compounds were docked against the active sites of both human α-amylase and human α-glucosidase crystal structure (PDB codes: 4W93 and 3L4W, respectively) [14,15]. The binding site was determined according to the enzyme's co-crystallized ligands (Montbretin A and Miglitol, respectively). The co-ordinates of the grid boxes were (x = −9.682; y = 4.274; z = −23.145 and x = 45.424; y = 92.375; z = 34.811). The size of the grid box was set to 20 Å. Exhaustiveness was set to 24. Ten poses were generated for each docking experiment. Docking poses were analyzed and visualized using Pymol software [13]. The full method is provided in the Supplementary Materials.

In Vitro Testing of the Antidibetic Activity of A. alopecuroides (AAE)
This research represents an extension of our previous work on halophytic plants with potential antidiabetic activity. In the previous publication, in vitro testing revealed the strong inhibitory activity of the hydroalcoholic extract (AAE) against α-glucosidase and α-amylase with IC 50 values 117.9 and 90.9 µg/mL, respectively, compared to 191.4 and 53.3 µg/mL of the standard drug Acarbose [7].

Discussion
This study is a continuation of our previous research dealing with the potential antidiabetic agents from halophytes. Previously, AAE was acknowledged for potent antidiabetic activity through both in vitro and in vivo investigations. The current study adds more information about the metabolic profiling of AAE and highlights the most promising metabolites expected to be responsible for the recorded antidiabetic activity.
Another biologically important and widely prevailing chemical class is the flavonoids that were represented by five compounds from different flavonoid subclasses. Narirutin (9) is a flavanone common in the citrus family and reported for potent antidiabetic activity using in vitro and docking studies [27]. Pelargonidin 3-O-rutinoside (10), an anthocyanin with potent antidiabetic activity depicted through the inhibition of α-glucosidase and α-amylase enzymes, was isolated from strawberries [28]. Biochanin A-β-D-glucoside (12) is an isoflavone previously isolated from Trifolium pratense L. [31], while Sophora isoflavanone A (14) is a pterocarpan previously identified from Sophora tomentosa [33]. Finally, Dracorubin (23) was recognized as the major red coloring matter in the tree Dracaena draco resin [45].

Molecular docking study of A. alopecuroides metabolites for inhibition of α-amylase and α-glucosidase enzymes
In order to highlight the probably bioactive metabolites in AAE, all the dereplicated compounds were subjected to molecular docking study against both human α-amylase and α-glucosidase enzymes. All the dereplicated compounds displayed binding energies within the range of −4.5 to −9.1 Kcal/mol with the two enzymes ( Table 2). The two alkaloids Eburnamonine (2) and Isochondrodendrine (24) as well as the two flavonoids Narirutin (9) and Pelargonidin 3-O-rutinoside (10) achieved the best binding scores with α-amylase enzyme. These compounds showed various binding modes inside the enzyme active site. Narirutin and Pelargonidin 3-O-rutinoside binding poses were comparable with that of the co-crystalized inhibitor Montbretin A ( Figure 4B,C,E), where they established multiple H-bonds with TYR-151, ASP-197, HIS-201, GLU-233, HiS-299, and ASP-300. In addition, Narirutin established further hydrophobic interactions with TRP-58 and TRP-59. Narirutin was previously reported to have a potent role in diabetes management and control of its complications. This effect was confirmed via in vitro and docking studies against eight target proteins including α-amylase and α-glucosidase. In this report, Narirutin displayed hydrogen bonding interactions with both enzymes [13,15,27]. Moreover, variable flavonoids were previously tested for α-amylase and α-glucosidase inhibitory activity using in vitro testing and molecular docking approaches. The ligand-enzyme complexes for these compounds were studied, and it was concluded that the interactions occur mainly through H-bonding [54,55]. the other side, the anthocyanin compound, Pelargonidin 3-O-rutinoside, was previously reported to be a potent novel α-glucosidase inhibitor that can improve postprandial hyperglycemia [28,59]. Herein, Pelargonidin 3-O-rutinoside showed a promising dual inhibitory activity against both enzymes α-amylase and α-glucosidase, expressed as binding energy (−8.5 and −8.4 kcal/mol, respectively), which was better than that of both co-crystalized inhibitors of the two corresponding enzymes (−8.1 and −8.0 kcal, mol, respectively). The current results augmented the previous finding for α-glucosidase inhibitory activity in addition to providing further proof of the α-amylase inhibitory effect. Accordingly, this study nominated Pelargonidin 3-O-rutinoside as a potential antidiabetic agent.  On the other side, the alkaloid Eburnamonine (2) established four hydrophobic interactions only inside the enzyme's active site with TRP-58 TRP-59, TYR-62, and LEU-165 without any H-bonds ( Figure 4A). Eburnamonine is an alkaloid that was previously isolated from several Vinica species and stated to contribute to the recorded antidiabetic effect of the total extract, via increasing hepatic utilization of glucose, suppressing the gluconeogenic enzymes, and regulation of insulin secretion, glucose, and lipid metabolism [17,56,57]. One more alkaloid, the isoquinoline alkaloid Isochondrodendrine (24), showed a remarkable result where it achieved the highest docking score (−9.1 kcal/mol) among all tested compounds. It established two H-bonds with THR-163 and ASP-300 together with a single hydrophobic interaction with TRP-59 ( Figure 4D). Notably, this is the first report for the α-amylase enzyme inhibitory potential of Isochondrodendrine (24). However, other alkaloids, e.g., Topetecan and Cathine, were previously studied, and docking results concluded potent inhibitory activity against α-amylase enzyme [58].
Regarding the α-glucosidase enzyme, the three flavonoids Pelargonidin 3-O-rutinoside (10), Sophora isoflavanone A (14), and Dracorubin (23) achieved the best scores for binding affinity. They showed different binding interactions inside the enzyme's active site ( Figure 5). Pelargonidin 3-O-rutinoside and Sophora isoflavanone A established interactions highly similar to that of the co-crystalized inhibitor Miglitol, where H-bonds were the predominant, e.g., with ASP-203, ASP-327, TRP-406, ASP-443, ASN-449, ARG-526, ASP-542, and HIS-600 ( Figure 5A,B). On the other hand, Dracorubin's major interactions were hydrophobic (e.g., with TRP-406, PHE-450, and LYS-480) in addition to a single Hbond with GLN-603 ( Figure 5C). It is worth mentioning that this is the first report on the anti-enzyme activity of both compounds Sophora isoflavanone A and Dracorubin. On the other side, the anthocyanin compound, Pelargonidin 3-O-rutinoside, was previously reported to be a potent novel α-glucosidase inhibitor that can improve postprandial hyperglycemia [28,59]. Herein, Pelargonidin 3-O-rutinoside showed a promising dual inhibitory activity against both enzymes α-amylase and α-glucosidase, expressed as binding energy (−8.5 and −8.4 kcal/mol, respectively), which was better than that of both co-crystalized inhibitors of the two corresponding enzymes (−8.1 and −8.0 kcal, mol, respectively). The current results augmented the previous finding for α-glucosidase inhibitory activity in addition to providing further proof of the α-amylase inhibitory effect. Accordingly, this study nominated Pelargonidin 3-O-rutinoside as a potential antidiabetic agent. Bridging the metabolomic profiling of A. alopecuroides with its biological activity.
In a research program dedicated to investigation of the biological potential and the phytochemical content of halophytes, A. alopecuroides was acknowledged for its characteristic antidiabetic activity [7]. Hence, it was crucial to explore the phytoconstituents in this species that might be responsible for such characteristic activity. In order to achieve this

Bridging the metabolomic profiling of A. alopecuroides with its biological activity.
In a research program dedicated to investigation of the biological potential and the phytochemical content of halophytes, A. alopecuroides was acknowledged for its characteristic antidiabetic activity [7]. Hence, it was crucial to explore the phytoconstituents in this species that might be responsible for such characteristic activity. In order to achieve this goal, the LC-HRMS/MS technique was employed. The current results addressed the richness of AAE with a wide variety of secondary metabolite classes. Among them, alkaloids and flavonoids are the most characteristic. Afterwards, the molecular docking technique was used to assess the antidiabetic potential of all dereplicated compounds. Docking results indicated the probable potential of most of the annonated compounds (binding energies ranging from −5 to −9); however, the most characteristic results were recorded by alkaloid and flavonoid constituents. Some of these constituents, e.g., Eburnamonine, Narirutin, and Pelargonidin 3-O-rutinoside, were previously reported for such activity, thus giving an interpretation for the observed antidiabetic activity of the crude extract. Other metabolites, such as Isochondrodendrine (−9.1 Kcal/mol, α-amylase), Sophora isoflavanone A (−9.1 Kcal/mol, α-glucosidase), and Dracorubin (−8.3 Kcal/mol, α-amylase), were noted for the first time as potential antidiabetic compounds. This finding adds more explanation for the observed activity of the total extract. Among the remaining dereplicated compounds, Pinoresinol glucoside was previously stated as a potent antidiabetic natural entity [32]. Herein, Pinoresinol glucoside displayed good inhibitory activity against α-amylase enzyme (−7.9 Kcal/mol) and α-glucosidase (−6.0 Kcal/mol). Other compounds such as Heliettin, 1,2-Dehydroreticuline, Epinorlycoramine, N-Feruloyltyramine, and Veraguensin also displayed good activity, expressed as binding energy in the range of −7.5 to −7.9 Kcal/mol. In conclusion, AAE contains a powerful mixture of phytoconstituents that could be considered, either individually or collectively, as a probable antidiabetic agents.