Green Synthesis, Characterization and Biological Evaluation of Silver Nanoparticles from Medicinally Important Plant Allium ursinum

Younas Khan
Government Degree College,  Alpurai District, Shangla KPK, Pakistan
Correspondence to: younaskhanbueri333@gmail.com

DOI: https://doi.org/10.70389/PJS.100066

Additional information

• Ethical approval: N/a
• Consent: N/a
• Funding: No industry funding
• Conflicts of interest: N/a
• Author contribution: Younas Khan – Conceptualization, Writing – original draft, review and editing
• Guarantor:Younas Khan
• Provenance and peer-review:
  Unsolicited and externally peer-reviewed
• Data availability statement: N/a

Keywords: Silver nanoparticles, Allium ursinum, Green synthesis, Antimicrobial activity, Biosynthesis characterization.

Peer Review
Received: 12 December 2024
Revised: 12 February 2025
Accepted: 13 February 2025
Published: 26 February 2025

Introduction

Nanoparticles have attracted considerable interest due to their unique size and shape-dependent properties, which are applicable in optical and chemical sensing, electronics, catalysis, and biomedicine.¹ Noble metal nanoparticles have been extensively reached, primarily due to their strong optical absorption in the visible spectrum, which arises from the collective excitation of free electrons.² Silver nanoparticles (AgNPs) have garnered considerable attention because of their diverse applications, including nonlinear optics,³ selective coating for solar energy absorption,⁴ optical receptors,⁵ chemical catalysts, and antibacterial agent.⁶ Silver is widely recognized for its inhibitory effects on various bacterial strains and microorganisms commonly found in medical and industrial settings.⁷ The common approach for synthesizing silver nanoparticles through chemical reduction involves forming colloidal dispersion in water or organic solvents.⁸

Conventional chemical and physical synthesis routes often involve toxic reagents, high energy consumption, and low yield, motivating the development of sustainable alternatives.⁹ The green synthesis approach employs nontoxic reagents, environmentally friendly solvents, and sustainable materials.¹⁰ Various biological methods for the green synthesis of silver nanoparticles have been documented, utilizing plant leaf extracts from species such as Alternanthera sessilis,¹¹ Decaschistia tribolata,¹² Youngia japonica,¹³ and Ocimum gratissmum.¹⁴ These methods use the natural phytochemicals present in plants, which act as reducing, stabilizing, and capping agents during the formation of nanoparticles.¹⁵ Allium ursinum is a perennial herbaceous plant belonging to the Alliaceae family.¹⁶ Allium ursinum, known for its rich phytochemicals such as sulfur compounds, polyphenols, flavonoids, phenolic acids, fatty acids, and terpenoids,¹⁷ has been used for medicinal purposes and is a promising candidate for nanoparticle synthesis.¹⁸ In this study, we utilize Allium ursinum leaves for the biosynthesis of AgNPs and investigate their antimicrobial efficacy.

General Experimental Condition

Plant Materials

Allium ursinum leaves (10 kg) were collected during the flowering season from District Shangla, Khyber Pakhtunkhwa, Pakistan, and authenticated by the University of Swat. The leaves were shade-dried for 2 weeks and ground into a fine powder. The powder was macerated in methanol, an eco-friendly solvent, for 15 days at room temperature with daily agitation. The resulting methanolic extract was then concentrated using a rotary evaporator set at 80 °C (80–95 RPM) to obtain a thick gummy crude extract.

Fractionation of Crude Alkaloids

A portion of methanolic extract was dissolved in 1.5 L of 0.5 N H2SO4 and shaken for 1 hour to obtain an acidic aqueous fraction (pH 1–2). This fraction was extracted thrice with a 2 L portion of chloroform to remove non-alkaloidal components. A rotary evaporator was used to concentrate the chloroform layer to yield a non-alkaloid acidic fraction, which tested negative with Dragendorff’s reagent. The remaining aqueous phase was basified to pH 8–10 using a 10% KOH solution and repeatedly extracted with chloroform until the extract became clear. The combined organic layer was concentrated to produce the basic alkaloidal fraction, confirmed by a positive Dragendorff’s test.¹⁹ Finally, the residual aqueous phase was neutralized to pH 7 using 0.5 N H2SO4 and stored for further use.

Synthesis of Nanoparticles (AgNPs)

Preparation of Stock Solution

• Silver nitrate solution: Dissolve 215 mg of AgNO3 in 500 ml distilled water (pH 5.3) and store it at room temperature.
• Crude alkaloid solution: Dissolve 200 mg of Allium ursinum crude alkaloid in 500 ml of distilled water (7.8) and mix thoroughly using a shaker.

Nanoparticle Synthesis Procedure

For the synthesis of AgNPs, stock solutions were mixed in varying ratios. In a typical synthesis, 1 ml of AgNO3 solution was added to 9 ml of a crude alkaloid solution in a 100 ml round-bottom flask. The mixture was maintained at 70 °C on a magnetic hotplate under constant stirring for 1 hour. The reaction’s progress was monitored by observing the color change of the solution from colorless to deep brown, indicating the formation of AgNPs.

Characterization Techniques

• UV-Vis spectroscopy: The formation and stability of silver nanoparticles were monitored by measuring the absorbance between 400 and 500 nm.
• Fourier transform infrared (FTIR) spectroscopy: FTIR analysis was used to identify the functional groups involved in reducing and stabilizing AgNPs.
• X-Ray Diffraction (XRD): XRD was used to determine the crystalline structure and estimate the average particle size of the AgNPs.
• Scanning and transmission electron microscopy (SEM and TEM): SEM and TEM analysis provide insight into the morphology and distribution of the nanoparticles.

Antimicrobial Evaluation

The antimicrobial activity of the synthesized AgNPs was evaluated using the minimum inhibitory concentration (MIC) method against bacteria strains, including Staphylococcus aureus, Vibrio cholerae, Salmonella typhi, and Escherichia coli. Furthermore, antifungal activity was assessed against various candida species. Zone of Inhibition was measured and compared to standard antibiotic chloramphenicol and antifungal agents like fluconazole.

Results and Discussion

Visual Confirmation of AgNPs

The synthesis process was visually confirmed by a change in the reaction mixture color from colorless, which is characteristic of AgNP formation due to the surface plasmon resonance effect, as shown in Figure 1.

UV-Vis Spectroscopy Analysis

UV-Vis spectroscopy revealed a distinct absorption peak between 400 and 500 nm, the maximum absorbance observed at the optimized mixing ratio (1 ml AgNO3 to 9 ml crude alkaloid solution). This peak is associated with the surface plasmon resonance of silver nanoparticles, confirming their formation, as depicted in Figure 2.

Stability Studies

Stability tests indicated that the AgNPs remained most stable within a temperature range of 25–45 °C. Moreover, the nanoparticles exhibited enhanced stability under basic pH conditions compared to neutral. Figure 3 illustrates the variation in pH.

Structural and Morphology Characterization

• XRD Analysis: the XRD pattern confirmed the crystalline nature of the AgNPs, with distinct diffraction peaks corresponding to t (110), (200), (221), and (310) planes. Figure 4 illustrates the average particle size calculated to be approximately 24 nm.

• FTIR Analysis: Figure 5 presents the FTIR spectra, showing broad peaks at 3375 cm−1 (indicative of O–H stretching from surface absorbed water) and other peaks at 1410 cm−1 and near 610 cm−1, corresponding to methanol and C–H stretching vibrations,20 respectively. The results confirmed the involvement of phenolic compounds, aromatic molecules, and proteins in the nanoparticle synthesis.²¹

• SEM and TEM observations: Figures 6 and 7 illustrate the SEM and TEM images, showing that the AgNPs are predominantly spherical with a size distribution ranging from 15–28 nm, demonstrating good dispersion and uniformity.

Antimicrobial Activity

Figure 8 depicts the antimicrobial evaluation demonstrating that AgNPs possess significant antibacterial activity. For instance, at a 50 µg/ml concentration, AgNPs produce inhibition zones of 26 mm against S. aureus, which was further enhanced when combined with chloramphenicol (Table 1). Similarly, synergistic effects were observed against V. cholerea and S. typhi. Although E. coli exhibited a consistent inhibition zone of 20 nm, the overall antibacterial performance of AgNPs was comparable to or better than that of standard antibiotics. The findings align with those obtained by Loo et al.²²

Table 1: Zone of inhibition produced by agnps, reference antibiotic chloramphenicol and agnps with chloramphenicol.
Bacterial Strains	Zone of Inhibition (mm)
	Chloramphenicol 50 mg/ml	AgNPs 25 mg/ml	AgNPs 50 mg/ml	AgNPs and Chloramphenicol 50 mg/ml
Bacillus subtilis MTCC1133	20	20	22	23
Bacillus cereus ATCC10987	30	25	26	26
Micrococcus luteus ATCC4698	28	21	21	30
Staphylococcus aureus MTCC96	19	23	26	28
Escherichia coli MTCC118	23	20	20	20
Vibrio cholerae ATCC14035	21	25	27	30
Salmonella typhi MTCC733	18	25	29	31
Klebsiella pneumoniae MTCC109	31	29	31	31
Fungal Strains	Zone of Inhibition (mm)
	Fluconazole 50 mg/ml	AgNPs 25 mg/ml	AgNPs 50 mg/ml	AgNPs& Fluconazole 50 mg/ml
Candida parapsilosis MTCC 2509	–	18	20	20
Candida tropicalis MTCC 184	27	27	29	30
Candida albicans MTCC 183	–	19	25	25

Antifungal Activity

Figure 9 presents the antifungal potential of AgNPs evaluated against several candida strains. While fluconazole showed limited efficacy against certain strains, the AgNPs at concentrations of 25 and 50 µg/ml demonstrated measurable inhibition zones. Combining AgNPs with fluconazole, in some cases, enhanced antifungal activity, suggesting a strain-dependent synergistic effect.

Conclusion

This study demonstrated that Allium ursinum leaf extract is an effective and environmentally friendly agent for the eco-friendly synthesis of silver nanoparticles. The biosynthesized silver nanoparticles were confirmed by a series of analytical techniques and displayed a crystalline, spherical morphology having an average diameter of around 24 nm. The silver nanoparticles exhibited significant antibacterial and antifungal activities, emphasizing their potential in antimicrobial therapies and pharmaceutical applications. The green synthesis approach presented here provides a sustainable alternative to conventional nanoparticle production methods.

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Cite this article as:
Khan Y. Green Synthesis, Characterization and Biological Evaluation of Silver Nanoparticles from Medicinally Important Plant Allium ursinum. Premier Journal of Science 2025;8:100066

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