S. littoreus was collected from Kunthukal Beach, Pamban, Tamil Nadu, India (latitude: 9.25323; longitude: 79.21829). The collected plants were authenticated, and the prepared herbarium sheets of the plantwas submitted to the Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph's College, Tiruchirappalli, Tamil Nadu, India. (Voucher nos. J.V 001 and J.V 002).

The young leaves of S. littoreus were separated from the plant, cleaned and shade-dried. The dried S. littoreus leaves were mechanically pulverized. The powdered sample of S. littoreus was extracted with methanol and chloroform through the Soxhlet apparatus (Borosil Scientific Ltd.)for 16 h. To concentrate the collected extract, it was subjected to a rotary evaporatorand weighed. The extraction process was repeated until obtaining 5 g of powdered form of the extracts (31).

The qualitative analysis of carbohydrates, proteins, amino acids, tannins, saponins, flavonoids, alkaloids, terpenoids, steroids, phenols, glycosides, quinones, coumarins and anthraquinones was performed in six different solvent-mediated (aqueous, acetone, ethanol, methanol, chloroform and ethyl acetate) extracts of S. littoreus. All the chemicals and glassware used for the phytochemical screening were sourced from Royal Scientific Suppliers.

Carbohydrates. A total of two drops of Molisch reagent were added to 2 ml S. littoreus extract. Subsequently, 2 ml concentrated H₂SO₄ were added on the sides of the tube. The formation of purple colour was an indicator of the presence of carbohydrates (32).

Proteins. A few drops reagent were combined with 2 ml S. littoreus extract. The presence of proteinswas indicated by the production of a white precipitate, which subsequently turns brick red in colour (33).

Amino acids. In a boiling water bath, 1 ml S. littoreus extract along with 1 drop of ninhydrin solution was heated. The development of a purple colour indicated the presence of amino acids (32).

Tannins. In a test tube, 0.5 g powdered S. littoreus extract was heated in 20 ml distilled water, filtered and treated with 0.1% iron III chloride (FeCl₃). The samples were then observed for the development of brownish-green or blue-black coloration, indicating the presence of tannins (33).

Terpenoids. In a test tube, 5 ml S. Littoreusextract were used along with 2 ml CHCl₃. Subsequently, 3 ml concentrated H₂SO₄ were gently added to the mixture to form a layer. In the case that terpenoids are present, an interface with a reddish-brown colour will form (33).

Alkaloids (Mayer's test). A test tube was filled with 1 ml S. littoreus extract and 1 ml potassium mercuric iodide solution (Mayer's reagent). When this mixture was shaken gently, aprecipitate will be formed, confirming the presence of alkaloids (33).

Quinines. To 10 mg S. littoreus extract in isopropyl alcohol, one drop of concentrated sulfuric acid was added. The presence of quinones was indicated by the formation of a red colour (33).

Steroids. A total of 5 ml S. littoreus extract was mixed with 2 ml chloroform and concentrated H₂SO₄. A red colour appeared in the lower chloroform layer, indicating the presence of steroids (34).

Coumarin. A total of 2 ml S. littoreus extract was mixed with 3 ml 10% NaOH. The presence of coumarin was indicated by a yellow coloration (35).

Saponins. A total of 20 ml distilled water and 2 g powdered S. littoreus extract were heated together in a water bath and then filtered. Subsequently, 10 ml of the filtered sample were mixed with 5 ml distilled water in a test tube and vigorously shaken to form a stable, long-lasting froth. This was followed by the addition of three drops of extra virginolive oil to the froth to create an emulsion, indicating the presence of saponins (34).

Phenols. A few drops of FeCl₃ and 1 ml water were added to a test tube along with ~2 ml of the S. littoreus extract. The presence of a blue, green, red, or purple colour indicates the presence of phenols (35).

Anthraquinones (Borntrager's test). A total of 1 ml S. littoreus extract was added to a mixture of diluted ammonia, chloroform, or benzene. The presence of anthraquinone derivatives causes the ammonical layer to change colour from pink to bright red (35).

Flavonoids. S. littoreus extract was added to a test tube along with a few drops of a 1% NH₃ solution. Yellow colour concludes the presence of flavonoids (36).

Glycosides. S. littoreus extract was used along with 2 ml acetic acid and 2 ml chloroform. Once the mixture was cooledat room temperature, concentrated H₂SO₄ was added. The steroidal glycosides can be identified by their green colour appearance (37).

The presence of alkaloids, terpenoids, phenolics, flavonoids and tannins was quantitatively estimated with their respective standard using the following methods:

Quantitative estimation of alkaloids. To estimate the quantity of alkaloids in S. littoreus, the extracts were treated with 5 ml bromocresol green and PBS. Atropine was used as a standard solution. Alkaloids were measured in milligrams of atropine equivalents per gram (mg AE/g) of chloroform extract of sample (38).

Quantification of terpenoids. A total of 500 g S. littoreus leaf powder was soaked in ice-cold 95% methanol and then centrifuged at 4,000 x g for 15 min at room temperature. The supernatant was mixed with 1.5 ml chloroform and 100 ml concentrated sulfuric acid. Following incubation at room temperature for 1.5-2 h in the dark, a brown precipitate appeared at the bottom, which was dissolved in 1.5 ml 95% methanol, and its absorbance was measured in the colorimeterat 538 nm (39).

Quantification of flavonoids. A colorimetric test was used to determine the quantity of flavonoids in the S. littoreus extract. To 1 ml of the extract, 0.3 ml of 10% aluminum chloride, 5% sodium nitrite and 2 ml of NaOH were added. The absorption spectrum of this mixture was measured using UV-visible instrumentat 510 nm. Similarly, a set of quercetin solutions was prepared and measured as standards. Flavonoid concentration was expressed as mg of quercetin equivalents per gram of extract (mg QE/g) (40).

Quantification of tannins. A total of 1 ml S. littoreus extract was mixed with 0.5 ml Folin-Ciocalteu reagent and 1 ml of 35% Na₂CO₃, followed by the addition of 7.5 ml distilled water. This mixture was then incubated at 30˚C for 30 min. Similarly, a set of tannic acid solutions was prepared, and the optical density was measured at 700 nm with an UV/visible spectrophotometer. The amount of tannins in the S. littoreus extract was expressed in milligrams of tannic acid equivalent per gram (mg TAE/g) (41).

Determination of total phenolics. A total of 1 ml S. littoreus extract was dispersed in 9 ml of distilled water along with 1 ml of Folin-Ciocalteu and 10 ml of 7% Na₂CO₃ solution. This was incubated at 30˚C for 2 h at room temperature. Gallic acid was used as standard. Gallic acid equivalents (mg GAE/g) were used to represent mg of total phenolic component per gram of extract (42).

The phytoconstituents present in the methanolic ethanol, ethyl acetate and chloroform extracts were screened using GC-MS analysis. The experimental condition of the instrument was a fused silica column filled with Elite-5MS (5% biphenyl, 95% dimethylpolysiloxane, 30 m, 0.25 mm ID, 250 m df) utilized in the analysis by the Clarus 680 GC, and the Helium carrier gas was used to separate the components at a constant flow rate of 1 ml/min. The injector temperature was set to 260˚C. The operating parameters of the mass detector were 240˚C for the transfer line, 240˚C for the ion source, 70 eV for the electron impact in the ionization mode, 0.2 sec for each scan, and 0.1 sec between scans. The component spectra were compared to a database of component spectrums maintained in the GC-MS NIST (2008) library (43,44). No standard compounds were used for the spectral comparison.

The physicochemical, pharmacokinetic and drug-likeliness properties of the compound SL-MHDC were predicted using the SwissADME server (http://www.swissadme.ch/) to optimize the compound based on its bioavailability, safety and efficacy (45,46).

Based on the GC-MS results of S. littoreus, the chemical structure of the phytocompound SL-MHDCwas retrieved from the PubChem database (PubChem CID: 550196) as an optimized 3D ligand molecule. The downloaded ligand was prepared using the Autodock MGL application (https://autodock.scripps.edu/) by detecting its torsion root and saved in pdbqt format. This was then used for docking with AutodockVina (47,48).

Target protein preparation. The target protein selected for the study was the structure of SARS-CoV-2 XBB.1. The structure was downloaded from the protein data bank (PDB ID: 8IOV) in PDB format. The chain B spike glycoprotein (RBD) of XBB.1 was used as a target receptor for the docking analyses. The protein preparation was performed by removing the water molecules and the heteroatom within the structure. The structure was then examined for the missing residues and it was saved in pdbqt format. Both the optimized protein and ligand were then subjected to docking analysis using Autodock 4.2.6 software (49) to identify the affinity of the GC-MS-derived compound towards the SARS-CoV-2 XBB.1. The optimal pose with the least energy of binding was used for further analyses.

Molecular dynamics simulations (MDS). MDS is a computational paradigm that enables atomistic interrogation of biomolecular systems, which serves as a cornerstone in pharmaco-reconnaissance. Thus, MDS were executed using Desmond module (Schrödinger Suite) (50) (https://www.schrodinger.com/platform/products/desmond/). The SARS-CoV-2 XBB.1 spike glycoprotein complexed with SL-MHDC was solvated via system builder by employing TIP4P aqueous solvation within an orthorhombic solvent box. Moreover, the cell size was 49.6x68.1x51.7 Å, as well as the solvent buffer extending 10 Å, which is beyond the protein in all Cartesian directions. As a corollary, OPLS_2005 force field was deployed to parameterize molecular energetics. Judiciously, electrostatic neutrality was achieved by the incorporation of counter ions (Na⁺ and Cl^-) that was followed by constrained energy minimization with the exorbitant of 2,500 iterations, as well as the convergence thresholds of 1 kcal/mol/Å. Furthermore, the present study exploited NPT ensemble with the Nose-Hoover thermostat algorithm which is lauded for thermal fluctuations set at a reference temperature of 300 K. A >30 nsec trajectory was generated and the post-simulation analyses encompassed with Root-mean-square deviation (RMSD) and the interrogation of SARS-CoV-2 XBB.1 integrated with SL-MHDC complex persistent was designated against the binding locus.

The preliminary phytochemical screening of S. littoreus was carried out to identify the presence of secondary metabolites, such as alkaloids, flavonoids, tannins, saponins, glycosides, quinones, coumarins, steroids, phenols, terpenoids and anthraquinones using six different solvents (aqueous, acetone, ethanol, methanol, chloroform and ethyl acetate). Among these, the chloroform extract revealed sevensecondary metabolites and one primary metabolitein S. littoreus followed by the acetone, methanol and ethanol extracts, which revealed sixsecondary metabolites and one primary metabolite. The ethyl acetate extract only revealed four compounds (Table I).

The total amount of terpenoids, phenols, tannins, alkaloids and flavonoids present in the S. littoreus extract was estimated using quantitative analysis with their respective standards. The findings indicated the presence of all five examined compounds in the S. littoreus plant, albeit in varying concentrations: Phenol (7.5 mg/g), terpenoids (5.05 mg/g), flavonoids (0.4 mg/g), tannin (0.2 mg/g) and alkaloids (0.19 mg/g). The concentrations of phenol, terpenoids, flavonoids, tannin and alkaloids in the S. Littoreus leaflet extract are illustrated in Fig. 1. Among these compounds, phenol and terpenoids accumulated at higher levels in the S. Littoreus leaflet. Additionally, the leaflet extract of S. Littoreus also contained trace levels of alkaloids, tannins and flavonoids.

Following the preliminary screening, GC-MS analysis of the ethanol, methanol, chloroform and ethyl acetate extracts of S. Littoreus leaves was performed; this revealed the presence of various active phytocompounds. A total of 33 different compounds were eluted from this plant. Among these 33 compounds, 1-methylene-2β-hydroxymethyl-3,3-dimethyl-4β-(3-methylbut-2-enyl)-cyclohexane (28.834%), hexadecane (19.962%), tetratetracontane (18.416%), octacosane (16.136%), octadecanal (15.833%), heptacosane (14.574%), 2-methyl-3-(3-methyl-but-2-enyl)-2-(4-methyl-pent-3-enyl)-oxetane (14.434%) had the high percentage area. The names, molecular formulas and area percentages of the screened phytoconstituents are presented in Table II, and the biological activities with the 2D structures of the compounds are presented in Table SI. The chromatogram images of the tested samples are illustrated in Fig. 2, Fig. 3, Fig. 4 and Fig. 5.

The compound selected from the GC-MS analysis was subjected to in silico drug-likeliness and pharmacokinetic prediction to investigate its absorption, distribution, metabolism and toxicity profiles, and to evaluate whether there are any violations in Lipinski's rule of 5. According to Lipinski's rule of 5, the molecular weight should be <500 g/mol, not >10 g/mol, with >5 hydrogen bond acceptors and donors, and a log P-value >5. It is important to consider any bioactive compounds as a lead molecule in the process of drug discovery. From the results of ADME analysis, it was identified that there were 0 violations in Lipinski's' rule of 5, the molecular weight of the compound was observed to be 222.37 g/mol, there was 1 hydrogen bond donor and acceptor, the MLogP of the compound was 3.56 (Table III). Additionally, the pharmacokinetic properties of the compounds were evaluated, including the calculation of inhibition, which includes the subfamilies of cytochrome P450, such as CYP2C19, CYP2C9, CYP1A2, CYP2D6 and CYP3A4, and BBB permeant. All these results, including the drug likeliness of the compounds are presented in Tables IV and V. This suggests that the compound has drug-like properties, as it meets the Lipinski's and Ghose's rules, which evaluate the properties for drug-likeliness, indicating that it falls within the range of properties commonly found in known drugs. The compound meets Veber's criteria suggesting its good oral availability potential. In addition, the compound meets the Egan rule, indicating it is less likely to be a P-glycoprotein substrate.

To evaluate the activity of the compound SL-MHDC against the SARS-CoV-2 XBB.1 spike RBD (PDB ID: 8IOV), both the target protein and the selected ligand were subjected to docking analyses. The computational docking was performed using the AutoDock tool, which generated binding modes in the lowest energy state. The binding energy of SL-MHDC with the XBB.1 was observed to be -4.86 kcal/mol. This interaction formed two conventional hydrogen bonds with the amino acid residues LYS444 (B) and ASN 448 (B) (Fig. 6) in the spike glycoprotein. The bond lengths were measured as 3.08 and 2.58 Å, respectively (Table VI). The other non-bonded interactions include the Vander der Waals interaction formed by the residues, such as LEU 441 (B), ASP 442 (B), ARG 509 (B), THR 345 (B), PHE 347 (B), THR 346 (B), ASN 450 (B), the Pi-sigma and Pi-Alkyl interaction was formed by TYR 451 (B) (Fig. 7). Hence, from the molecular docking it could be inferred that the compound SL-MHDC S. littoreus can be used as a putative inhibitor potent drug against the spike glycoprotein of XBB.1.

In order to assess conformational stability and interaction fidelity of the SARS-CoV-2 XBB.1 spike glycoprotein complexed with the phyto-ligand SL-MHDC, explicit MDS were effectuated for a 30 nsec. The simulation encompassed with the canonical analyses including RMSD profiling and protein-ligand contact mapping (Fig. 8).