Introduction

The nanotechnology field is currently expanding more in the manufacturing market, with a wide quest for innovative nanomaterials along with manufacturing technologies¹. Living cells have long been recognized as the best examples of equipment that operates at the nano level and executes a multitude of operations that vary from energy production to distillation of specific materials with high efficiency². Nanomedicine is an emerging interdisciplinary field that integrates nanoscale technologies with medical science, leading to broad applications of nanomaterials in healthcare. Medicine is no longer only the domain of physicians; Nanoscale elements and gadgets serve as tools for detection, disease control and treatment for ease of pain and general wellness, preservation and advancement³. The field of nanotechnology can help us better comprehend live cells and molecular interactions. Several nanoparticle-mediated therapeutic techniques for various diseases and pharmaceutical uses have been clinically approved⁴. Oligodynamic silver possesses antibacterial activity that extends far beyond its virotoxicity with deadly effects spanning all microbiological domains^5-6. The utilization of metallic nanoparticles in the delivery of medicines, pharmaceutical development, and innovative treatment methods has announced conflict against numerous awful illnesses⁶¹. They leverage the human system’s inherent circulatory system and method of absorbing drugs by sick tissues⁶. Chemically generated methodology produces a large number of nanoparticles quickly, it necessitates the use of sealing substances to stabilize their dimensions. The compounds utilized during the manufacturing and stabilization of nanoparticles are hazardous and manufacture unwanted metabolites⁷. The rise of demand in natural methods for nanoparticle synthesis originates from the demand for ecologically friendly chemical methods for nanoparticle formation. Green synthesis methods are employed to minimize the production of byproducts, hence providing a more environmentally friendly and secure procedure⁸. As a result, there is a growing need for “Green Nanotechnology”⁹. Moreover, the use of plant products minimizes the expense of bacterial separation and media for cultivation, boosting the cost-effectiveness of microbial nanoparticle synthesis¹⁰. Nanobiotechnology is now a widely popular subject of research in the realm of substance science. Nanostructures have entirely different or improved features depending on their dimension, dispersion and forms¹¹. Silver-based compound’s antimicrobial action is used in healthcare to reduce sickness during healing from burns and arthroscopy and additionally to minimize germs colonization of implants, tubes, coronary transplants, dental implants, surgical metals, and the skin of people¹². The nanoparticles of silver have applications in catalysis, biochemical detection, biosensing, photographic science and technology, including medicine because of their distinctive attributes¹³. The nanoparticles of silver offer significant possibilities for ecological applications including antimicrobial properties. Silver metal’s antimicrobial abilities enable them to be suitable for usage in a variety of common items, such as fabrics, alimentary vessels, appliance parts, and healthcare equipment¹⁴. Silver is a potent antimicrobial agent with minimal toxicity¹⁵. The primary common use for silver and nanoparticles of silver is within healthcare applications, including tropical lotions that stop illness in injuries and untreated cuts. Due to its attractive physiochemical properties, nanoparticles of silver serve a significant function in both science and medicine¹⁶. Silver compounds are known to have considerable suppressive and microbiological capabilities as well as a broad spectrum of antibacterial action for ages. It is also used to prevent and treat illnesses with a particular focus on infections¹⁷. Silver nanoparticles are known to have fungicidal effects as well as antiviral, anti-angiogenic and antiplatelet actions¹⁸. In the pursuit of advancing sustainable and cost-effective methodologies for nanoparticle synthesis, this research endeavors to explore the green synthesis of silver nanoparticles through the plant extracts in conjunction with silver nitrate¹⁹. The integration of various plant extracts as both reducing and stabilizing agents represents a departure from traditional chemical and physical techniques, offering a more environmentally friendly⁶⁶ and economically viable alternative²⁰. By replacing chemical compounds and intricate synthetic procedures with biologically derived components, this study aims to contribute to the field of green synthesis²¹. Plant extracts not only provide a cheaper and more straightforward approach to the synthesis of silver nanoparticles but also align with the global shift towards eco-friendly practices²². The investigation encompasses the utilization of multiple plant extracts to discern their unique contributions to replace conventional chemical techniques²³.

Material and Methodology

Materials

All the plant leaves of Azadirachta indica, Mangifera indica, Eucalyptus and Fenugreek were collected from a residential area of Ahmedabad city (latitude 23°01′17.83″ North, longitude 72°34′46.96″ East), India. Silver nitrate (99.9% Assay, Finar Chemicals Ltd.) was used as provided. Throughout all the experiments water was subjected to distillation and subsequently purified utilizing the Millipore Company’s Milli-Q filtration device²⁴. All the bacterial strains [Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 1688), Staphylococcus aureus (MTCC 96), Streptococcus pyogenes (MTCC 442)] and fungal strains [Candida albicans (MTCC 227), Aspergillus niger (MTCC 282) and Aspergillus clavatus (MTCC 1323)] purchased from Institute of Microbial Technology in Chandigarh, India. Ciprofloxacin and Norfloxacin standard drugs were used for screening antibacterial activity and Nystatin and Griseofulvin standard drugs were used for screening of antifungal activity. 

Experimental method

Experimental procedures were conducted under supervision at a steady temperature ranging from 25-30°C. The experimentation room was equipped with air conditioning, ensuring a stable and consistent temperature, with negligible fluctuations in humidity observed throughout the experiments²⁵. 

Plant collection

The following plant’s leaves were collected from a residential area of Ahmedabad city, India.

Azadirachta indica

Family: Meliaceae; Genus: Azadirachta; Species: Indica; Scientific name: Azadirachta indica; Common name: Neem²⁶

Mangifera indica

Family: Anacardiaceae; Genus: Mangifera; Species: Indica: Scientific name: Mangifera indica; Common name: Mango²⁷

Eucalyptus

Family: Myrtaceae; Subfamily: Myrtoideae; Genus: Eucalyptus; Species: Eucalyptus obliqua; Scientific name: Eucalyptus teriticornis; Common name: Nilgiri²⁸

Fenugreek

Family: Fabaceae; Subfamily: Faboideae; Genus: Trigonella; Species: foenum-graecum; scientific name: Trigonella foenum-graecum; Common name: Methi²⁹ 

Preparation of plant extracts

Selected Plant leaves were collected from the residential area of Ahmedabad city, Gujarat (India). All the samples were thoroughly washed with tap water then distilled water and dried in the absence of sunlight³⁰. 25 grams of minced leaf material was carefully taken in a 250 mL round bottom flask and 100 mL of demineralized water. ⁶⁴ This mixture was heated in a water bath for 1 hour at 100°C temperature³¹. After 1 hour mixture was cooled to room temperature and then filtered through Whatman filter paper number one. After filtration extracts were collected in plastic bottles shown in Figure 1.

Figure 1: Plant leaf extracts Click here to view Figure

Preparation of 1 mM silver nitrate solution

Accurately weighed 0.169 grams of silver nitrate (99.9% purity) was dissolved in Milli-Q water and then diluted to a final volume of 100 mL³².

Synthesis of Silver Nanoparticles

One millimolar silver nitrate (AgNO₃) aqueous solution was filled in the 50 mL burette. Five milliliters of plant extracts were taken in different four sterilized glass beakers for each plant extract. It was placed on a Magnetic Stirrer. Silver nitrate was added dropwise into the plant extract. The color changed from yellow to dark brown after a few minutes, indicating the synthesis of silver nanoparticles shown in Figure 2. The beaker was always wrapped with aluminum foil. Synthesized silver nanoparticles were further utilized for characterization and antimicrobial testing³³.

Figure 2: a) Plant leaves extract before the addition of silver nitrate and b) Plant leaves extract after the addition of silver nitrate.  Click here to view Figure

Characterization Techniques

The samples were analyzed using Fourier-transform infrared spectroscopy (PERKIN–ELMER Spectrophotometer). The appropriate level of humidity remained the same during the collection of FTIR spectra³⁴. The Jasco ultraviolet (UV) spectrophotometer was used to do an Ultraviolet/visible spectroscopy examination.  JEOL JSM-7900F FESEM was employed for obtaining sample micrographs³⁵. To ensure representative compositional datasets reflecting Materials have been ground in small particles to obtain the mean volume content of the components before conducting XRD, Powder X-ray diffraction (PXRD) structures have been identified using a Bruker D8 DISCOVER X-ray diffractometer with Cu Kα (λ = 1.5405 Å) System within the 2θ scale of 5° to 80°, with an image acquisition pace is 2° per min³⁶. By employing these methods, the shape, crystallinity, and stability of the synthesized nanoparticles were determined, showcasing their suitability for various applications³⁷.

Antimicrobial effects

Well diffusion method was followed for the screening of antimicrobial activities³⁸. Ciprofloxacin and Norfloxacin standard drugs were used for the antimicrobial study of Silver Nanoparticles samples synthesized using different plant extracts. AgNO₃ and Deionized water were used for the baseline condition³⁹. The broth of Mueller Hinton was employed as a source of nutrients to cultivate and reduce an antibiotic solution to examine microbes⁴⁰. The suspension amount for the experimental bacteria was adjusted to 10⁸ CFU [Colony Forming Unit] per milliliter based on absorbance. Typical isolates were utilized to test the antimicrobial and antifungal abilities of the strains^63,41. Bacterial and fungal strains were purchased from the Institute of Microbial Technology, Chandigarh.

Results and Discussion

The nanoparticles of silver were formed utilizing a biosynthesis that utilized several leaf extracts as a provider of bio-reducing substances that are responsible for the breakdown of metallic ions into silver molecules. After about a full day of growth, the specimen was exposed to various characterization procedures⁴², with the following results 

UV/VIS spectrophotometric analysis

Figure 3: UV-visible spectrum from silver nanoparticles from different plant extract Click here to view Figure

Synthesized silver nanoparticles were characterized by UV-visible spectroscopy. The ultraviolet-visible (UV-Vis) absorption spectra of the silver nanoparticles were investigated within the wavelength range of 300 to 800 nm. Figure 3 illustrates the absorption peaks of the silver nanoparticles synthesized, predominantly observed between 350 and 450 nm⁴³. In the UV–visible spectroscopy of silver nanoparticles at different time durations are total of 4 different plant extracts are taken for the synthesis of silver nanoparticles. In Figure 4 among the four graphs, Mangifera indica leaves plant extract gives the highest absorption peak at 380 nm compared to Azadirachta indica, Fenugreek, and Eucalyptus leaves plant extract⁴⁴.

Figure 4: UV–visible spectroscopy of Silver Nanoparticles at different time durations. Click here to view Figure

FTIR analysis

The spectral band observed between 3490 and 3500 cm⁻¹ in the FT-IR spectra of silver nanoparticles corresponds to the stretching vibrations of O-H bonds in hydroxyl groups present in alcohols and phenols, or N-H bonds in amide functional groups. Notably, the peak observed around 1600-1650 cm⁻¹ signifies the presence of C-H stretching vibrations (Figure 5)⁷⁰. The signal at 1041 cm⁻¹ represents glycoside or ether (C-O-C) bonds in the sample⁴⁵. Easter groups present in the sample, such as -NH, -OH, C=O, and -C-O-C-, may be in charge of the bioreduction of Ag⁺¹ to Ag⁰ nanoparticles⁴⁶. The stretching frequency for silver nanoparticles was determined to be around 400-450 cm⁻¹. 

Figure 5: FT-IR of synthesized silver nanoparticles. Click here to view Figure

X-ray diffraction (XRD) analysis

To acquire compositional data that accurately represents the overall composition of the structures, initially, the samples were finely processed into powder before performing X-ray diffraction (XRD) analysis⁴⁷. We then used a Bruker D8 DISCOVER X-ray diffractometer with Cu Kα (λ = 1.5405 Å) radiation to generate powder X-ray diffraction (PXRD) arrangements with a scan rate of 2 degrees per minute in the 2θ values ranging from 10 – 80⁴⁸. In Figure 6 XRD patterns of the Neem, Mango, Methi and Nilgiri mediated Ag nanoparticles are shown.  The X-ray diffraction (XRD) pattern of silver nanoparticles synthesized using different extracts of Neem, Mango, Methi and Nilgiri exhibits distinct peaks at 2 θ values. These values correlate with the Bragg reflections of specific crystallographic planes, confirming the face-centered cubic (FCC) lattice systems of the silver particles. The peaks are observed at approximately 38, 44, 64, and 77 degrees, these relate to the levels of crystallography (111), (200), (220), and (311)⁴⁹. Peak intensities vary among the different samples, as evidenced by the above figure 6. These findings indicate the successful synthesis of Silver Nanoparticles in the prepared sample. The XRD patterns of the prepared samples agree with the previously reported XRD patterns⁵⁰.

Figure 6: a) Azadirachta indica, b) Mangifera indica, c) Fenugreek and d) Eucalyptus are the PXRD patterns of the silver nanoparticles synthesized by various plant extracts.  Click here to view Figure

Scanning electron microscopic (SEM) analysis

The size and shape of prepared AgNPs have been identified using Field emission scanning electron microscopy (FESEM). Using ImageJ software particle size for every sample is measured. Figure 7 depicts the morphological properties of silver nanoparticles synthesized with various plant extracts, displaying diversity in micrometer-scale parameters. As shown in Figure 7 a) was observed that the morphology of silver nanoparticles, synthesized through neem mediation, exhibited a predominantly spherical shape, interspersed with cuboidal and rectangular forms⁵¹. The particles displayed a polydisperse distribution and tended to aggregate into intricate irregular structures, lacking well-defined morphological boundaries. As shown in Figure 7 b), SEM images unveil the presence of flake-like structures in the synthesized silver nanoparticles through mango mediation their size ranged from 42 to 201 nm. The spherical shape-like morphology is evident from the SEM image as shown in Figure 7 c) having particle sizes ranging from ~12 to 46 nm. As shown in Figure 7 d), synthesized silver nanoparticles through Nilgiri mediation, the SEM image illustrates the presence of spherical agglomerated particles having particle sizes ranging from ~68 to 106 nm. Therefore, SEM analysis confirms the presence of silver nanoparticles⁵². 

Figure 7: a) Azardica indica, b) Fenugreek, c) Mangifera indica and d) Eucalyptus are the SEM images of the silver nanoparticles in micrometer parameters synthesized by different plant extracts. Click here to view Figure

Antimicrobial Activity

The antibacterial activity of silver nanoparticles was studied against pathogenic bacterial strains of gram-negative and gram-positive using the well diffusion method. For screening of antibacterial and antifungal activities: Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 1688), Staphylococcus aureus (MTCC 96), Streptococcus pyogenus (MTCC 442), Candida albicans (MTCC 227), Aspergillus niger (MTCC 282) and Aspergillus clavatus (MTCC 1323) strains were used. Ciprofloxacin and Norfloxacin standard drugs were used for screening antibacterial activity and Nystatin and Griseofulvin standard drugs were used for screening of antifungal activity. The Institute of Microbial Technology in Chandigarh, India provided all microorganisms^53-54. The antimicrobial capability was evaluated using the Broth Dilution Technique. It is a non-automated on-site microbial resistance study. It is done using containers for testing shown in Figure 8.

Macrodillution Method in Tubes

Microdilution format using plastic trays

Figure 8: Broth Microdilution Format using plastic trays which is a standard method of Microdilution. Click here to view Figure

Table 1: Antibacterial Activity

Table 1 denoted as A (Azadirachta indica), B (Mangifera indica), C (Eucalyptus globulus) and D (Fenugreek). Antimicrobial and antifungal activities were assessed, with Table 1 indicating that sample B, synthesized from Mango (Mangifera indica) leaves extract, exhibited the highest antimicrobial activity⁶⁸, while Table 2 reveals that sample C, synthesized from Nilgiri (Eucalyptus) leaves extract, observed the highest antifungal activity⁶⁷.

Table 2: Antifungal Activity Table.

The inherent advantages of the biosynthesis technique extend beyond economic considerations, encompassing its profound eco-friendliness⁵⁵. The diminished reliance on chemical compounds not only mitigates environmental impact but also aligns with the imperative of sustainable practices, contributing to the well-being of future generations. The consequential reduction in chemical usage is particularly significant, given the ubiquitous applications of silver nanoparticles in pharmaceuticals, cosmetics, dyes, and other industries⁵⁶. The affordability of the biosynthesis process translates directly into cost-effective end products, such as medicines and cosmetics, making them more accessible to a broader demographic⁵⁷. The collective adoption of this biosynthesis technique over chemical and physical methods holds transformative potential, not only benefiting human health but also mitigating the ecological footprint associated with traditional synthesis approaches. Thus, advocating for the widespread implementation of biosynthesis emerges as a crucial strategy for fostering a more sustainable and harmonious coexistence between human activities and the environment⁵⁸.

Conclusion

Reported research work has shown the successful synthesis of silver nanoparticles by green methods from various plant extracts aiming to replace conventional chemical compounds and techniques with eco-friendly biological methods. The utilization of natural, low-cost biological reducing agents in green nanotechnology has been demonstrated, a green synthetic route for silver nanoparticles⁵⁹. Synthesized silver nanoparticles were characterized by UV-visible spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD) and Scanning electron microscopy (SEM). The characterization data confirms that the silver nanoparticles have a size of less than 100 nm. The application involved the evaluation of synthesized silver nanoparticles for their potential as highly effective antibacterial and antifungal agents⁶⁵. The microbiological analysis results indicate strong action against specific gram-negative bacterial strains and moderate activity against selected fungus strains. The demonstrated effectiveness of this green synthesis approach highlights its significance in promoting environmentally conscious practices. 

Acknowledgment

The authors expressed sincere gratitude to the Rai University, Ahmedabad and Pandit Deendayal Energy University, Gandhinagar for providing laboratory facilities to complete this research work.

Conflict of Interest

All authors declare no conflict of interest.

Funding Source

This research did not receive any grant. 

Authors’ Contribution

Kinjal Gohil is a research scholar; she has performed all experiments, collected all experimental data, and drafted a manuscript.

Sureshkumar Dhakhda is a corresponding author as well as a research guide; he has provided complete research guidance to research scholars. He has coordinated with the research scholar to generate all experimental data, including methodologies for sampling plants, the synthesis of silver nanoparticles, and the characterization and study of antimicrobial activities. Also provide guidance to scholars on the writing of the paper, correction of the drafted paper, and revision of the manuscript as per the reviewer’s comments.

Vipul Patel has helped the scholar to generate X-ray diffraction (XRD) spectra and interpret the XRD spectra of synthesized silver nanoparticles.

Ajay Rathod has helped to generate and interpret the scanning electron microscopic data of synthesized silver nanoparticles.

Pradeep Kumar Singh has helped to research scholar for the study of antimicrobial activities.

Data Availability Statement

Not applicable because we have already shared all data in manuscript.

Ethics Approval Statement

This study did not involve the use of humans or animals.

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