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

Abstract

In this study, the morphological properties, antioxidant activities, and phytochemical content of <i>Spiranthes spiralis</i> 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 <i>S. spiralis</i> seeds, offering promising perspectives for future pharmacological and biotechnological applications.