BIBECHANA Vol. 22, No. 3, December 2025, 248-257 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Eco-friendly synthesis of silver nanoparticles using Withania somnifera root extract: characterization, phytochemical profiling, and evaluation of antimicrobial activity Ratna Bahadur Thapa1, Shankar Datt Ojha2, Sujan Dhungana2, Laxmi Tiwari2, Devendra Khadka3,4, Milan Babu Poudel5, Megh Raj Pokhrel4, Janaki Baral2, Bhoj Raj Poudel2* 1Department of Chemistry, Damak Multiple Campus,T.U., Damak Nepal 2Department of Chemistry, Tri Chandra Multiple Campus, T.U., Kathmandu, Nepal 3Department of Chemistry, Tribhuvan Multiple Campus, T.U., Tansen, Palpa, Nepal 4Central Department of Chemistry, T.U,Kirtipur, Kathmandu, Nepal 5Department of Convergence Technology Engineering, Jeonbuk National University, Jeonju, jeollabuk-do 54896,Republic of Korea ∗Corresponding author. Email: bhoj.poudel@trc.tu.edu.np Abstract Silver nanoparticles (Ag NPs) were synthesized using a green chemistry approach, utilizing Withania somnifera (Ashwagandha) root extract, whose phytochemicals acted as eco-friendly, non-toxic reducing agents. The synthesized AgNPs were characterized using X-ray diffraction (XRD), UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). XRD analy- sis confirmed the crystal structure of the Ag NPs and estimated their size to be 10 nm. UV-visible spectroscopy showed a characteristic absorption peak, confirming the formation of AgNPs. FTIR analysis identified functional groups associated with the phytochemicals in the root extract, which may contribute to nanoparticle stabilization. SEM images revealed the spherical morphology of the Ag NPs, while EDS analysis confirmed the presence of sil- ver in the synthesized nanoparticles. The phytochemical examination of Ashwagandha found secondary metabolites, including phenolic, alkaloid, and flavonoid compounds. The synthe- sized Ag NPs were assessed for their antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans, demonstrating significant efficacy markedly against Gram-positive bacteria. This finding highlights the role of Ashwagandha-mediated Ag NPs in sustainable NP formation and antimicrobial applications. Keywords Antimicrobial activity, phytochemical analysis, root extract, Ag NPs, Withania somnifera. Article information Manuscript received: March 9, 2025; Revised: August 5, 2025; Accepted: August 8, 2025 DOI https://doi.org/10.3126/bibechana.v22i3.76471 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 248 http://nepjol.info/index.php/BIBECHANA bhoj.poudel@trc.tu.edu.np https://doi.org/10.3126/bibechana.v22i3.76471 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Ratna Bahadur Thapa et al./ BIBECHANA 22 (2025) 248-257 249 1 Introduction Nanoscience involves the investigation and modi- fication of materials on a nanoscale, generally de- fined as being between 1 and 100 nanometers (nm). Nanoscience has rapidly evolved and gained con- siderable attention in research with industry world- wide [1]. Nanoparticles (NPs) exhibit distinct elec- trical, optical, chemical, and catalytic character- istics that set them apart from larger bulk sub- stances, making them highly important for many applications [2,3]. Bio-nanotechnology is the back- bone of nanotechnology; it combines biotechnol- ogy and nanotechnology to develop eco-friendly and biosynthetic methods for synthesizing nanomateri- als [4]. Metallic NPs, including AgNPs, gold, tita- nium, copper, and zinc, are widely studied for their superior performance in various applications. Ag- NPs are widely recognized for their potent antimi- crobial activity, which makes them highly valuable across many fields, including medicine, food science, agriculture, and environmental remediation. Their effectiveness stems from their ability to inhibit mi- crobial growth, contributing to advancements in nu- merous applications [5, 6]. The process of synthesizing AgNPs has ad- vanced over time, with earlier chemical reduction techniques frequently using harmful chemicals that present health and environmental hazards [7]. In response, green or eco-friendly synthesis methods have gained attention, utilizing natural materials such as plant extracts, microbes, and biopolymers to create NPs without harmful chemicals, mak- ing the process more secure and environmentally friendly [8]. AgNPs exhibit multifunctional proper- ties that have led to their widespread applications in medicine, catalysis, and environmental remedia- tion [9,10]. Compared to metals like gold and plat- inum, silver offers cost-effective and scalable syn- thesis, particularly when biosynthetic routes are adopted. Furthermore, biosynthetic methods us- ing plant-derived compounds such as polyphenols and proteins as reducing agents enhance the en- vironmental friendliness and cost-efficiency of Ag NPs formation [11]. However, challenges related to scalability, cost, and labour intensity remain in NP synthesis. Withania somnifera is widely recognized as Ash- wagandha (Figure 1). It contributes to Ayurvedic medicine and is renowned for its numerous posi- tive health effects, including anti-swelling, oxida- tive stress-reducing, adaptogenic, and antimicro- bial [12]. With its ability to thrive in arid re- gions and poor soils, Ashwagandha is widely cul- tivated in India, Nepal, the Middle East, and parts of Africa [13]. The utilization of plentiful natural resources, including botanical extracts, further em- phasizes the potential of green synthesis techniques. The plant is rich in various bioactive entities that enhance its therapeutic benefits [14]. Among many plant-based sources, Ashwagandha has emerged as an intriguing candidate for the synthesis of AgNPs. Figure 1: Arial part and root of Withania somnifera (Ashwagandha) plant. The eco-friendly Ag NPs synthesis using With- ania somnifera root extract presents a sustainable alternative to conventional methods, harnessing the plant’s natural phytochemicals for both reduction and stabilization. This method supports environ- mental sustainability and can elevate the antimi- crobial properties of AgNPs by leveraging the syn- ergistic interaction with bioactive compounds de- rived from plants [15]. This finding explores the synthesis of AgNPs using Withania somnifera root extract. Research highlighted the characterization, phytochemical profile, and antimicrobial properties of the NPs. Various analytical techniques, includ- ing XRD, SEM, and UV-visible spectroscopy, will be utilized to assess the size, morphology, and Ag NPs' stability. The phytochemical composition of Withania somnifera compounds will be examined to lead to the formation of NPs. Additionally, Ashwagandha-based AgNPs of antimicrobial activ- ity will be assessed against bacterial, fungal, and viral pathogens, aiming to evaluate their potential applications in medical and biotechnological fields. The findings of this research will further promote the use of plant-based materials in NP synthesis, offering novel solutions for creating safe, efficient, and sustainable nanomaterials. 2 Materials and Methods 2.1 Materials In the laboratory experiments, distilled water and analytical-grade chemicals were utilized. Silver nitrate (AgNO3), and sodium hydroxide (NaOH) were obtained from Thermo Fisher Scientific India Pvt. Ltd., India. 2.2 Collection and preparation of Ashwa- gandha root extract The roots of Ashwagandha plant were collected from their natural habitat in Tapoban forest of Ratna Bahadur Thapa et al./ BIBECHANA 22 (2025) 248-257 250 Marma Rural Municipality-4, Darchula district, Sudurpashchim Province, Nepal (29.738° N, 80.821° E) between March and April 2024. The plant was harvested during the cooler morning hours. The roots were carefully washed with clean water, then sliced into smaller segments and allowed to dry in a shaded area for two weeks. After the drying pe- riod, the roots were processed into a fine powder using a mechanical grinder. For the extraction, 10 grams of the powdered Ashwagandha were soaked with 100 mL distilled water in a beaker. The solu- tion was heated to 60° C and stirred for 30 minutes with a magnetic stirrer. Afterwards, the mixture was allowed to cool to a suitable temperature, fil- tered using Whatman filter paper, and stored at 4°C for later use. 2.3 Phytochemical screening The hydro-methanolic root extract, prepared by the cold percolation of 30 g of powder, yielded 12 g of crude methanolic extract, which was subsequently used for the qualitative screening of phytochemi- cals. The analysis followed established protocols incorporating slight modifications based on insights from earlier studies [16–18]. 2.4 Synthesis of AgNPs AgNPs were synthesized using a green method un- der optimized conditions. A 0.001 M AgNO solu- tion was prepared by dissolving 0.17 grams of AgNO in 1000 mL of distilled water. In a typical reaction, 50 mL of this solution was mixed with 20 mL of freshly prepared Ashwagandha root extract, added dropwise under continuous magnetic stirring at 600 rpm. The reaction was conducted at ambient tem- perature (~25 °C) and a neutral pH of 7.0, main- tained by not adding any acid or base. The to- tal reaction time was 30 minutes, during which the color change from pale yellow to dark brown indi- cated the formation of AgNPs. These parameters were selected based on reported optimal conditions for plant-extract-mediated silver nanoparticle syn- thesis. 2.5 Characterization of AgNPs 2.5.1 UV-vis spectroscopy When materials absorb ultraviolet and visible light (200–600 nm), they produce the ultraviolet-visible spectrum. The AgNPs produced using Ashwa- gandha root extract were characterized by a UV- visible spectrophotometer (Specord 200 Plus, Ana- lytik, Jena, Germany). 2.5.2 XRD analysis The crystallographic information of synthesized Ag- NPs was investigated using a D2 phaser, Bruker with Cu K radiation. The scanning was performed over a 2 range of 30°-90° utilizing a step size of 0.2003 2.5.3 FTIR analysis FTIR analysis was conducted using a PerkinElmer spectrometer with Spectrum IR (Version 10.6.2 to determine the functional groups found in the root extract responsible for the synthesis of Ag NPs within the spectral range of 500-4000 cm1. 2.5.4 FE-SEM and EDS analysis The morphology and elemental composition of Ag- NPs were analyzed using a field emission scanning electron microscope (FE-SEM, Hitachi, Japan) equipped with an energy-dispersive X-ray spec- troscopy (EDS) detector. 2.6 Antimicrobial effectiveness The antimicrobial effectiveness of AgNPs and root extract was evaluated against three specific micro- bial strains: Escherichia coli (ATCC 8739), which is a Gram-negative bacterium; Staphylococcus au- reus (ATCC 6538P), a Gram-positive bacterium; and Candida albicans (ATCC 2091), a fungal or- ganism. The assessment utilized the agar-well dif- fusion technique to compare their effectiveness with that of a standard antibiotic. Each culture was in- cubated under optimal conditions, ensuring precise measurement of inhibition zones and clear differen- tiation between microbial responses. All antimicro- bial experiments were conducted in triplicates. The data are expressed as mean ± standard deviation (n=3). 2.6.1 Media preparation A liquid broth (LB) medium was made by mixing 13 grams of LB powder (from Sisco Research Labora- tories Pvt. Ltd., India) into 1 litre of distilled water. This mixture was subsequently sterilized in an au- toclave set at 121°C and 15 psi for 25 minutes. After sterilization, it was allowed to cool to 50 °C. Once cooled, 5 mL portions of the medium were asep- tically distributed into pre-sterilized 15 mL Falcon tubes. The tube was then inoculated with a bacte- rial culture and incubated for 1 day for bacterial cell growth. To ensure accuracy and reproducibility, it is crucial to maintain aseptic techniques throughout the process and to verify that the incubation con- ditions are optimal for the specific bacterial strains being studied. Ratna Bahadur Thapa et al./ BIBECHANA 22 (2025) 248-257 251 2.6.2 Media Plated and Antimicrobial As- say Preparation Muller Hinton Agar (MHA) plates were made by dissolving 39 grams of MHA powder (Sisco Re- search Laboratories Pvt. Ltd., India) in one litre of distilled water. After mixing, the solution was sterilized in an autoclave at 121°C and a pressure of 15 psi for 25 minutes. Following autoclaving, the medium was allowed to cool to approximately 50°C before being dispensed into sterile Petri dishes, with each plate receiving 25 mL of the prepared agar. Before the antimicrobial assay, each plate (stored in the refrigerator) was labelled with the correspond- ing sample name. By using a sterile cotton swab, a 150 µL aliquot of bacterial suspension was spread evenly on the surface of the agar. Wells were then created in the agar to accommodate the test sam- ples and standards. For plant extracts, samples were loaded with 50 mg/mL concentration dissolved in DMSO, while NPs were added at approximately 30 mg per well. A kanamycin solution (5 mg/mL; 10 µL) was used as a positive control to assess bac- terial strain comparability. Similarly, for antifungal evaluation, itraconazole (20 mg/mL; 10 µL) was in- troduced into the designated wells as a standard reference. The plates were incubated at 37°C for 24 hours, and the effects were evaluated. 2.7 Data analysis The data obtained was analyzed and plotted using Origin 2024b software. 3 Results and Discussion 3.1 Phytochemical screening The phytochemical components in the Ashwa- gandha root extract that contributed to the re- duction and stabilization of AgNPs were analyzed qualitatively. Results of phytochemical screening indicate that hydro-methanolic root extract is rich in secondary metabolites (test result in Table 1). Alkaline reagent tests were done in the presence of flavonoids [17], and alkaloids were found using Hager’s test [18]. The test showed positive results for saponins, as indicated by the Froth test [17]. Terpenoids and steroids were also detected through positive results in the Salkowski test [18]. Tan- nins were presented as evidenced by the lead ac- etate test [18]. Additionally, quinone was identified through the concentrated hydrochloride (HCl). The presence of glycosides and phenolic compounds was confirmed using sodium hydroxide and ferric chlo- ride tests [16]. These compounds, extracted with polar solvents, have an inherent polarity that aids in NP formation and stabilization. Table 1: Phytochemical screening of the hydro-methanolic root extracts of Ashwagandha S. N Secondary metabolites Tests Result 1 Flavonoid test Alkaline reagent test + 2 Alkaloids test Hager’s test + 3 Saponins test Froth test + 4 Terpenoids and Steroids test Salkowski test + 5 Tannins Lead acetate test + 6 Quinone test Conc. HCl + 7 Glycosides NaOH + 8 Phenolic FeCl3 + Note: “+” indicates the presence of the phytochemical content. 3.2 Green synthesis of AgNPs The environmentally friendly formation of AgNPs can be achieved using extracts from plants that are abundant in various bioactive substances, in- cluding phenolic acids, terpenoids, and proteins. These compounds serve as natural reducing agents in the synthesis process. These biomolecules aid in converting metal ions into stable NPs, offering a greener alternative that minimizes the use of harm- ful chemicals. These compounds help reduce metal salts to nanoscale particles, such as converting Ag+ to Ag° in Ag NPs synthesis. Ashwagandha, known for its antioxidant properties, is particularly effec- tive in reducing silver ions to AgNPs. In this pro- cess, an aqueous Ashwagandha extract is combined with AgNO under constant stirring. The bioactive compounds present in plant extract assist in the re- duction of silver ions (Ag+) by providing electrons, which results in the formation of AgNPs. This pro- cess often involves a noticeable colour change due to SPR, typically shifting the solution's colour to a shade of brownish-blue [19]. The neutral pH (~7) and ambient temperature maintained during the Ratna Bahadur Thapa et al./ BIBECHANA 22 (2025) 248-257 252 synthesis likely favored the reduction kinetics and stabilization of AgNPs by phenolic and flavonoid constituents in the extract. The 30-minute reac- tion time aligns with other rapid green synthesis protocols reported in the literature [20]. Figure 2 illustrates the green synthesis route of AgNPs using Withania somnifera root extract, showing the key steps: preparation of plant extract, reduction of Ag ions, and stabilization of nanoparticles by bioactive phytochemicals. Figure 2: Representative diagram of green synthe- sis of Ag NPs. 3.3 Characterization of AgNPs 3.3.1 UV-vis spectroscopy analysis Ag NPs synthesized using Withania somnifera root extract were confirmed through UV-vis spec- troscopy, which displayed a broad absorption spec- trum between 300–600 nm. A clear peak at 433 nm is observed, as illustrated in Figure 3(a). The optical absorption peak observed at 433 nm cor- responds to surface plasmon resonance (SPR), a hallmark feature of metallic AgNPs. The SPR peak arises from collective oscillation of conduction electrons on the nanoparticle surface in resonance with incident light [20]. The peak position varies based on the morphology and size of the NPs, with smaller particles generally exhibiting a blue shift. The broad spectral profile indicates a certain level of polydispersity, a common trait in green synthesis methods [21]. Figure 3: (a)Ultraviolet-visible absorption spectra (b) Tauc plot indicating an apparent optical energy gap (~2.25 eV), derived from quantum confinement effects in AgNPs. The energy gap of Ag NPs differs from that of macroscopic silver due to quantum confinement ef- fects [22]. In bulk form, silver behaves as a metal with no band gap since the conduction and valence bands overlap. However, when silver is reduced to the nanoscale, especially at smaller particle sizes, a shift in optical properties resembling a band gap can develop. For Ag NPs, this energy gap typi- cally ranges between 2.0 eV and 4.0 eV, affected by the surrounding and size conditions of the particle. This optical gap, observed through techniques like UV-visible spectroscopy, is linked to plasmonic res- onances rather than the electronic bandgap found in semiconductors. However, band gap governs the material's electrical properties and is essential in determining whether a material behaves as a con- ductor, semiconductor, or insulator [23]. AgNPs en- ergy gap (Eg) determined through the Tauc method [24,25]; (αhν)1/m = C(h− Eg) Here, = absorbance coefficient, C = constant, h = Planck's constant, = photon frequency, Eg = optical band gap, and m = ½ for direct band gap semiconductors. Additionally, an optical energy gap of 2.25 eV was calculated using the Tauc method (Figure 3 (b)). While this value mimics a "bandgap," it is not a true electronic bandgap as seen in semicon- ductors. Instead, it reflects quantum confinement effects and plasmonic behavior associated with the small size of AgNPs. As AgNPs are metallic, their conduction and valence bands overlap in bulk; how- ever, at the nanoscale, localized surface effects and interaction with light can produce apparent optical transitions that resemble a bandgap [21,22]. 3.3.2 FTIR analysis The analysis using FTIR was conducted to deter- mine the functional groups that play a role in the synthesis and stabilization of Ag NPs. In Figure 4, the FTIR spectra of the plant extract is shown in Ratna Bahadur Thapa et al./ BIBECHANA 22 (2025) 248-257 253 black, while those of the synthesized AgNPs are rep- resented in red. In the FTIR spectrum of the plant extract, several distinctive peaks are observed, with a prominent absorption band at 3307 cm¹. The presence of hydroxyl groups in the extract has been indicated by the vibrations associated with O–H stretching [26]. The peak observed at 2940 cm¹ cor- responds to asymmetric C–H stretching, suggest- ing that alkyl groups are present. Furthermore, the bands identified at 1607cm¹ and 1408 cm¹ are associated with C=C and C–H vibrations, respec- tively. Additionally, the spectrum feature at 1028 cm¹ is attributed to C–O stretching, a characteris- tic often associated with alcohols or phenolic com- pounds [27]. A band at 608 cm¹ was associated with C–H bending vibrations. The presence of Ag NPs resulted in significant changes, with all spectral bands showing weaker intensity in comparison to the original plant ex- tract. This indicates that Ag NPs were successfully formed. Notably, the reduced intensity of the O– H stretching vibration at 3307 cm-1 suggests that hydroxyl groups played a role in the synthesis of the NPs. Moreover, the shifts observed in the C=C (1593 cm-1) and C–O (1016 cm-1) absorption peaks provide further evidence of the involvement of these functional groups during the formation process. It is believed that various functional groups such as hydroxyl, and carbonyl are integral to not only the reduction of silver ions but also the stabilization of the NPs [28]. Figure 4: (FTIR spectra of AgNPs (black) and root extract (red). 3.3.3 XRD analysis XRD is a powerful method used to examine the size and crystalline structure of materials by ana- lyzing the diffraction patterns produced when X- rays interact with a sample. The crystalline struc- ture of Withania somnifera-based Ag NPs was an- alyzed using XRD as shown in Figure 5. The ob- served diffraction peaks at 2 angles of 37.8°, 44.3°, 64.4°, 77.2° and 81.2° correspond to the Miller in- dices (111), (200), (220), (311) and (222) respec- tively. These peaks suggest the presence of a face- centred cubic (FCC) crystal structure. These re- sults were validated by comparing them to the JCPDS database (file number: 01-087-0717) [29]. Some of the observed peaks were attributed to par- tial oxidation of silver, which occurred during long- term storage before characterization. The average crystallite size of powdered AgNPs was estimated using Scherrer’s equation [30], D = kλ βcos θ In this eqn, the following variables are defined: D refers to the size of the crystallite or grain, K is the dimensionless shape factor, which is set at 0.9, represents the wavelength of the X-ray radia- tion being utilized, denotes Bragg’s angle expressed in radians, and indicates the full width at half max- imum, also measured in radians. The most intense diffraction peak appeared at 37.8°. The size of the crystallites in the AgNPs was measured to be 10.57 nm. This calculation was based on the XRD data, which includes detailed information on peak posi- tions and angles, crucial for determining the crys- tallite size. Figure 5: XRD patterns of Ag NPs. 3.3.4 SEM/EDS analysis The surface features and elemental percentage of the formed Ag NPs were examined through SEM and energy-dispersive X-ray spectroscopy (EDS). SEM image captured at a scale bar of 0.5 m is presented in Figure 6, showing spherical parti- cles with consistent size distribution and notice- able agglomeration of the Ag NPs. When Ag NPs are heated indirectly, they tend to cluster together, leading to the electronic coupling between the metal Ratna Bahadur Thapa et al./ BIBECHANA 22 (2025) 248-257 254 particles. This interaction causes a shift in the SPR to higher wavelengths, accompanied by a higher intensity in the UV-visible spectrum, in contrast to the behaviour of individual particles [31]. The synthesis method, purification steps, and drying conditions are key factors affecting the agglomer- ation, dispersion, stability, and size distribution of Ag NPs [32]. Silver was found to be the most abundant el- ement, comprising 56.9%, followed by carbon at 29.7%, and oxygen at 13.5%. A prominent peak confirmed the presence of silver metal. The peaks corresponding to oxygen and carbon, as seen in Figure 6, are attributed to organic components from the proteins or enzymes in the extract, as well as the carbon tape used during the measurement process [33]. 3.4 Antimicrobial Evaluation The agar diffusion method was used to perform an- timicrobial activity, adhering to established guide- lines [34]. The Zone of Inhibition (ZOI) served as the primary indicator to gauge antimicrobial effec- tiveness. The size of the ZOI created on bacterial cultures was assessed to determine the antimicro- bial activity of plant extracts, as shown in Table 2. The ZOI for each microbial strain was measured in millimeters and reported as mean ± SD (n = 3). ATCC (American Type Culture Collection) was the source for the microbial reference strains that play an essential role in standardizing and distributing these materials, ensuring reproducibility and relia- bility across scientific research and industrial appli- cations. Figure 6: SEM image and EDS spectrum of synthe- sized Ag NPs. Table 2: Antimicrobial screening of Withania somnifera extracts Strain Reference culture Type Control (c+) (mm) Ag NPs (mm) Plant extract (mm) E. coli ATCC8739 Gram-negative 25.0 ± 0.4 21.3 ± 0.4 0.0 S. aureus ATCC6538P Gram-positive 26.1 ± 0.4 22.4 ± 0.4 0.0 C. albicans ATCC2091 Fungi 27.2 ± 0.4 20.2 ± 0.4 0.0 Note: ZOI = Zone of inhibition in mm. Kanamycin (positive control, c+), concentration of 5 mg/mL. DMSO was used as a negative control and showed no activity. The antimicrobial activity of Ashwagandha ex- tract on E. coli was tested using the ATCC 8739 reference strain, with a positive control for com- parison. The positive control produced a ZOI of 25 mm, while Ag NPs exhibited a ZOI of 21 mm against the strain, shown in Figure 7(a), which suggests that Ag NPs demonstrate considerable an- tibacterial properties against E. coli. The antimi- crobial effect of Ashwagandha extract against S. aureus was assessed using the ATCC 6538P refer- ence strain, with a positive control for comparison. The ZOI for positive control was 26 mm, while Ag- NPs showed a ZOI of 22 mm against this strain, as shown in Figure 7(b). These findings demonstrate that Ag NPs have significant antibacterial activity against S. aureus. Antimicrobial activity of AgNPs possesses notable antimicrobial activity against C. albicans which was tested using the ATCC 2091 reference strain, with a positive control for compar- ison. The ZOI for the positive control was 27 mm, while AgNPs showed a ZOI of 20 mm against this strain, as depicted in Figure 7(c). Figure 7: Zone of Inhibition for (a) E. coli, (b) S. aureus, and (c) C. albicans. 3.5 Antibacterial Mechanism of AgNPs AgNPs exhibit antimicrobial properties through both direct and indirect mechanisms. A key fac- tor in their effectiveness is the continuous release of Ag, which plays a crucial role in bacterial inhi- bition. These ions have a strong affinity towards proteins, allowing them to attach to bacterial cell walls and membranes, Figure 8. This interaction disrupts membrane integrity, increasing permeabil- ity and leading to structural instability. Once sil- ver ions enter the cell, they disrupt the activity of respiratory enzymes, leading to the production of Ratna Bahadur Thapa et al./ BIBECHANA 22 (2025) 248-257 255 reactive oxygen species (ROS) and hindering the synthesis of ATP. The oxidative stress caused by ROS damages essential cellular components, includ- ing lipids, proteins, and DNA [35]. Additionally, silver ions bind to DNA, disrupting replication and transcription, which hampers cell division and ulti- mately leads to bacterial death [36]. They also in- terfere with ribosomal function, inhibiting protein synthesis and disrupting cellular processes [37,38]. Figure 8: Mechanism for the antimicrobial activity of Ag NPs.. Beyond ion release, Ag NPs themselves con- tribute to bacterial inhibition. When bacteria en- counter certain substances, they gather on their surfaces and infiltrate the cell wall. This infil- tration disrupts the internal structures and causes the leakage of cellular materials, ultimately lead- ing to cell death. Moreover, Ag NPs interfere with bacterial signaling pathways by dephosphory- lating tyrosine residues on peptide substrates, dis- rupting essential regulatory functions. This disrup- tion inhibits bacterial proliferation and promotes cell death. Through this combination of silver ion activity, nanoparticle interaction, and interference with cellular functions, Ag NPs demonstrate broad- spectrum antimicrobial efficacy [39,40]. 4 Conclusions Ag NPs synthesis using Ashwagandha root ex- tract provides an environmentally friendly and ef- fective method for NP formation. The synthesized nanoparticles were characterized through UV-Vis spectroscopy, FTIR, XRD, and SEM/EDS; this led to the formation of NPs that were spherical and crystalline in shape, measuring 10.57 nm in size. Ag NPs are stabilized by phytochemicals, including alkaloids, saponins, and phenolic compounds found in the extract. The NPs exhibited enhanced an- timicrobial effectiveness against Gram-positive and Gram-negative bacteria, displaying larger inhibi- tion zones and reduced MIC values compared to the extract on its own. These results underscore the potential of Ashwagandha-derived Ag NPs for medical and environmental purposes, highlighting the significance of green synthesis in promoting sus- tainable nanotechnology. Future studies should aim to refine synthesis conditions and investigate addi- tional applications to fully exploit the capabilities of these NPs. Future research will focus on finding the Quantitative phytochemical profiling. This study had some limitations, lack of quantitative phyto- chemical data and potential variability in extract composition. To gain a better understanding of the mechanistic role of bioactives in the synthesis of Ag NPs, future studies should incorporate quantitative estimation of the total phenolic/flavonoid content and HPLC/GC-MS profiling. Acknowledgments The first author (R.B. Thapa) acknowledges fund- ing support from Mini Research Grant (Grant No. 2/2081), Tribhuvan University, Institute of Science and Technology (IoST), Dean’s office, Kathmandu, Nepal Conflicts of Interest The authors declare that there are no conflicts of interest associated with the publication of this re- search paper. References [1] I. Khan, K. Saeed, and I. Khan. Nanoparticles: Properties, applications and toxicities. Arab. J. Chem., 12(7):908–931, 2019. [2] X. Cui et al. Photothermal nanomaterials: A powerful light-to-heat converter. Chem. Rev., 123(11):6891–6952, 2023. [3] Y. Fu et al. Applications of nanomaterial tech- nology in biosensing. J. Sci-Adv. Mater. Dev., 9(2):100694, 2024. [4] A. Karnwal et al. Gold nanoparticles in nanobiotechnology: From synthesis to biosens- ing applications. ACS Omega, 9(28):29966– 29982, 2024. [5] A. Dhaka, S. Chand Mali, S. Sharma, and R. Trivedi. A review on biological synthesis of silver nanoparticles and their potential ap- plications. Res. 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Introduction Materials and Methods Materials Collection and preparation of Ashwagandha root extract Phytochemical screening Synthesis of AgNPs Characterization of AgNPs UV-vis spectroscopy XRD analysis FTIR analysis FE-SEM and EDS analysis Antimicrobial effectiveness Media preparation Media Plated and Antimicrobial Assay Preparation Data analysis Results and Discussion Phytochemical screening Green synthesis of AgNPs Characterization of AgNPs UV-vis spectroscopy analysis FTIR analysis XRD analysis SEM/EDS analysis Antimicrobial Evaluation Antibacterial Mechanism of AgNPs Conclusions