Biosynthesis and assessment of antibacterial and antioxidant activities of silver nanoparticles utilizing Cassia occidentalis L. seed

This research explores the eco-friendly synthesis of silver nanoparticles (AgNPs) using Cassia occidentalis L. seed extract. Various analytical techniques, including UV–visible spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectroscopy (EDX), were employed for comprehensive characterization. The UV–visible spectra revealed a distinct peak at 425 nm, while the seed extract exhibited peaks at 220 and 248 nm, indicating the presence of polyphenols and phytochemicals. High-resolution TEM unveiled spherical and oval-shaped AgNPs with diameters ranging from 6.44 to 28.50 nm. The SEM exhibiting a spherical shape and a polydisperse nature, thus providing insights into the morphology of the AgNPs. EDX analysis confirmed the presence of silver atoms at 10.01% in the sample. XRD results unequivocally confirm the crystalline nature of the AgNPs suspension, thereby providing valuable insights into their structural characteristics and purity. The antioxidant properties of AgNPs, C. occidentalis seed extract, and butylated hydroxytoluene (BHT) were assessed, revealing IC50 values of 345, 500, and 434 μg/mL, respectively. Antibacterial evaluation against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli demonstrated heightened sensitivity of bacteria to AgNPs compared to AgNO3. Standard antibiotics, tetracycline, and ciprofloxacin, acting as positive controls, exhibited substantial antibacterial efficacy. The green-synthesized AgNPs displayed potent antibacterial activity, suggesting their potential as a viable alternative to conventional antibiotics for combating pathogenic bacterial infections. Furthermore, potential biomedical applications of AgNPs were thoroughly discussed.


Pathogenic bacteria
The fabrication of nanoparticles derived from noble metals has garnered significant attention in recent decades, with gold and silver emerging as primary candidates for synthesis.Among these, silver nanoparticles (AgNPs) have garnered particular interest due to their exceptional attributes including conductivity, catalytic activity, stability, and antimicrobial properties 1,2 .Notably, AgNPs serve as effective antibacterial, antiviral, and antifungal agents, mitigating surgical infections.Moreover, in contemporary research, AgNPs have emerged as promising candidates for anticancer therapeutics 3,4 , facilitating both diagnosis and treatment across various anticancer potential and apoptosis studies against Pa-1 (Human ovarian teratocarcinoma) cell line 5 .
The use of AgNPs in various fields, particularly in medicine and antimicrobial applications, has gained significant attention due to their unique properties.Pathogenic bacterial infections continue to pose a significant threat to public health, and the growing issue of antibiotic resistance underscores the need for alternative antimicrobial agents.AgNPs have demonstrated noteworthy antibacterial properties, and their green synthesis

UV-visible spectroscopy
The biologically synthesized AgNPs from Cassia occidentalis L. seeds extract were analyzed using a UV/Visible Spectrophotometer (Shimadzu 1800) to determine their absorption maxima within the range of 300-600 nm.The resulting data were plotted as wavelength (X-axis) against absorbance (Y-axis) on a graph.

Scanning electron microscopic analysis
The shape, morphology, and distribution of the synthesized AgNPs were assessed using a SEM.For SEM analysis, a minute amount of AgNPs was placed on conductive carbon tape affixed to an aluminum stub, followed by gold sputtering for 3-4 min.

Transmission electron microscopic analysis
The size and surface morphology of the synthesized AgNPs derived from Cassia occidentalis L. seeds extract were characterized using a transmission electron microscope.A droplet of the AgNPs solution was deposited onto a carbon copper grid, and images were captured at magnifications ranging from 6000 to 8000× using a Hitachi instrument (Model: S-3400N) operated at 80 kV voltage.

Energy dispersive spectroscopy analysis
This method is employed for assessing the elemental composition of substances, such as silver nanoparticles.The sample is inserted into a scanning electron microscope fitted with an EDX.Through EDX analysis, researchers gain crucial insights into the elemental makeup of AgNPs, facilitating the characterization and comprehension of their properties across diverse applications.

X-ray diffractometric analysis
The crystalline structure, lattice parameters, and grain size of the synthesized AgNPs were assessed using XRD.The powdered sample of AgNPs was carefully placed in a cavity slide and gently compressed to create a uniform surface.The XRD instrument, operated with data scan software, employed a scan rate of 1.2° per minute.Spectra were recorded within the 5° to 80° range using a CuKα filter (λ = 0.15418 nm) in 2θ/θ scanning mode.The size of the nanoparticles was determined utilizing Scherrer's formula.

Evaluation of antioxidant effect of AgNPs from C. occidentalis
To assess the antioxidant effects, 2 mL of 100 μM DPPH dissolved in methanol was mixed with 2 mL of different concentrations of AgNO 3 , C. occidentalis, and AgNPs.These mixtures were allowed to stand at room temperature for 30 min.Butylated hydroxytoluene (BHT) served as the positive control.Afterward, the absorbance of the samples was measured at 520 nm using a spectrophotometer.The DPPH free radical scavenging percentage was calculated using the following formula: Here, the test/control sample comprised 2 mL of DPPH and 2 mL of AgNO 3 , C. occidentalis, AgNPs, and BHT at various concentrations, while the control consisted of 2 mL of methanol.

Analysis of antibacterial properties
For the extract and nanoparticle sensitivity tests, E. coli, B. subtilis, and S. aureus were employed.The antibacterial properties were studied using agar disc/well diffusion techniques.A Pasteur pipette was used to form 6 mm wells on the culture medium with consistent spacing in the well diffusion method.In the disc diffusion technique, 6 mm blank discs were used on agar culture medium.The wells and discs were then filled with 60 μL of different dilutions of AgNO 3 , C. occidentalis extract and AgNPs.Tetracycline (10 mg/mL) and ciprofloxacin (10 mg/mL) were employed as positive controls in this investigation, with distilled water performing as a negative control (PC-1 and PC-2).After 24 h of incubation at 37 °C, the growth inhibition zone was measured.

Statistical analysis
The experiments were replicated three times, and the data obtained were entered into STATASTICA 7.0 (STASOFT) for analysis.

Synthesis of AgNPs from C. occidentalis
During the synthesis of silver nanoparticles, the addition of AgNO 3 to the prepared extract induced a noticeable shift in the solution's color, turning it into a yellowish brown color, which indicated the formation of AgNPs.The pH of the reaction mixture was recorded as 8.0.This change in color serves as a primary indicator of nanoparticle formation in the suspension.Following this, the resulting mixture underwent centrifugation at 12,000 rpm for 20 min to facilitate phase separation.The sediment obtained was then washed three times with deionized water and once with ethanol to remove any residual biological impurities effectively.

UV-visible spectrophotometer
The AgNPs from C. occidentalis were synthesized in solution and confirmed in the 200-700 nm range using a UV-visible spectrophotometer (Shimadzu UV-1800).The spectra of C. occidentalis seed extract are shown in DPPH free radical scavenging (%) = (control − test) × 100.
Fig. 1A, whereas the spectra of an aqueous solution containing AgNPs are shown in Fig. 1B.The color of AgNPs in aqueous solution was yellowish brown due to that also depends upon the size of particles.There was a single strong peak at 425 nm in AgNPs, but two peaks at 220 and 248 nm in the extract spectra, suggesting he presence of polyphenols and phytochemicals in the solution.

SEM analysis of AgNPs
Surface morphological and nanostructural analyses were conducted using SEM, as depicted in Fig. 2. The SEM micrographs revealed the presence of numerous small aggregates of AgNPs, exhibiting a spherical shape and a polydisperse nature, thus providing insights into the morphology of the AgNPs.

TEM analysis of AgNPs
The size and morphology of the nanoparticles were determined using TEM.TEM images depicted the AgNPs as round, spherical, and occasionally oval-shaped, with slight agglomeration observed at specific locations, as illustrated in Fig. 3A,B.Synthesized AgNPs have particle diameters ranging from 6.44 to 28.50 nm.The AgNPs histograms in C. occidentalis are shown in Fig. 3C.The size of the particles differs significantly.

EDX analysis
The elemental composition of AgNPs is presented in Fig. 4, with EDX measurements conducted at 1-10 keV revealing the presence of Ag (10.01%),P (0.65%), S (0.45%), Cl (0.46%) and C (88.43%).The elemental peaks of Ag were identified at both 1 and 3 keV, providing comprehensive insights into the composition of the studied nanoparticles.

XRD analysis
The XRD analysis was employed to investigate the crystalline nature and composition of AgNPs, as well as the phase purity of the synthesized AgNPs.As illustrated in Fig. 5, the XRD pattern exhibited well-defined

Antibacterial potential of AgNPs
The antibacterial efficacy of plant-derived AgNPs has been thoroughly explored against various microorganisms, as documented in previous studies 37,38 .In this investigation, three pathogenic bacteria were employed to evaluate the antibacterial properties of AgNPs, plant extract, and standard antibiotics, namely tetracycline and ciprofloxacin (PC-1 and PC-2), as summarized in

Possible mechanism of the antibacterial activity of silver nanoparticles
Antibacterial properties of AgNPs arise from various mechanisms, as illustrated in Fig. 8.

A. Disruption of cell membrane:
AgNPs can interact with and disrupt the cell membrane of bacteria.This interaction destabilizes the membrane integrity, leading to leakage of cellular contents and eventual cell death.

B. Generation of reactive oxygen species (ROS):
AgNPs can induce the generation of reactive oxygen species (ROS) within bacterial cells.ROS, such as superoxide radicals and hydrogen peroxide, cause oxidative damage to proteins, lipids, and DNA, ultimately leading to bacterial cell death.
C. DNA damage: AgNPs can penetrate bacterial cells and interact with DNA, leading to DNA damage.This interference with DNA replication and transcription processes can inhibit bacterial growth and viability.

D. Protein denaturation:
AgNPs can interact with proteins in bacterial cells, leading to their denaturation and loss of function.This disruption of essential cellular processes can impair bacterial growth and survival.

E. Inhibition of enzymatic activity:
AgNPs can inhibit the activity of essential bacterial enzymes, such as those involved in energy metabolism and cell wall synthesis.This disruption of enzymatic activity can compromise bacterial viability and survival.with AgNO 3 at a ratio of 1:9 (v/v).The shift from light yellow to dark brown served as an indicator of the surface plasmon resonance (SPR) of metallic silver, indicating the formation of AgNPs.This synthesis process suggested that the plant extract, rich in diverse phytoconstituents, acts as both reducing and capping agents 39 .The pH of the reaction mixture was recorded as 8.0 at that point, aligning with findings from other studies.The highest SPR absorption, observed at 425 nm, indicated that the reaction was concluded when the color transitioned from light yellow to dark yellowish brown.The excitation of the UV-visible band imparts a yellowish-brown color to AgNPs in aqueous solution, with the specific shade dependent on the particle size 29 .In previous studies, the SPR range of silver nanoparticles, typically between 410 and 450 nm, was associated with spherical nanoparticles 40,41 , As per publications, another study 42 identified a peak at 461.02 nm during the synthesis of AgNPs using the seed extract of C. occidentalis.Similarly, the utilization of Pyrostegia venusta and Passiflora vitifolia leaf extracts, containing a variety of phytochemicals, has been proposed for AgNPs synthesis 43,44 .
The SEM micrographs revealed the presence of numerous small aggregates of AgNPs, exhibiting a spherical shape and a polydisperse nature, thus providing insights into the morphology of the AgNPs.Anandalakshmi et al.   www.nature.com/scientificreports/ reported similar shapes, observing even-shaped, spherical AgNPs in SEM images derived from biosynthesized AgNPs from Pedalium murex leaf extract 45 .Hemalata et al. also noted comparable shapes in SEM images of biosynthesized AgNPs from a Cucumis prophetarum leaf extract 46 .
The TEM images depicted the AgNPs as round, spherical, and occasionally oval-shaped, with minor agglomeration observed at specific sites.Particle sizes ranged from 6.44 to 28.50 nm.As per other reports, AgNPs synthesized from D. indica exhibited a spherical morphology with size ranges of 10.0 to 23.24 nm.Although aggregation was evident in the AgNPs, a small fraction displayed dispersion and variations in size 39 .Previous studies indicated that AgNPs derived from I. balsamina and L. camara leaf extracts exhibited spherical shapes with size ranges of 10-30 nm and a polydisperse nature 47 .
The EDX spectra of AgNPs indicated that the sample comprised 10.01% of silver, with a significant peak observed at 3 keV, suggesting the reduction of Ag + ions to Ag°.Additionally, the EDX spectrum revealed the presence of carbon, sodium, chlorine, and other elements, along with the identification of supplementary metallic elements.Similarly, AgNPs derived from R. serrata flower buds extract exhibited a prominent signal for silver, as well as elemental peaks corresponding to phytomolecules, with additional peaks of carbon and oxygen observed 48 .The EDX analysis of AgNPs demonstrated a notable signal for silver, along with other elemental peaks.These additional peaks, apart from silver, may be attributed to the presence of phytomolecules on the external surface of the nanoparticles, playing a crucial role in capping and stabilization.Peaks indicating carbon, oxygen, and other elements may be attributed to atmospheric moisture content.
X-ray diffractometry confirmed the face-centered cubic crystal structure of AgNPs.The XRD patterns exhibited reflection peaks at 33.15°, 39.02°, 45.65°, 65.19°, and 78.90° 2 theta, corresponding to the 101, 111, 200, 220, and 311 Bragg's plane faces, respectively.These results indicate the crystalline nature of the AgNPs suspension, consistent with findings from other studies.Another investigation into the production of silver nanoparticles from C. sativus revealed a similar XRD pattern, with crystalline phases associated with inorganic plant extract components present on the surface of the synthesized AgNPs 5 .
The antioxidant potential of the synthesized AgNPs, aqueous C. occidentalis seed extract, butylated hydroxytoluene (BHT), and AgNO 3 was investigated using the DPPH free radical assay, a widely recognized method for assessing antioxidant activity.DPPH, being a stable compound, serves as a valuable tool in evaluating antioxidant capacity, as it readily accepts hydrogen or electrons.The IC 50 value obtained from this assay serves as an indicator, with lower values indicating stronger DPPH scavenging activity.Our findings revealed that both the synthesized AgNPs and the aqueous extract possess significant free radical scavenging abilities.Interestingly, AgNPs exhibited remarkable scavenging activity, comparable to that of BHT, and surpassed C. occidentalis seed extract and AgNO 3 .These findings are consistent with previous research demonstrating the considerable antioxidant properties of Ag nanoparticles, which effectively neutralize various free radicals, including DPPH 49,50 .Antioxidants play a crucial role in combating free radicals 51 .The DPPH antioxidant assay is a well-established method known for its ability to assess the capacity of compounds to reduce free radicals 52,53 .Stable free radical scavengers, such as DPPH, exhibit an absorbance at 517 nm and undergo a color change from violet to yellow during the reduction process 54 .Free radicals induce cellular damage, posing health risks to both humans and animals 55 .
According to the findings, AgNPs are a good material for use as antibacterial agents against pathogenic bacterial species as also evident by previous research [56][57][58] .Recent findings indicate that silver and copper nanoparticles possess biocidal properties, making them suitable for use as antibacterial coatings on consumer goods 59 .Research has demonstrated that silver nanoparticles can serve as effective antibacterial agents against both gram-positive and gram-negative bacterial infections 60,61 .Silver nanoparticles interact with the bacterial cell wall in a natural manner, disrupting its integrity and causing the breakdown of phosphodiester linkages, ultimately leading to the bacterium's demise.Additionally, silver ions bind to crucial biological components such as sulfur, oxygen, and nitrogen, thereby impeding bacterial growth 62 .

Potential biomedical applications
The versatility of AgNPs in diverse applications, including anti-diabetic, antiviral, antifungal, antibacterial, DNA cleavage, anti-aging, dye degradation, environmental assay indicators, plant growth, and antioxidants, as well as their protective role, is illustrated in Table 2.For example, AgNPs have exhibited promise in regulating glucose levels, presenting therapeutic advantages in diabetes management.Research suggests that AgNPs can modulate insulin signaling pathways, potentially enhancing insulin sensitivity and cellular glucose uptake, thus offering a novel approach for treating diabetes 63,64 .AgNPs also demonstrate significant antiviral properties by disrupting viral attachment and entry into host cells.This mechanism has been investigated against viruses like HIV, influenza, and herpes simplex virus, indicating potential applications in the development of antiviral agents and coatings for medical equipment to mitigate viral transmission 65,66 .The antifungal efficacy of AgNPs has been proven against various fungal pathogens, including Candida species.This suggests their potential utility in antifungal formulations for the treatment of fungal infections, especially in topical applications [67][68][69] .Renowned for their potent antibacterial properties, AgNPs demonstrate effectiveness against both Gram-positive and Gram-negative bacteria.This renders them promising candidates for the development of antimicrobial coatings, wound dressings, and antibacterial agents in medical settings 70,71 .The capability of AgNPs to cleave DNA strands holds significant implications for genetic and molecular research, offering potential applications in targeted drug delivery, gene therapy, and as a tool for understanding DNA structure and function 72,73 .The anti-aging properties of AgNPs, attributed to their capacity to scavenge free radicals and alleviate oxidative stress, present opportunities for potential utilization in skincare formulations.This application could aid in diminishing signs of aging, including wrinkles and fine lines 74 .In environmental contexts, AgNPs have shown the capability to degrade synthetic dyes, proving valuable in environmental remediation endeavors.Potential

Figure 3 .
Figure 3. TEM micrograph of the AgNPs using C. occidentalis at the scale bar corresponds to (A) 20 nm at 100,000× and (B) 20 nm at 40,000×.(C) Particle size histogram (nm) of AgNPs.

Figure 7 .
Figure 7. Antibacterial potential of AgNPs and aqueous extracts of C. occidentalis, positive controlantibiotic tetracycline and ciprofloxacin against B. subtilis, E. coli and S. aureus.

Figure 8 .
Figure 8.A hypothetical illustration of the possible mechanisms of antibacterial activities of silver nanoparticles against bacterial cells.

Table 2 .
Impact of silver nanoparticles from Cassia occidentalis L. and seed extract on multiple functions, including anti-diabetic, antiviral, antifungal, antibacterial, dna cleavage, anti-aging, dye degradation, environmental assay indicators, plant growth and antioxidants and protective role. S.