The dried roots of S. oblonga were collected in Mysore District, Karnataka. Prof. G. R. Shivamurthy, Chairman of Botany, Department of Botany, University of Mysore, Mysuru, authenticated the plant specimen. The collected root material was gently rinsed with distilled water and 10 gm was then weighed out and placed in a Soxhlet extractor for 16 h of consecutive solvent extraction. The obtained aqueous root extract of S. oblonga (SOR) was filtered using 0.2M filters (Evans and Trease, 1989). The qualitative phytochemical screening of SOR was performed using standard procedures (Harborne, 1984).

Using green synthesis methods, AgNPs were successfully synthesized using SOR as a capping and reducing agent and 100 mL of silver nitrate (AgNO₃) as a precursor (Adeleke et al., 2017). To synthesize AgNPs, a volumetric flask was filled with 2 mM 100 mL AgNO₃ and 30 mL of SOR. The AgNPs were synthesized in a magnetic stirrer at 600 rpm at 80°C for 3–4 h. A color change from colorless to brown indicated the synthesis of silver nanoparticles. The UV–Visible spectra of the reaction solution verified the reduction of Ag + ions, confirming the synthesis of silver nanoparticles. Finally, silver nanoparticles were separated from the mixture by centrifugation at 5,000 rpm for 15 min and were washed with methanol, freeze-dried with a Lyophilizer, and kept at 4°C for further use (Senguttuvan et al., 2014).

Understanding and controlling NP manufacturing and application requires the characterization of silver nanoparticles (Abou El-Nour et al., 2012). The synthesis of silver nanoparticles was affirmed by sampling the reaction mix at frequent intervals and scanning the absorption maximum with UV-Vis spectra between 350 and 700 nm using a HITACHI U-2900 Double beam spectrometer. The functional biomolecules in S. oblonga that were responsible for the metal reduction and nanoparticle stabilization were classified using FTIR RX1-Perkin Elmer in the 4,000–400 cm⁻¹ wavelength range. XRD was used for the determination of the crystalline nature of the AgNPs, and patterns of the AgNPs were estimated using the Smart Lab 3Kw. A DLS particle size analyzer was used to estimate the size distribution and average size of AgNPs. Energy dispersive X-RAY analysis was utilized to estimate the elemental makeup. SEM was used for examining the structure of AgNPs (HITACHI S-3400N).

The antibacterial activity of the SOR-AgNPs was determined by using the agar disc diffusion method (Saratale et al., 2017). The antimicrobial property of the SOR-AgNPs was determined against Bacillus subtilis, Escherichia coli, Bacillus cereus, Salmonella typhi, and Staphylococcus aureus. Inoculum containing around 150 × 10⁴ CFU/mL was swabbed uniformly onto the nutrient agar (NA) coated plates and left to incubate in a sterilized atmosphere. A 6 mm sterilized disc was loaded with various dilutions of SOR-AgNPs (1, 5, 10, and 15 μg/mL) solutions, while another disc emersed in 1 μg/mL of ampicillin was considered a positive control. To determine the zone of inhibition, the Petri dishes were incubated at 36°C for 2 days. The average was derived by repeating the experiments three times.

The mixture of α -amylase comprised of 0.5 mL of sodium phosphate buffer (0.02M, pH 6.9) with α-amylase (1U/mL) and differing concentrations of SOR-AgNPs (20–100 μg/mL) that had been pre-incubated at 37°C for 20 min. After incubation, 2.5 mL of 1% starch in phosphate buffer was supplemented and incubated at 37°C for 15 min. This reaction was halted by adding 1 mL of dinitro salicylic acid reagent and heated for 10 min then cooled, and the absorbance was determined at 540 nm (Chowdhury et al., 2014). The activity was calculated with a 0.01 increase in absorbance per mL/min.

The method developed by Chowdhury et al. (Macovei et al., 2021) was used to cap the protein, with minor modifications. The protein(s) attached to the surface of SOR-AgNPs were isolated by washing them with sterile distilled water and boiling them for 10 min with 1% SDS (sodium dodecyl sulfate). Electrophoresis was carried out in 12% SDS-PAGE at a constant voltage of 70 V for 120 min. Coomassie Brilliant blue dye was used to stain the gel for observation.

The outcomes were calculated as mean ± SD of three trials (p < 0.01). The DPPH assay’s IC₅₀ values were calculated and statistically analyzed.

The phytochemical screening was done qualitatively using SOR, which contained lead phytoconstituents, such as alkaloids, flavonoids, saponins, phenols, tannin, phytosterols, and vitamin C, proteins, amino acids, and terpenoids. These phytoconstituents are important to lead molecules involved in nanoparticle production. When the findings were compared to earlier studies, it was discovered that Phyto-functionalized silver nanoparticles contained more phytoconstituents from conifer bark extract (Melkamu and Bitew, 2021).

UV-Visible spectroscopy was used in this investigation to monitor the process of silver ion bioreduction to AgNPs. It was used to monitor the creation and optimization of AgNPs by monitoring absorbance in the 300–700 nm scanning range. The development of a distinctive Surface Plasmon Resonance (SPR) peak of 448 nm confirms the presence of AgNPs (Sharifi-Rad et al., 2021). Owing to the excitation of electrons and variations in electronic energy levels, the synthesized SOR-AgNPs will have a dark-brown appearance in an aqueous solution, conforming to the formation of the nanoparticles (Jyoti et al., 2016).

SEM analysis of the morphology (viz size and shape) of SOR-AgNPs showed multiform nanoparticles having a spherical, crystal, and rode-like appearance. As depicted in Figure 1, the shapes had an average size range of between 59 and 78 nm. Even within the aggregates, the nanoparticles were not in direct contact, indicating that the nanoparticles were stabilized by a capping agent. According to the previous report, SEM demonstrated the spherical nature of silver metal particles generated in Urtica dioica Linn leaf extract (Blois, 1958).

1st lane, molecular size marker; 2nd lane, NP (10 g/mL); 3rd lane, NP (20 g/mL); 4th lane, NP (30 g/mL); and 5th lane, NP (40 g/mL) were demonstrated in the SDS-PAGE study of capping protein surrounding AgNPs generated from the aqueous root extract of S. oblonga (Figure 2). In the aqueous root extract of S. oblonga, there is a strong protein band (30 kDa) that is primarily involved in protein capping and offers stability for the bio-reduction of AgNO₃ to AgNPs.

The influence of stirring time on the bioproduction of AgNPs is depicted in the diagram (Figure 4). With increasing stirring time, the color intensity and monodispersity increased. The emergence of an SPR peak in the 400–450 nm wavelength range correlates to AgNPs, which absorb heavily at the 426 nm wavelength (Figure 3). When compared to previous reports, the stability of AgNPs rises after 72 h of stirring time.

The existence of protein stabilizing molecules was shown by FT-IR analysis, which revealed a peak range of 500–4,000 cm⁻¹, all of which corresponded to distinct functional groups. The results of the FTIR study demonstrate that various functional groups have sharp absorption peaks at 2327, 2352, 2117, 1999, 1600, 1312, and 1183 cm⁻¹ (Figure 4). The different absorption peaks, such as those at 1000–1300 cm⁻¹, which could be aldehyde or ketone; 1999 cm⁻¹, which could be aromatics; and 2300 cm⁻¹, which could be the amide bond of proteins, are caused by carbonyl stretching in proteins and by the interaction of SOR-AgNPs through green synthesis, and the secondary structure was unaffected during the reaction with silver ions or post binding with AgNPs. The carbonyl group has a strong silver binding ability, indicating the formation of SOR-AgNP covering layers and acting as a capping mediator to prevent agglomeration and give the medium strength, according to this study. These findings support the existence of proteins that act as reducing and stabilizing agents (Anandalakshmi et al., 2016).

EDX determines the quantitative and qualitative properties of the elements involved in AgNP production. The peak in the silver region at 3 KeV (80 in mass) is usually for the absorption of metal silver nanocrystalline owing to SPR, as can be seen in Figure 5. Additional signals from atoms N, O, C, and Cl were documented. Similar results were confirmed in the elemental profile of synthesized NPs using banana peel extract, showing greater counts at 3 keV because of silver and affirming the development of AgNPs (Ibrahim, 2015).

The XRD pattern of the biosynthesized AgNPs is depicted in Figure 6. The (4.202), (2.799), (2.371), and (1.955) reflections of metallic silver were revealed by the four diffraction peaks at 28.00˚, 32.23˚, 38.25˚, and 46.48˚. A strong and crisp diffraction peak positioned at 32.23˚ was observed, which can be linked to the (2.799). The strong peaks clearly show that the SOR-AgNPs produced have a face-centered cubic (FCC) shape. The bioorganic phase crystallizes on the silver nanoparticles’ surfaces, according to other peaks. The results were compared to the synthesis of AgNPs from the seed extract of Tectona grandis and an XRD pattern of AgNPs, both of which corroborated the crystalline nature of AgNPs. The four different diffraction peaks at four values of 38.05, 44.23, 64.41, and 76.66 may be indexed to the face-centered cubical structure of silver (111), (200), (220), and (311) reflection planes (Rautela et al., 2019).

The diameter of SOR-AgNPs is 181.5 nm, according to the size distribution histogram of DLS (Figure 7). The sample’s Zeta potential analysis revealed that the positive polarity of the particles favors drug targeting (Clogston and Patri, 2011).

Antioxidants are compounds that fight free radicals. Antioxidants are natural or manmade chemicals that can prevent or postpone oxidant-induced (ROS, RNS, free radicals, and other unstable molecules) cell damage (Azeez et al., 2017). ROS is involved in the etiology of several degenerative disorders, along with cardiovascular disease and cancer. In this study, the radical-scavenging effects were investigated using DPPH with a distinctive absorption at 517 nm. When compared to the traditional BHT scavenging assay, the DPPH scavenging assay showed efficient SOR-AgNPs inhibition (butylated hydroxytoluene). The antioxidant activity (DPPH method) reveals that SOR-AgNPs had an IC₅₀ value of (80.64 g/mL), whereas conventional BHT is 60 g/mL, as shown in (Figure 8A). According to a previous study, green synthesis of silver nanoparticles using Prosopis farcta fruit extract exhibited potential antioxidant activity in comparison with ascorbic acid, which was considered the standard (Salari et al., 2019). For Nitric oxide radical scavenging activity (Figure 8B), the SOR-AgNP activity was significant (96.58 g/mL) when compared to standard BHT (Sudha et al., 2017). SOR-AgNPs extract has significant reducing power activity, and the increasing SOR-AgNP concentration continuously increased reducing power activity. SOR-AgNPs (81.09 g/mL) and normal BHT (54.71 g/mL) exhibit nearly identical reducing power activity, as shown in (Figure 8C). Similarly, in the hydroxyl scavenging assay, scavenging potential increased with an increase in the concentration of BHT and SOR-AgNPs (Figure 8D). The presence of phytoconstituents in the leaf extract resulted in the activity of reducing power (Bhakya et al., 2016).

The carbohydrate-hydrolyzing enzyme α-amylase was substantially inhibited by SOR-AgNPs. The level of enzymatic activity was dramatically reduced as the concentration of SOR-AgNPs increased (Figure 9A). The EC₅₀ values for SOR-AgNP inhibition of α-amylase were 58.38 g/mL, and previous research has described the green synthesis of AgNO₃ utilizing the leaf extract of Calophyllum tomentosum with antidiabetic action (Govindappa et al., 2018).

The antibacterial efficacy of SOR-AgNPs is powerful against bacterial species. AgNPs showed antibacterial activity in different degrees, as indicated by the inhibition zone diameter, but SOR-AgNPs had much stronger antibacterial activity than AgNPs (Figures 9B, C). When compared to previous research, the results show that aqueous callus extracts of Fagonia indica silver nanoparticles have a stronger antibacterial effect (Adil et al., 2019).