Chemical Fingerprinting of Spiranthes spiralis L. Methanol Seed Extract: Spectroscopic, Chromatographic, and Computational Approaches

In this study, the morphological properties, antioxidant activities, and phytochemical content of Spiranthes spiralis L. seed methanol extracts were characterized and analyzed in silico. Microscopic analysis revealed a fusiform seed shape characterized by prominent basal cells with thick, slanted ridges and polygonal testa structures. Fourier transform infrared spectroscopy (FTIR) identified distinct absorption bands corresponding to O–H, –CH₂–, C=O, and other functional groups, indicating the presence of phenolic compounds, proteins, and polysaccharides. The total phenolic content was measured at 24.65 ± 1.43 mg GAE/g dry weight, while flavonoid and tannin contents were determined to be 43.98 ± 3.10 mg QE/g dry weight and 2.13 ± 0.13 mg GAE/g dry weight, respectively. Antioxidant activity, assessed via DPPH radical scavenging, yielded an IC₅₀ value of 0.21 mg/mL, indicating a strong antioxidant potential associated with the phenolic content. The gas chromatography–mass spectrometry (GC–MS) analysis revealed 20 bioactive compounds, with 2,2-dimethoxybutane and hydrazinecarbothioamide among the major constituents. Molecular docking indicated high binding affinities to the GPR52 receptor, with 4,4,6,6-tetramethyl-1,3-dioxane displaying the lowest binding energy (−6.3 kcal/mol). Absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions revealed mixed toxicity profiles, as hydroxyacetic acid, hydrazide, and hydrazinecarbothioamide were flagged for potential mutagenicity. Further computational analysis, including Molecular Electrostatic Potential (MEP) and non-covalent interaction (NCI) and reduced density gradient (RDG) mapping, supported the presence of well-defined electrostatic regions and weak interaction zones. Additionally, the highest occupied molecular orbital (HOMO)–lowest unoccupied molecular orbital (LUMO) and global reactivity descriptors suggested that hydrazinecarbothioamide exhibits high electrophilicity and reactivity. At the same time, 2,2-dimethoxybutane was found to be more chemically stable and less reactive. Overall, the findings emphasize the phytochemical richness and bioactive potential of S. spiralis seeds, offering promising perspectives for future pharmacological and biotechnological applications.