Phytochemical Analysis, Isolation, and Characterization of Gentiopicroside from Gentiana kurroo and Cytotoxicity of Biosynthesized Silver Nanoparticles Against HeLa Cells

Gentiana kurroo Royle, a critically endangered Himalayan herb, is valued in treating leucoderma, syphilis, bronchial asthma, hepatitis, etc. The current investigation performed quantitative and qualitative phytochemical analysis of G. kurroo root extracts prepared in chloroform, methanol, and ethyl acetate. The phenolic and flavonoid contents were the highest in methanol and chloroform extract, respectively. Several pharmacologically important compounds were identified through gas chromatography-mass spectrometry. Antioxidant analysis revealed methanolic extract to be the most efficient scavenger of 2,2-diphenyl-1-picrylhydrazyl (IC₅₀ = 114 µg mL^-1), hydrogen peroxide (IC₅₀ = 109.9 µg mL^-1), and superoxide (IC₅₀ = 74.63 µg mL^-1) radicals. Gentiopicroside was isolated from the methanolic root extract through silica-gel column-chromatography, and the characterization of concentrated fractions was achieved employing various analytical techniques. Pertaining to silver nanoparticle (GkAgNPs) synthesis, different physicochemical parameters were optimized and it was observed that root extract treated with silver-nitrate (0.5 mM) at 60 °C and incubated in dark for at least 120 min after initial color change, yielded GkAgNPs optimally. GkAgNPs were anisotropic and polydisperse and exhibited characteristic surface plasmon resonance (424 nm), crystalline face-centered cubic geometry, size (50-300 nm), and zeta-potential (- 16.3 mV). FT-IR spectra indicated the involvement of phenols and flavonoids in AgNPs synthesis. GkAgNPs were evidenced as strongly cytotoxic (IC₅₀ = 1.964 µg mL^-1) against HeLa cells and also showed deformed cellular morphology, a significant reduction in viable cell counts and colony-forming efficiency (4.08%). The findings suggest potential applications in drug development for treating serious human diseases. To the best of our knowledge, this study represents the first report on the isolation of gentiopicroside, the bio-fabrication of GkAgNPs using G.kurroo root extract, and their strong bioefficacy against HeLa cells.