Isoorientin 2''-O-rhamnoside from Crotalaria verrucosa: Deep Eutectic Solvent Based Extraction, Silver-Nanoconjugates Functionalization and in Vivo anti-biofilm Effects against MRSA

Crotalaria verrucosa known as Brihatapushpi/Shanapushpi in Ayurveda, is a treasure trove of phytochemicals like phenolics, saponins and alkaloids, and these bioactives are responsible for its diverse therapeutic properties. This study embarks on developing a sustainable method to isolate a natural flavonoid, harboring a rare C-glycoside moiety - Isoorientin 2’’-O-rhamnoside (IO), from C. verrucosa and aims to tap the antimicrobial potency of IO, by conjugating IO with zero-valent silver nanoparticles. Extracts of C. verrucosa were prepared using ethanol-water solvents. Deep eutectic solvents (DES) were synthesized having Bicine as hydrogen bond donor (HBD) and Choline chloride, acetyl choline chloride, CTAB, TBAB and TBAC as hydrogen bond acceptors (HBA) to extract the phytochemicals. Bicine – Choline chloride DES gratifyingly precipitated a single flavonoid C-glycoside (IO). Box-Behnken (BBD) based response surface methodology optimization was executed to enhance the yield of IO from C. verrucosa extract. High yield of IO was obtained at the optimized conditions of Bicine [HBD, 2.023 mM], Choline chloride [HBA, 2.082 mM] and Ultrasonication [46.3 min]. Further, IO was used to biogenically synthesize silver nanoparticle conjugates (IOAgNPs), and high yield of IOAgNPs were obtained with optimum BBD conditions as silver nitrate 3.658 (mM), IO 3.019 (mM), with 48.7 min of sunlight exposure. IOAgNPs exhibited effective antibiofilm activity against drug-resistant pathogen MRSA. IO@AgNPs demonstrated 1.9-fold reduction in an in-vivo zebrafish infection study and no variations were observed in liver structure when treated with IO@AgNPs, making them promising for pharmaceutical and biomedical applications.