“Chemical Analysis and Biological Activities of Crude Extracts and Green Synthesized Silver Nanoparticles of Medicinal Plants from Mustang and Kaski Districts of Nepal

Abstract

Medicinal plants contain numerous secondary metabolites with significant biological activities. Due to diverse geographical and climatic conditions, several indigenous plants that comprise unique phytochemicals having a wide spectrum of biological assets are found in Nepal. This study aims to synthesize, characterize, and evaluate the biological activities of silver nanoparticles (AgNPs) by using some of the active medicinal plants of the study area. Methanol extracts of the selected plants were evaluated for antioxidant, antibacterial and α-amylase inhibition activities by using the DPPH radical scavenging, agar well diffusion, and CNPG3 methods respectively. The chemical profiling of essential oil isolated from the aerial parts of Ephedra pachyclada and Ayenia grandifolia was performed by the GC-MS analysis. This study exposed the phytochemical and biological activities of methanol extract, chemical profiling of essential oil, and green synthesis of AgNPs by using an aqueous extract of A. grandifolia for the first time. Among the 22 plants evaluated, Rubus ellipticus, E. pachyclada, Pyrus pashia, Drynaria coronans, Mimosa rubicaulis, and Ziziphus mauritiana extracts exhibited significant antioxidant properties with the highest activity of A. grandifolia (IC50 = 12.87 ± 0.14 µg/mL). The GC-MS analysis of the essential oil (EO) of stem barks of A. grandifolia contained di-n- octyl phthalate (28.39%), 2,6,11 trimethyl dodecane (15.77%), 4,6 dimethyl dodecane (12.79%), and o-guaiacol (7.07%). The methanol extracts of R. ellipticus and P. pashia exhibited the highest antibacterial activity. The resazurin microtiter assay method revealed the MIC and MBC of the methanol root extract of R. ellipticus as 3.12 and 12.5 mg/mL respectively against Staphylococcus aureus ATCC 25923. The methanol stem bark extract of P. pashia exhibited the highest α-amylase inhibition activity with an IC50 value of 24.22 ± 0.10 µg/mL. From the preliminary investigation, A. grandifolia, R. ellipticus, P. pashia, and Z. mauritiana which exhibited the highest biological activities were used for the fabrication of AgNPs. Each of the plant extracts and AgNO3 (1 mM) in the ratio of 1:9 by volume were mixed with constant stirring at lab temperature (25 ± 2ºC), and neutral pH with constant stirring over a magnetic stirrer. The change of color into light brown within an hour was considered a visual indication of the growth of AgNPs which was further confirmed by the appearance of sharp SPR peaks in the UV-visible spectra. The UV-visible spectra at different reaction conditions of temperature, pH, and concentration were used to optimize the fabrication of AgNPs. FTIR spectra of the extract and the AgNPs were examined to detect the functional groups responsible for the reduction, capping, and stabilizing of AgNPs. The face-centered crystalline nature of the silver nanoparticles was established by the X-ray diffraction patterns by matching the diffractogram with the Joint Committee on Powder Diffraction Standards (JCPDS file no: 03-0921). It was further confirmed by the selected area electron diffraction (SAED) pattern having four discrete rings corresponding to the crystal planes at 110, 200, 220, and 311. The energy dispersive X-ray (EDX) analysis showed the presence of silver in the highest proportions and trace quantities of oxygen, chlorine, calcium, and carbon in the AgNPs. The surface morphology and nearly spherical shapes were determined by field emission scanning electron microscopy (FESEM) images. Transmission electron microscopy (TEM) was used to confirm the topographical, compositional, and morphological status of the AgNPs. Further, TEM images were used to find the sizes of the synthesized AgNPs which ranged from 28.05 ± 11.8 nm of A. grandifolia extract-mediated AgNPs to 16.73 ± 4.94 nm of Z. mauritiana extract-mediated AgNPs. The antioxidant activities of AgNPs synthesized by using Z. mauritiana (with an IC50 value of 37.02 ± 1.0 µg/mL) and A. grandifolia (with an IC50 value of 142.77 ± 10.75 µg/mL) were found to be nearly twice as potent as their respective crude extracts. The AgNPs synthesized using A. grandifolia demonstrated notable antibacterial activity, whereas its crude extract showed no such activity. The process of transforming plant material into AgNPs not only enhanced their antioxidant and antibacterial properties but also indicated the potential biomedical applications of these plant-based nanoparticles. Further investigation involving the synthesis of controlled-sized AgNPs from other plants, toxicity testing, and exploring potential applications would greatly benefit humanity.