Green Synthesis of Gold and Silver Nanoparticles Using Invasive Alien Plant Parthenium hysterophorus and Their Antimicrobial and Antioxidant Activities

Abstract

Due to the high energy demands and environmental hazards of physical and chemical methods, it is now essential to produce nanoparticles using plant sources as reducing and stabilizing agents. In this study, silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) were biosynthesized using an aqueous extract of Parthenium hysterophorus aerials as a reducing and stabilizing agent. The synthesized nanoparticles were characterized using UV–Vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and transmission electron Microscopy (TEM). UV–Vis spectroscopy indicates that the peaks of AgNPs and AuNPs are at 422 and 538 nm, respectively. The results of the DLS analysis showed that both Au and AgNPs are monodispersed and stable and have mean hydrodynamic sizes of 53.55 nm and 68.12 nm, respectively. According to an XRD analysis, the generated AgNPs and AuNPs are face-centered cubic crystals with average crystalline diameters of 33.4 nm and 30.5 nm, respectively. TEM image depicted that the synthesized NPs mainly have spherical shapes with particle size in the range of 3.41–14.5 nm for AuNPs and 5.57–26.3 nm for AgNPs. These biologically produced AuNPs and AgNPs were investigated for their antibacterial, antifungal, and antioxidant effects. Both AuNPs and AgNPs were found to strongly influence the growth of bacterial pathogens, with a maximum zone of 22.3 and 19.7 mm in Escherichia coli and a minimum zone of 11.7 and 10.3 mm in Salmonella enterica, respectively. The synthesized AuNPs and AgNPs reduce the numbers of viable fungi by 51.06% and 47.87%, respectively. The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay revealed that the synthesized AuNPs and AgNPs have significant radical scavenging ability with 88.75% and 86.25% inhibition and 33.62 μg/mL and 42.86 μg/mL of IC₅₀, respectively. Therefore, an aqueous extract of aerial parts of P. hysterophorus can be a suitable precursor for synthesizing AuNPs and AgNPs, with numerous applications. Due to their smaller size, AuNPs have better antimicrobial and antioxidant activities than AgNPs. This study supports the conservation by a utilization strategy of invasive alien plant species control and management (such as P. hysterophorus) for biodiversity conservation and environmental sustainability.

1. Introduction

The field of nanotechnology is expanding and has enormous potential to enhance human welfare. It primarily focuses on producing nanoparticles (NPs) of diverse sizes, shapes, chemical compositions, and controlled dispersion for enhancing human life (Mohanpuria et al., 2008) [1]. Physical, chemical, and biological techniques are generally used to synthesize NPs. Simple, well-defined NPs can be formed using chemical and physical techniques, but they are expensive and detrimental to the environment [2,3] (Bhattacharya and Rajinder 2005; Bachheti et al., 2022). Due to these issues, nanotechnologists are working to develop environmentally friendly procedures for producing NPs. Green synthesis is economical, easy to scale up, and is environmentally sustainable for large-scale NPs synthesis because it does not require the use of toxic chemicals, high temperatures, or power [4] (Dhuper et al., 2012). Additionally, costly and unfriendly reagents and solvents can be avoided [5] (Roy and Das 2015). Metal nanoparticles (MNPs) have recently received much attention due to their unique optical, magnetic, and catalytic characteristics [6,7] (Suganthy et al., 2018; Bachheti et al., 2021). In particular, because of their elite features such as controllable size, dispersity, durability, biocompatibility, and good adsorbing power, gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) are considered to be the most promising nanoproducts for biological applications [8,9,10,11] (Wang et al., 2021; Manjari et al., 2017; Husen et al., 2019; Singh et al., 2018).

Biological approaches are widespread and favored for the reasons stated above. As a result, many commercial AgNP and AuNP products are currently on the market [12] (Vance et al., 2015). These NPs are produced using a variety of physicochemical and biological techniques. Microbe-mediated synthesis is not commercially viable among the numerous biological NPs synthesis methods due to the need for extreme aseptic conditions and maintenance. Plant extracts are preferable to microorganisms for this application since they are simpler to improve, constitute less of a biohazard, and do not require the maintenance of cell cultures [13] (Kalishwaralal et al., 2010). Thus, it is an intriguing concept to examine the viability of an aggressive invasive plant species, P. hysterophorus, for the production of NPs in Ethiopia. P. hysterophorus is a widespread invasive plant species in this area that lowers agricultural yields, displaces beneficial pasture species, and lowers cattle productivity by reducing the amount of grazing land available [14,15] (Mamgain 2016; McConnachie and Strathie 2011).

P. hysterophorus is an invasive alien plant species believed to be endemic to Southern and Northern America. It was initially noted in Ethiopia in 1988 near Desse in northeastern Ethiopia and in Diredawa and Harerge in eastern Ethiopia [16] (Tamado and Milberg 2000). It was distributed as a contaminant of grain and other crop items and by farm equipment to other countries. Its concern has been raised in Australia, India, China, Kenya, Ethiopia, and the West Indies. The upright ephemeral herb P. hysterophorus has deep tap roots, branching stems that eventually turn woody and hairy, leaves that are 3–20 cm long and 2–10 cm wide, and flowers. It can reach heights of 1.5–2 m. It grows in various environments, such as disturbed and degraded fields, banks of streams, and rivers, and has a bluish or grayish-green appearance [17] (Guyana and Paraguay 2014). It can only reproduce through seeds, and because a single plant typically yields 15,000 seeds, it is known to be highly prolific.

In this study, as an alternative to traditional industrial feeds, we propose to investigate the ability of the synthesis of NPs to overcome probable ecological hazards. This economical approach to managing invasive alien plants will also pave the door for job development, establishing a local manufacturing base, accelerating economic growth, environmental sustainability, poverty alleviation, cost savings and safeguarding biodiversity [18,19] (Rawat, 2017; Rawat et al., 2023). It is important to remember that separate papers and reviews on this herb can be collected from the literature databases. For instance, reports are available on its allelopathic properties, reduction in chlorophyll content of crop plants, allergic study, biodiversity loss, for xylanase production, as compost, bioethanol production, and the removal of heavy metals [20,21,22,23,24] (Singh et al., 2003; Lakshmi and Srinivas 2007; Morin et al., 2009; Akhtar et al., 2010; Akter and Zuberi 2009).

P. hysterophorus is well-known for its phytochemicals, especially alkaloid, steroid, phenolic compound, flavonoid, protein, amino acids, sesquiterpene, lactones, and parthenin that contribute to its allelopathic capacity, antioxidant activity, and allergic reactions [25] (Marimuthu and Ravi 2014). Titanium dioxide, silver, zinc oxide, and copper oxide NPs have all been produced due to the biochemicals of P. hysterophorus [26,27,28] (Thandapani e al. 2017; Ahsan et al., 2020; Datta et al., 2017. There are reports on the synthesis of AgNPs, and their biological activities separately. However, there is no comparative study on the antimicrobial and antioxidant activities of AgNPs and AuNPs synthesized using P. hysterophorus. Hence, this study aimed to investigate the synthesis of AuNPs and AgNPs using an aqueous extract of the aerial parts of P. hysterophorus, and to compare their antimicrobial and antioxidant activities.

2. Materials and Methods

2.1. Materials

The experiment only utilized analytical-grade chemicals. Silver nitrate solution (99.9%), AgNO₃, tetrachloroauric acid trihydrate (99.9%), HAuCl₄ · 3H₂O (both are products of LobaChemie Pvt. Ltd., Mumbai, India), sodium hydroxide, NaOH (Blulux, Haryana, India), potassium bromide, KBr (Uvasol, Frankfurt, Germany), ascorbic acid (99%), C₆H₈O₆ (Sigma Aldrich, Darmstadt, Germany), methanol (99%), CH₃OH (Sisco research laboratories, Mumbai, India), ethylenediaminetetraacetic acid (99%), EDTA (Labort Fine chem Pvt. Ltd., Surat, Gujarat, India), 2,2-diphenyl-1-picrylhydrazyl, DPPH (Himedia Laboratories Pvt. Ltd., Gujarat, India), Muller Hinton agar (Oxoid CM, Hampshire, UK), buffered peptone water (Tecno Pharma Chem, Gujarat, India), plate count agar (Himedia Laboratories Pvt. Ltd., Gujarat, India), and others were used.

2.2. Sample Collection and Plant Extraction

P. hysterophorus aerial parts were collected in February 2020 from Addis Ababa Science and Technology University, Addis Ababa, Ethiopia. The plant material was properly cleaned with distilled water after being repeatedly washed with tap water to eliminate all dirt and undesirable visible particles, and then air-dried entirely in the shade. The dried plant material was ground with a mortar and pestle into powder form. The plant extract was produced by mixing 10 g of the powdered material with 100 mL of double distilled water, then the mixture was boiled for 20 min at 80 °C with a magnetic stirrer. After boiling, the extract was cooled and filtered through filter paper three times to remove impurities. After centrifuging the extract, the clear supernatant solution was collected and stored at 4 °C [29] (Latha et al., 2019).

2.3. Green Synthesis of AgNPs and AuNPs Using Extracts of P. hysterophorus

In order to synthesize AgNPs and AuNPs, 10 mL of P. hysterophorus aerial part extract was added to 90 mL of each 1.0 mM aqueous AgNO₃ and HAuCl₄ 3H₂O solution and stirred continuously at 70 °C for 60 min. The reduction process began with a color change and the solution turned to deep red and deep purple for AgNPs and AuNPs, respectively, confirming the formation of these NPs. The reaction product was submitted to a UV–Vis analysis, showing the absorbance in the 300 to 800 nm wavelength range. The upper phase of these NPs was removed after a 30 min centrifugation at 4500 rpm and three washes with water and ethanol to remove the impurities. The pellets produced were dried for 8 h at 100 °C in an oven. The dried AgNPs and AuNPs were stored in a light-proof container up until use [30] (Khan et al., 2020). The synthesis of AgNPs was optimized to produce the NPs with the optimal size and morphology. Five main factors, including reaction time (15, 30, 45, 60, and 100 min), metallic ion concentration (0.5, 1, 2, 3, 4, and 5 mM), pH (4, 6, 8, 10, and 11), extract concentration (5, 10, 15, 20, and 25 %) and reaction temperature (25, 40, 50, 60, 70, and 80 °C) were investigated by UV–Vis measurement to determine the optimal conditions. The optimizations were performed by one-variable-at-a-time method.

2.4. Characterization of Nanoparticles

The UV–Vis Spectrophotometer (JASCO V-770) was employed to examine UV–Vis spectra between 300 and 800 nm at a scanning rate of 40 nm/min [31] (Balasubramanian et al., 2020). An X-ray diffractometer (Rigaku, Ultima, IV, USA) was used to investigate AgNPs and AuNPs. The samples were centrifuged for 15 min at 4500 rpm to form pellets, which were subsequently ground into powder. This powder was stacked in the X-ray diffraction (XRD) cubes and examined using the XRD apparatus following the earlier study [32] (Zangeneh and Zangeneh 2020). FTIR spectrometer, Nicolet iS50R (Thermo Fisher Scientific, Waltham, MA, USA), was used to determine the FTIR analysis. The dried extract, AgNPs, and AuNPs were combined and pelleted with KBr (FTIR grade), and then FTIR scans were performed with a resolution of 0.09 cm⁻¹ over a wavelength range of 4000–400 cm⁻¹.

To determine the AgNPs and AuNPs’ particle size distribution, the powder was diluted with double distilled water (1 mg/100 mL) and ultrasonically dispersed. On the other hand, the particle size distribution was measured using a dynamic light scattering (DLS) instrument called Zetasizer Nano ZS ZEN3600 (Malvern, Worcestershire, UK). After homogenization and preparation for DLS measurement, the solution was added to the device [33,34] (Zhang et al., 2016; Qidwai et al., 2018). JEOL 2100F transmission electron microscopy (TEM) was used to examine the morphology and particle size of the NPs. This was conducted by coating a drop of NP-containing solution to carbon-coated copper grids, vacuum-drying them for an entire night, and then loading them into a specimen holder [35] (Soliman et al., 2023).

2.5. Antibacterial and Antifungal Activities

Antibacterial and antifungal activities were conducted in Ethiopian Conformity Assessment Enterprise (ECAE), microbiological laboratory. The agar-well diffusion method was used to conduct the antibacterial tests on a variety of Gram-positive (S. aureus and E. faecalis) and Gram-negative (E. coli and S. enterica) bacteria [36] (Roy at al., 2019). The bacterial strains were subcultured in a Petri dish and kept in broth before testing. The Mueller Hinton (MH) agar plates were first loaded with 100 mL of plant extract, AgNPs, and AuNPs, with 100 mL of distilled water acting as the negative control. The plates were sealed and kept at 37 °C for 24 h while incubating face up. Finally, a measurement of the inhibition zone’s diameter (in mm) was made in triplicate.

A plate-counting technique was used in addition to the agar-well diffusion method to evaluate fungicidal and bactericidal activities. Around 1.5 × 10⁷ CFU of E. coli, S. enterica, S. aureus, E. faecalis, and A. niger were incubated in 1 mL of AgNPs and AuNPs. Controls were prepared using the same procedures without the NPs. Samples of 1 mL were placed on a nutrient agar plate after being treated for 1 h with the AgNPs and AuNPs. After that, the plate was incubated for 48 h at 37 °C. The colonies were then counted to assess the AgNPs and AuNPs’ activity against bacteria and fungus to that of control plates [37] Chamakura et al., 2011).

2.6. Antioxidant Activity

Antioxidant activity of synthesized AgNPs and AuNPs was carried out using DPPH assay. Samples comprising 20, 40, 60, 80, and 100 µg/mL were made from the stock solution of the NPs (1 mg/mL) and mixed with 1 mL of 0.1% DPPH. At room temperature, the reaction mixture was incubated for 30 min. Any antioxidant reacts with DPPH to reduce it, which reduces the color intensity. There was a decrease in absorbance at 517 nm. Ascorbic acid was used as a positive control [38] (Velammal et al., 2016). The experiment was performed three times, and the scavenging activity was determined as a percentage of inhibition using the formula below (absorbance is ‘A’):

Percentage of Inhibition = (A of control − A of sample/A of control) × 100

2.7. Statistical Analysis

All experiments were executed three times, and the means and standard errors were obtained. One-way ANOVA was used to assess the statistical significance of differences between values of the treated and untreated (control) groups. Differences with p < 0.05 were accepted as statistically significant.

3. Results and Discussion

3.1. Green Synthesis of Silver and Gold Nanoparticles

Focusing on a greener method for the synthesis of NPs has been suggested in various papers over the past few years. AgNPs and AuNPs production were observed after the extract was mixed with the AgNO₃ and HAuCl₄ solution. The formations of AgNPs and AuNPs were preliminarily confirmed by the change of color when silver nitrate and auric chloride trihydrate solution were mixed with plant extracts. The light yellow to dark purple color shift, which is unique to AuNPs and resembles the collective oscillations of electrons in NPs upon interaction with light, confirmed the reduction in Au³⁺ to Au⁰ [39] (Singh et al., 2016). As a result, the reaction mixture’s appearance of a dark purple color indicated the formation of AuNPs. On the other hand, the complete color shift from colorless to deep red indicated the formation of AgNPs. The intense red color is due to the excitation of the localized surface Plasmon resonance in the NPs. Ag⁺ and Au³⁺ were reduced and stable NPs were produced within 60 min at 70 °C of the reaction. This implies that the reducing agents reduced the silver and gold salts in the reaction mixture from the P. hysterophorus extract.

3.2. Optimization of Silver Nanoparticle Synthesis

The optimization technique is critical to ensure the stability of any preparation process and the quality of MNPs generated under ideal circumstances, particularly when using aqueous plant extracts as reducing and capping agents [40] (Doan et al., 2020). In order to find the optimum experimental conditions for the synthesis of AgNPs using the P. hysterophorus aqueous aerial extract as a reducing and stabilizing agent, it was necessary to investigate the effects of pH, temperature, reaction time, P. hysterophorus, and precursor silver ion concentrations. Position, symmetry, and the narrowness or broadness of the localized surface Plasmon resonance (LSPR) band were all considered in this study to pick the best parameters. According to [41] Velgosová et al. (2016), the morphology, size, and aggregation of NPs can all be affected by the characteristics of the LSPR band. A symmetrical and narrow LSPR band often implies the existence of uniformly shaped NPs with a small size range. In addition, the LSPR band may shift or change in shape, absorbance capacity may rise or fall, and the maximum absorbance wavelength values may vary [42] (Sohrab et al., 2016).

3.3. The Effect of Plant Extract Concentration

Various extract concentrations were used to investigate the effect of P. hysterophorus aerial extract concentration on the production of AgNPs. The assessment for extract concentration was performed by mixing 10 mL of 5%, 10%, 15%, 20%, and 25% (v/v) P. hysterophorus aerial extract with 90 mL of 1 mM AgNO₃ solution at 60 °C and incubating for 1 h. The UV–Vis absorption spectra of AgNPs produced using various extract concentrations are shown in Figure 1A. A blue shift from 426 nm to 416 nm was noticed as the extract concentration rose from 5 to 15% (v/v). The absorption peak also became sharper. The peak’s sharpness suggests that the produced AgNPs are spherical and have a uniform distribution. Additionally, the blue shifts demonstrated a decrease in the size of the produced AgNPs [43] (Mat Yusuf et al., 2020). However, the absorption band significantly shifted toward the red area when the extract concentration was above 15% (v/v), showing at 418 nm for 20 and 25% (v/v). According to earlier research, this is most likely caused by the NPs’ agglomeration and the rapid reduction in silver ions [44] (Shaik et al., 2018). In accordance with the findings, 15% (v/v) of the extracts were chosen to optimize the next parameter.

3.4. The Effect of AgNO₃ Concentration

AgNO_3′s effect on the production of AgNPs was evaluated by varying the AgNO₃ concentration (Figure 1B). AgNO₃ concentrations of 0.5, 1, 2, 3, and 5 mM were examined for an hour at 60 °C in the presence of 15% (v/v) P. hysterophorus aerial extract. The results showed that different maximum wavelengths and peak intensities were obtained at different concentrations. Comparing the peaks obtained, it was found that 1 mM AgNO₃ was the best concentration of the precursor to be used to synthesize the AgNPs with a sharp peak and a more blue shift as compared to the (0.5, 2, 3, 4, and 5 mM). This finding is consistent with the amount of AgNO₃ used to fabricate AgNPs from an aqueous extract of Prunus persica leaves [45] (Patra and Baek 2016). The metal ion concentration significantly influences the synthesis of MNPs. Since no additional reducing agent is present after the optimum concentration of AgNO₃, there is an excess of silver ions with an increase in AgNO₃ concentration. As a result, the peak’s symmetry was distorted, flattened, and the NPs were aggregated, indicating the non-uniformity in particle size. This investigation concludes that the optimum silver nitrate concentration suitable for NPs synthesis is 1 mM because the peak is sharper and blue-shifted, and then used for the next experiment.

3.5. The Effects of pH

The reaction mixture’s color and the SPR bands’ intensities were found to be pH-dependent. In the production of AgNPs, the effect of various pH levels was investigated. HNO₃ and NaOH solutions of 0.1 N each were used to adjust the pH. The resulting solutions’ absorbance was measured spectrophotometrically. The intensity of the absorption peaks increased and the spectra became intense and sharp as the pH of the solution was raised from acidic to basic (pH 4–10). According to earlier studies, the acid medium stimulates the synthesis of AgNPs with large particle sizes, whereas the AgNPs were produced with small sizes in the alkaline medium [46] (Aboelfotoh et al., 2017). Figure 1C displays absorption spectra for various pH values (4, 6, 8, 10, and 11). Larger-sized NPs are produced at low pH levels, which have little effect on the amount of AgNPs formed in the solution. In the acidic condition, the NPs’ sizes increase as the peak becomes broader. The increase in pH results in a narrow peak at pH 10. Thus, the optimum pH for maximizing NPs’ synthesis was 10. Therefore, we assume that the preferred media for controlling particle size is basic.

3.6. The Effect of Temperature

It was also investigated how temperature affected the synthesis of AgNPs. Different temperatures, including 25 °C, 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C, were evaluated, while the concentrations of metal ions, plant extract, pH, and reaction time (1 mM, 15%, pH 10, and 1 h) were held constant. According to the results of these analyses, a temperature of 70 °C was optimum for the synthesis of AgNPs. AgNPs’ absorbance rose with temperature, reaching its peak at 70 °C (Figure 1D). A prior investigation on the synthesis of AgNPs utilizing the leaves and stems of Clinacanthus nutans revealed that the production of AgNPs increased proportionally with temperature [43] (Mat Yusuf et al., 2020). The highest absorbance peaks of the produced AgNPs were blue-shifted from 434 nm at 25 °C to 412 nm at 70 °C, indicating that the size of NPs decreases with increasing temperature. In accordance with the previous article, the synthesis of smaller NPs was facilitated because as the temperature was raised, the rate at which the reactants were being consumed during the synthesis process also increased.

3.7. The Effect of Reaction Time

The formation of AgNPs in the reaction mixture was observed using UV–Vis spectra at various time intervals, along with a change in the color of the solution. AgNPs’ UV–visible spectra are depicted as a function of time in Figure 2.

The graph shows that the UV–visible spectra’s intensities increase in the first stage (0–60 min) as reaction time increases and shifts toward the blue, indicating an increase in AgNPs concentration and the synthesis of small and spherical NPs. There is a slight shift in the maximum wavelength toward larger values when the reaction time is longer than 60 min, indicating the formation of larger AgNPs and the agglomeration of NPs. Therefore, the best reaction time for the biosynthesis of AgNPs was 60 min [47] (Kumar et al., 2017).

3.8. Characterizations of the Synthesized Nanoparticles

3.8.1. UV–Vis Spectroscopy Analysis

UV–Vis spectroscopy is an essential technique for confirming the fabrication of AgNPs and AuNPs by referring to the optical properties and electronic structures of the produced NPs. Each NP surface has an oscillating electron cloud that can absorb electromagnetic waves of a specific frequency. This phenomenon, known as localized surface plasmon resonance (LSPR), is recorded as electromagnetic wavelengths by a UV–Vis spectrophotometer [43] (Mat Yusuf et al., 2020). UV–Vis spectroscopy was used to detect the reduction in Ag and Au ions after exposure to the aerial extract of P. hysterophorus. According to the UV–Vis spectrophotometric investigation, the final reaction mixture had maximal absorption peaks at 422 nm and 538 nm, typical for AgNPs and AuNPs, respectively (Figure 3A). According to [48], Arya et al. (2018), AgNPs with absorbance maxima of around 420 nm often has a spherical shape. Therefore, the AgNPs produced here were spherical because the absorption maxima were close to 420 nm.

3.8.2. FTIR Spectral Analysis

The main functional groups in plant extract were identified using FTIR spectra. Several papers explain the roles of these essential biomolecules in the stability and capping of NPs [49,50] (Niraimathi et al., 2013; Krishnaraj et al., 2010). Their potential role in the reduction process during the synthesis of AgNPs and AuNPs and the stability of the NPs was also investigated. For the plant extract, the spectra revealed absorption peaks at 3281 cm⁻¹ and 1636.79 cm⁻¹, confirming the presence of capping and stabilizing agents. The analysis of the FTIR spectra in Figure 3B revealed that the major peaks of the P. hysterophorus extract were shifted in AgNPs and AuNPs. The interaction of bulk AgNO₃ and HAuCl₄ with those chemical functional groups of the plant extract is indicated by the slight change in the vibrational bands of AgNPs and AuNPs from the corresponding vibrational bands of P. hysterophorus extract. In the case of NPs, a shift in the peak was observed from 3281 to 3330 and 3327 cm⁻¹ and 1636 to 1637 cm⁻¹ for Au and AgNPs, respectively, signifying the binding of silver and gold ions with hydroxyl (OH) groups and carboxylate groups of the extract [51,52] (Kumar et al., 2011; Prabhu and Poulose 2012). The bioreduction in Au³⁺ and Ag⁺ to AuNPs and AgNPs may be mediated by major functional groups such as C–O, N–H, and O–H in various chemical classes such as flavonoids, triterpenoids, polyphenols, proteins, and pigments in the plant extract [53].(Venu 2011).

3.8.3. Particle Size and Zeta Potential Measurement

Dynamic light scattering (DLS) analysis measured the hydrodynamic size of the produced AgNPs and AuNPs. According to DLS measurements, the produced AgNPs and AuNPs were monodispersed and had an average hydrodynamic size of 68.12 ± 0.535 nm for AgNPs and 53.55 ± 0.483 nm for AuNPs (Figure 4). AgNPs synthesized in this study have a smaller hydrodynamic size than the AgNPs synthesized by [27], Ahsan et al. (2020), which have a hydrodynamic size of 187.87 ± 4.89 nm. AgNPs and AuNPs had polydispersity indices (PDIs) of 0.471 ± 0.003 and 0.395 ± 0.001, respectively (

Table 1. Particle size, polydispersity index, and zeta potential values of the synthesized AgNPs and AuNPs.

Samples	Polydispersity Index (PDI)	Particle Size (nm)	Zeta Potential (mV)
AgNPs	0.471 ± 0.003	68.12 ± 0.535	−25.7 ± 0.602
AuNPs	0.395 ± 0.001	53.55 ± 0.483	−30.4 ± 0.327

).

The DLS uses a small volume of samples to estimate the average hydrodynamic size of NPs in liquid suspension. The Brownian motion theory is used in this measurement to determine the size of the particles. The random movement of particles in a gas or suspension is known as Brownian motion. The average size of the NPs was determined by analyzing the dynamic variation of the light scattering intensity and velocity movement of the particles in suspension [54] (Murdock et al., 2007).

The estimated PDI values lie within the ranges of 0 and 1, where 0 denotes monodispersed and 1 denotes polydispersed [54] (Murdock et al., 2007). With little particle aggregation, this finding demonstrates that the produced AgNPs and AuNPs were in a monodisperse phase.

The electric charge on an NP’s surface is measured and quantified using zeta potential. The values of the zeta potential can determine the stability of the MNPs. Figure 4B,D and Table 1 display a negative zeta potential in AgNPs and AuNPs. Polyphenolic compounds and other bioactive substances adhering to the surface of AgNPs and AuNPs may cause a negative charge [55] (Moldovan et al., 2016). Particles are prevented from aggregating by a strong repulsive force produced by a high zeta potential [43] (Mat Yusuf et al., 2020). These high negative zeta potential values demonstrated that the colloidal solutions of AgNPs and AuNPs had a good stability.

3.8.4. XRD

As shown in Figure 5A,B, XRD analysis was used to determine the crystallite size and crystalline nature of AgNPs and AuNPs. Five Bragg’s reflection peaks were observed in the XRD pattern of powdered AgNPs at 2θ values of 38.18°, 44.25°, 64.18°, 77.38°, and 81.11°. The Joint Committee on Powder Diffraction Standards (file no. 04-0783) states that the face-centered cubic (FCC) structure of the biosynthesized AgNPs has five main lattice planes with the numbers (111), (200), (220), (311), and (222).

In the case of AuNPs, XRD peaks at 38.46°, 44.43°, 64.20°, and 77.24° were seen. These peaks corresponded to the (111), (200), (220), and (311) planes, respectively, and are related to the FCC structure of the gold metal, which is supported by the JCPDS file sno. 04-0784. These results agree with an earlier report [9] (Manjari et al., 2017).

Using the Debye–Scherrer equation, it was possible to determine the average crystal size of AgNPs and AuNPs from the FWHM (full-width half maximum) of Bragg’s reflections [56] (Borchert et al., 2005):

$$D=\frac{K\lambda }{\beta \cos \theta }$$

where D is the average NPs crystal size, K is Scherrer’s constant, equal to 0.9, $\lambda$ is the X-ray source’s wavelength, equal to 0.15406 nm, and $\beta$ is the peak’s full width at half maximum (FWHM) in radians at Bragg’s angle, $\theta$. The AgNPs and AuNPs’ average crystalline diameters were found to be 33.4 and 30.5 nm, respectively.

3.8.5. TEM

The morphological characteristics of the biosynthesized AgNPs and AuNPs, including their size, shape, and other features, were examined using transmission electron microscopy (TEM). TEM images of the NPs are shown in Figure 6A,B. According to the TEM pictures, the sizes for AuNPs are in the range of 3.41–14.5 nm, and for AgNPs, they are 5.57–26.3 nm (Figure 6A,B). Using TEM and DLS images to determine the NPs’ sizes, they disagreed. This is due to the NPs accumulating, which decreases the accuracy of the DLS analysis because there are larger particles present, increasing light scattering and changing the measured particle size to larger values [57] (Souza et al., 2016). The majority of the NPs were found to be spherical, as evidenced by the TEM images.

3.9. Application of the Synthesized Silver and Gold Nanoparticles

3.9.1. Antibacterial Activity

Method One

E. coli (ATCC 25922), E. faecalis (ATCC 51299), S. aureus (ATCC 25923), and S. enterica (ATCC 13311) were used to test the antibacterial activity of the synthesized AuNPs and AgNPs. Zones of inhibition in AgNPs were 10.3–19.7 mm in size, whereas zones in AuNPs were 11.7–22.3 mm (

Table 2. Antibacterial activity of AuNPs and AgNPs on some human pathogenic bacteria by disc diffusion assay.

No.	Microorganisms	Zone of Inhibition Produced (mm)
		AuNPs	AgNPs	PE
1	E.coli	22.3 ± 0.58	19.7 ± 0.58	15.7 ± 1.15
2	S. enterica	11.7 ± 1.53	10.3 ± 1.53	9.7 ± 1.53
3	E. faecalis	19.3 ± 0.58	18.3 ± 2.52	12.3 ± 2.08
4	S. aureus	16.0 ± 1.00	14.7 ± 0.58	13.7 ± 0.58

, Figure 7A,B). E. coli was where the antibacterial impact was most noticeable. Bacteria and fungi are adversely affected by AuNPs. They adhere firmly to the surface of bacteria, resulting in significant cell injury, total destruction of flagella, accelerated biofilm development, and biofilm aggregation [58] (Ghosh et al., 2008). Compared to AgNPs, AuNPs are smaller. The smaller size of AuNPs is thought to be the cause of their increased activity. Smaller particles produce more antibacterial activity [59] (Sathiyaraj et al., 2021). The results indicated that the AgNPs and AuNPs synthesized using P. hysterophorus effectively encounters the growth of tested pathogens.

Additionally, compared to other studies, the antibacterial activity in the current study was significantly higher. Zones of inhibition smaller than 15.5 mm in diameter were observed for Trianthema decandra AgNPs and AuNPs that exhibited efficacy against E. coli, S. aureus, and E. faecalis [60] (Geethalakshmi and Sarada 2012). The outcomes of the current study point to improved antibacterial properties. The present study’s results indicate enhanced antibacterial effects.

Method Two

Biosynthetic AgNPs and AuNPs significantly inhibited bacterial growth. Figure 8 shows the number of live bacteria in the culture medium at the same concentration of AgNPs and AuNPs, proving that both AuNPs and AgNPs have a significant antibacterial effect. As shown in

Table 3. Antibacterial and antifungal activity of AuNPs and AgNPs by plate count method.

	N Before	AuNPs		AgNPs
		N After	% Reduction	N After	% Reduction
E. coli	1.1 × 108	8.3 × 105	99.24	1.2 × 106	98.91
E. faecalis	3.0 × 108	1.8 × 107	94	2.7 × 107	91
S. aureus	2.4 × 108	4.0 × 107	83.33	5.6 × 107	76.67
S. enterica	1.8 × 107	7.8 × 106	56.67	8.4 × 106	53.33
A. niger	9.4 × 104	4.6 × 104	51.06	4.9 × 104	47.87

, AgNPs and AuNPs reduce the number of viable E. coli by 98.91 and 99.24%, S. aureus by 76.67 and 83.33%, E. faecalis by 91 and 94% and S. enterica by 53.33 and 56.67%, respectively. Hence, both AgNPs and AuNPs are highly effective against E. coli. Compared to AgNPs, AuNPs have better bacteria-killing ability. S. enterica is more resistant to both NPs than E. coli, S. aureus, and E. faecalis as shown in Figure 8.

Antifungal Activity

The plate count method investigated the antifungal activity of AgNPs and AuNPs produced by P. hysterophorus extract against Aspergillus niger (ATCC 64974). According to the findings, AuNPs are more effective in reduction than AgNPs. When treated with AgNPs and AuNPs, the number of live fungi decreased by 47.87 and 51.06%, respectively, as shown in Figure 9 and Table 3, demonstrating that both AgNPs and AuNPs have potent antimicrobial activities.

Antioxidant Activity

Antioxidants, also called radical scavengers, can reduce DPPH free radicals [61] (Sharad et al., 2014). Reactive oxygen species can harm cells, but antioxidants protect against this by neutralizing free radicals before they can cause harm and repairing some of the damage already inflicted on particular cells. Free radical scavengers such as phenolic compounds and vitamins are abundant in medicinal plants. P. hysterophorus is believed to be a potential antioxidant since it contains several crucial chemical compounds. The antioxidant activity of the produced AgNPs and AuNPs was investigated using the DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay method. The stable free radical that is DPPH has a purple color and is nitrogen-centered. It is converted into a stable molecule (DPPH-H) with a yellow color when it interacts with an antioxidant that can donate an electron to the DPPH radical [62] (Hristea et al., 2006). A UV spectrophotometer can be used to measure purple color deterioration at 517 nm.

As the concentration of ascorbic acid, plant extract, AgNPs, and AuNPs increased, the free radical scavenging activity also increased. Figure 10 displays the percentage of free radicals that were inhibited. Plant extract inhibits 68.75% of free radicals at a concentration of 100 μg/mL, whereas standard ascorbic acid, AuNPs, and AgNPs showed 91.25, 88.75, and 86.25% inhibition, respectively. Standard ascorbic acid is the most effective antioxidant at this concentration, followed by AuNPs, AgNPs, and plant extract. According to [38], Velammal et al. (2016), the antioxidant activity of AuNPs and AgNPs demonstrated up to 87.34 and 78.17% of inhibition, respectively. In this work, we found that at a concentration of 100 μg/mL, AuNPs and AgNPs have an inhibition of 88.75 and 86.25%, respectively. Thus, the present study has a better antioxidant activity at low concentrations compared to the above report.

DPPH activity of plant extract, AgNPs, and AuNPs showed IC₅₀ values of 71.25, 42.86, and 33.62 μg/mL, respectively. AgNPs synthesized using Mangifera indica seed aqueous extract have an IC₅₀ value of 544 μg/mL [63] (Donga and Chanda 2021). Therefore, in comparison to the above report, the current study has a better radical scavenging activity at lower concentrations.

4. Conclusions

In this study, the extract of the aerial part of P. hysterophorus was used as a reducing, stabilizing, and capping agent in the biosynthesis of AuNPs and AgNPs. This approach was simple, economical, ecofriendly, non-toxic, and energy-efficient. The biosynthesized NPs were characterized by UV–Vis, DLS, XRD, TEM, and FTIR. The synthesized AuNPs and AgNPs have been found to have a maximum absorbance at a wavelength of 538 and 422 nm, respectively. FTIR spectra revealed that biomolecules act as reducing and capping agents for the biosynthesis of AgNPs and AuNPs. DLS and zeta potential analyses revealed that the synthesized AgNPs and AuNPs are stable with a particle size of 68.12 and 53.55 nm, respectively. TEM images also depict that the nanoparticles are spherical in shape. Both AgNPs and AuNPs have desirable antibacterial activity against E. coli, E. faecalis, and S. aureus and also have potential antioxidant activity. The percentage of the inhibition of free radicals is found to be 88.75% and 86.25 %, with an IC₅₀ of 33.62 μg/mL and 42.86 μg/mL, respectively. Due to their smaller size compared to AgNPs, AuNPs show superior antibacterial and antioxidant properties. The overall results show that biologically produced AgNPs and AuNPs have potential antibacterial and antioxidant properties, indicating that nanoparticle synthesis could be one of the utilization of the noxious invasive alien plant Parthenium hysterophorus. The present study is useful for the conservation by the utilization strategy of invasive alien plants’ control and management, thereby contributing to biodiversity conservation and environmental sustainability.