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Green Synthesis of Zinc Nanoparticles Using Phaseolus vulgaris

Extract: Evaluation of Antimicrobial, Anticancer, and in Silico

Properties

Nishant Solanki

, Dr. Sandesh Chibber

Department of Biotechnology, Mehsana Urban Institute of Sciences, Faculty of Science, Ganpat

University, Mehsana - Gandhinagar Highway, Ganpat Vidhyanagar – 384012 Gujarat, (India)

Assistant Professor Department of Biotechnology, Mehsana Urban Institute of Sciences, Faculty of

Science, Ganpat University, Mehsana - Gandhinagar Highway, Ganpat Vidhyanagar – 384012 Gujarat,

(India)

Corresponding Author

DOI: https://doi.org/10.51584/IJRIAS.2025.101100056

Received: 25 November 2025; Accepted: 02 December 2025; Published: 12 December 2025

ABSTRACT

This study reports the green synthesis of zinc nanoparticles (ZnNPs) using fresh Phaseolus vulgaris leaf extract

as a natural reducing and stabilizing agent. A visible color change and a UV–Vis absorption peak at ~340 nm

confirmed ZnNP formation. SEM and TEM analyses revealed predominantly cubical nanoparticles with an

average size of 67 nm. DLS showed a hydrodynamic diameter of 148.7 nm and a zeta potential of –21 mV,

indicating good colloidal stability. The ZnNPs exhibited strong antimicrobial activity against Escherichia coli,

Bacillus cereus, and Staphylococcus aureus, with effects dependent on concentration and exposure time.

Cytotoxicity assays on MCF-7 breast cancer cells showed dose-dependent inhibition, accompanied by

morphological changes such as membrane blebbing and nuclear fragmentation. Molecular docking studies

indicated interaction of ZnNPs with Human Serum Albumin (HSA) near Subdomain IB, involving residues like

Arg-196 and His105, with a binding energy of –1.64 kcal/mol. These results suggest ZnNPs have potential for

biomedical applications.

Keywords: Zinc nanoparticles; Green synthesis; Phaseolus vulgaris; UV–Vis spectroscopy; Scanning Electron

Microscopy (SEM); Transmission Electron Microscopy (TEM); Dynamic Light Scattering) DLS.

INTRODUCTION

Nanotechnology is a branch of science that aims at the design, development, production and characterization of

1–100 nm-sized matter by examining it at atomic and molecular levels (Ensafi et al., 2010, 2013; Bakhsh Raoof

et al., 2011; Karaman et al., 2021). With nanotechnological studies operating in all areas of life, interest in

nanostructures has started to increase day by day, and new products and materials have been developed by

placing these structures in large material components and systems (Dave and Chopda, 2014; Dreher, 2003).

Metal oxides have an important place in many fields such as physics, chemistry, and materials science

(Taherkhani et al., 2014; Karimi-Maleh et al., 2014a,b; Alavi-Tabari et al., 2018). They find applications such

as; drug delivery (Hu, 2010), tissue/tumour imaging (Jain et al., 2008), photothermal therapy (Jain et al., 2008),

catalysis (Xiao and Xia., 2010; Alanazi et al., 2010), optoelectronics (Mohanpuria et al., 2008), water

purification (Pradeep, 2009, Karimi-Maleh et al., 2021; Mehdizadeh et al., 2020; Rad et al., 2020; Hassandoost

et al., 2019; Orooji et al., 2020), SERS detection (Cai et al., 2008; Philip et al., 2008) and biosensing (Zheng et

al., 2010; Hu et al., 2007). The use of metal and metal oxide nanoparticles such as silver, aluminium oxide, iron

oxide, silica oxide, titanium oxide and zinc oxide continues to increase day by day. Zinc oxide (ZnO)

nanoparticles attract a lot of attention due to their widespread use and nanotechnological properties and are

produced by researchers in various ways (Bijad et al., 2013; Alavi-Tabari et al., 2018; Karimi-Maleh et al.,

2014a,b; Taherkhani et al., 2014). In the present study, the combined spectroscopic, light scattering, and

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microscopic analyses unequivocally confirm the successful synthesis of cubic zinc nanoparticles with nanoscale

dimensions, moderate polydispersity, stable colloidal dispersion, and surface morphology conducive to enhanced

reactivity. These physicochemical properties make the ZnNPs suitable candidates with antimicrobial and

anticancer property which is clearly demonstrated. Moreover, the bioinformatics findings have expanded the

potential therapeutic applications of ZnNPs.

MATERIALS AND METHODS

Zinc acetate hexahydrate [Zn(CH₃COO)₂·2H₂O] (analytical grade), Fresh Phaseolus vulgaris leaves, Distilled

water, Whatman No. 1 filter paper.5 g of P. vulgaris leaves were thoroughly washed, dried, and ground into

powder [1]. The powder was mixed in 100 mL of distilled water at 60°C for 30 minutes [2], [3], [4]. The mixture

was filtered and cooled to room temperature. To synthesize ZnNPs 50 mL of 0.1 M zinc acetate solution was

mixed with 50 mL of P. vulgaris extract under continuous stirring at 60°C and 8.5 pH. The color change from

light yellow to pale brown was observed within 3 hours of continuous stirring, which indicated the formation of

ZnNPs. The mixture was then incubated for 24 hours, centrifuged at 10,000 rpm for 15 minutes, and the pellet

was washed and dried. LCMS was performed to separate remaining biomass from the mixture obtained through

centrifugation and the pure sample was characterized further [5], [6], [7], [8].

Characterization

The synthesized zinc nanoparticle (ZnNP) sample was systematically characterized using complementary

spectroscopic, light scattering, and electron microscopic techniques to evaluate optical, dimensional, surface

charge, and morphological properties.

3.1. UV–Visible Spectroscopy: The UV–Visible absorption spectrum of the ZnNP colloidal dispersion,

recorded in the wavelength range of 200–800 nm using a 1 cm path length quartz cuvette, exhibited a welldefined

and intense surface plasmon resonance (SPR) peak at 341 nm as shown in fig. 1. This distinct absorption feature

is characteristic of zinc nanoparticles and arises from the coherent oscillation of conduction band electrons upon

interaction with incident photons. The sharpness and symmetry of the peak, with negligible spectral broadening,

indicate a relatively narrow particle size distribution, which is consistent with wellcontrolled nucleation and

growth processes in the plant-mediated synthesis route [9], [10], [11], [12].

Figure 1: UV-Visible Spectrophotometry

3.2. Dynamic Light Scattering (DLS): Particle size distribution analysis was performed using a Malvern

Zetasizer (Ver. 6.34) equipped with a 4.65 mm measurement position and glass cuvette with square aperture,

under dispersant conditions of deionized water (refractive index 1.330, viscosity 0.8872 cP) at 25 °C. The

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Zaverage hydrodynamic diameter was determined to be 148.7 nm with a polydispersity index (PDI) of 0.426,

indicating moderate polydispersity. The intensity-weighted size distribution profile revealed three distinct

populations: Peak 1 at 7.3 nm (86.1% intensity), Peak 2 at 53.5 nm (8.2% intensity), and Peak 3 at 385.5 nm

(5.7% intensity) as depicted in fig. 2, suggesting the predominance of smaller particles with minor fractions of

larger aggregates. The intercept value of 0.864 indicated a good signal-to-noise ratio in the correlation function.

The relatively high contribution of Peak 1 supports the presence of primary nanoparticles, whereas Peaks 2 and

3 likely represent agglomerated forms or secondary assemblies [13], [14], [15], [16].

Figure 2: Dynamic Light Scattering Graph

3.3. Scanning Electron Microscopy (SEM): SEM analysis was conducted at an accelerating voltage (EHT)

of 5.00 kV, a working distance (WD) of 11.7 mm, and a magnification of 50,000×. The micrographs revealed

nanoparticles with an average size of approximately 55 nm as shown in fig. 3, smaller than the hydrodynamic

diameter from DLS due to the absence of the solvation and capping layers in electron microscopy imaging. The

particle surfaces appeared relatively rough, likely resulting from organic phytochemical capping agents derived

from the plant extract, which not only stabilize the nanoparticles but may also enhance surface reactivity by

increasing the effective surface area [17], [18], [19] .

Figure 3: Scanning Electron Microscopy image

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3.4. Transmission Electron Microscopy (TEM): TEM imaging confirmed nanoscale dimensions and

revealed that the nanoparticles predominantly exhibited a cubic morphology with well-defined edges and planar

facets. This shape is indicative of anisotropic growth along specific crystallographic planes, possibly mediated

by the selective adsorption of phytochemicals to certain facets during nucleation and growth as shown in fig. 4..

The cubic geometry is of particular significance in influencing the nanoparticles’ surface energy distribution and

interaction profiles, which may impact catalytic and biological performance. Selected area electron diffraction

(SAED) is expected to reveal distinct diffraction rings corresponding to the face-centered cubic (fcc) crystalline

structure of metallic zinc [20], [21], [22], [23], [24], [25], [26].

Antimicrobial activity:

In the present antimicrobial study, both concentration-dependent and time-dependent effects of the synthesized

nanoparticles were evaluated to determine their efficacy against selected microbial strains (E.coli, B. cereus, S.

aureus) as shown in fig, 5 and fig. 6 respectively [33], [34]. A series of concentrations from 0 to 100 µg were

tested using broth microdilution methods to assess the minimum inhibitory concentration (MIC) and establish a

dose-response relationship. The results demonstrated a significant increase in antimicrobial activity with

increasing concentrations, indicating a strong concentration-dependent effect as shown in table 1. [8], [10].

Additionally, time-kill assays were conducted over intervals ranging from 0 to 7 days to evaluate the bactericidal

kinetics as shown in table 2. The findings revealed that microbial inhibition was also time-dependent, with

prolonged exposure resulting in greater reduction in viable cell counts. These observations suggest that both the

concentration and exposure duration play crucial roles in determining the antimicrobial potential of the tested

agents, supporting their use in controlled therapeutic applications [10], [34], [35].

Figure 6: Time Dependent study of Zn NPs against (a) E. coli (b) B. cereus (c) S. aureusP value is calculated <

0.02

Data presented ± SEM

Anticancer activity:

In the anticancer study, a concentration-dependent cytotoxicity assay was performed to evaluate the effectiveness

of the synthesized nanoparticles against MCF-7 which is human breast cancer cell line [28]. Various

concentrations were administered to cultured cells, and cell viability was assessed using the MTT assay after 24

and 48 hours of treatment. The results revealed a decrease in cell viability with increasing nanoparticle

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concentration, indicating a strong dose-dependent anticancer effect as shown in fig. 7 [36], [37]. The IC₅₀ values

were calculated to determine the effective concentration required to inhibit 50% of the cancer cells.

Morphological changes such as cell shrinkage, membrane blebbing, and nuclear fragmentation were observed

at higher concentrations, further confirming apoptosis induction. These findings suggest that the nanoparticles

exhibit potent anticancer activity in a concentration-dependent manner, highlighting their potential as therapeutic

agents in cancer treatment [38], [39], [40].

Figure 7: Anticancerous activity (MTT Assay)

Bioinformatics Study:

To explore the interaction between zinc nanoparticles (ZnNPs) and the target protein, Human Serum Albumin

(HSA), molecular docking was employed using two complementary approaches: (1) Blind docking and (2)

Sitespecific docking. This dual-method strategy enabled a comprehensive evaluation of potential binding regions

and interaction strength, further aiding in identifying the bioactive orientation of ZnNPs.[41], [42]

In the blind docking approach, the web-based tool CB-Dock2 was utilized, which predicts potential binding

cavities automatically by analysing the whole surface of the receptor protein. This method is especially useful

when the exact active or binding site is unknown. Upon docking, seven distinct binding cavities were identified

on the HSA molecule as being favorable for ZnNP binding. Among these, the highest-ranking cavity was selected

based on binding energy, cavity volume, and docking score, ensuring a non-biased prediction of potential

interaction hotspots. The volume of the selected cavity was 1385 Å³, indicating a well-defined pocket large

enough to accommodate the nanoparticle cluster.[43]

For more precise interaction assessment, site-specific docking was performed using AutoDock Vina, targeting

the known subdomains of HSA involved in ligand binding. Subdomain IB and IIA, particularly residues around

the Sudlow’s sites, are known to mediate interaction with a wide range of ligands. The docking of ZnNPs at this

region provided a focused understanding of nanoparticle–protein interactions at biologically relevant sites.

In the case of ZnNP–HSA interaction, the docked conformation revealed a binding pocket located near

Subdomain IB, with a 7.1 Å distance from the geometric center of the ligand to the side chain of Arg-196, a

residue known to contribute to ligand stabilization via hydrogen bonding or electrostatic interactions.

Surrounding this site, a group of amino acid residues including LEU103, SER104, HIS105, and LYS106 formed

the immediate binding environment, likely contributing to hydrophobic and polar interactions as shown in fig.

8. Additionally, ASP108 and SER109 were identified near the aliphatic region of the nanoparticle’s contact

surface, potentially stabilizing the interaction through electrostatic and hydrogen bonding forces.[43], [44]

The binding energy calculated from Auto Dock Vina for this conformation was –1.64 kcal/mol. While this energy

value suggests a relatively weak interaction compared to typical small molecule ligands, it is consistent with the

behaviour of larger, inorganic ligand systems like metal nanoparticles, which often rely on surface adsorption

and non-covalent, multi-residue interactions rather than deep pocket binding. The observed interaction pattern

indicates that ZnNPs may adsorb onto the protein surface via multiple low-affinity interactions, which

cumulatively provide sufficient binding stability for biological activity.[45], [46]

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Figure 8: Molecular docking of ZnNPs – HSA complex

DISCUSSION

The synthesized zinc-based nanoparticles were comprehensively characterized using multiple analytical

techniques to confirm their physicochemical properties and biological potential. UV-Visible spectrophotometry

revealed a distinct absorption peak at 341 nm, a characteristic signature associated with the surface plasmon

resonance (SPR) of zinc nanoparticles. This result aligns with previously reported absorption maxima for zinc

based nanostructures, confirming the successful synthesis of zinc nanoparticles [3], [47].

Further particle size analysis was performed using Dynamic Light Scattering (DLS), which estimated the

hydrodynamic diameter of the nanoparticles to be approximately 70 nm. Additionally, the zeta potential

measurement yielded a value of –26 mV, indicating that the synthesized nanoparticles possess adequate surface

charge to maintain colloidal stability. A zeta potential below –25 mV typically reflects strong electrostatic

repulsion among particles, which minimizes aggregation and suggests good long-term stability in suspension

[5], [47].

Scanning Electron Microscopy (SEM) provided more insight into the surface morphology and size distribution,

revealing nanoparticles with an average size of 55 nm and a rough surface texture, which may increase the

effective surface area and enhance biological interactions. Transmission Electron Microscopy (TEM) further

confirmed the cubic shape of the nanoparticles and supported the SEM and DLS findings regarding nanoscale

dimensions [8], [48].

The biological evaluations of the synthesized nanoparticles revealed promising antimicrobial and anticancer

properties. In the antimicrobial assays, the nanoparticles demonstrated both concentration-dependent and time

dependent effects against selected bacterial strains including Escherichia coli, Bacillus cereus, and

Staphylococcus aureus. The broth microdilution method confirmed a clear dose-response relationship, with

increasing concentrations (up to 100 µg/mL) showing progressively higher bacterial inhibition. The observed

Minimum Inhibitory Concentrations (MICs) validate the effective antimicrobial potential of the nanoparticles.

Moreover, time-kill kinetic studies showed enhanced bactericidal effects with prolonged exposure (up to 7 days),

indicating that extended contact time increases nanoparticle efficacy. These dual-dependent outcomes suggest a

robust and adaptable antimicrobial profile suitable for diverse biomedical applications, including sustained

release antimicrobial coatings and wound dressings[3], [47], [49].

In the anticancer assessment, the nanoparticles exhibited significant cytotoxicity against MCF-7 human breast

cancer cells, as determined through MTT assays conducted at 24 and 48-hour intervals. A concentration

dependent decrease in cell viability was noted, with IC₅₀ values supporting their effective anticancer dose range.

Morphological changes such as cell shrinkage, membrane blebbing, and nuclear fragmentation at higher

concentrations are hallmark indicators of apoptosis induction, further substantiating the anticancer mechanism

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of action. These observations are consistent with other reports where metal nanoparticles trigger oxidative stress

and programmed cell death in cancerous cells [28], [32], [36], [39], [50].

Overall, the results affirm that the green-synthesized zinc nanoparticles are not only structurally stable and

morphologically uniform but also biologically active, displaying potent antimicrobial and anticancer capabilities

[51], [52]. Their nanoscale size, high surface area, and surface charge collectively contribute to their

effectiveness. These multifunctional characteristics position the synthesized nanoparticles as promising

candidates for biomedical applications such as nanomedicine, targeted drug delivery, and therapeutic coatings,

aligning well with the goals of sustainable and green nanotechnology [5], [48], [53], [54], [55], [56], [57], [58].

The moderate binding energy, combined with a large cavity volume (1385 Å³) and participation of several surface

residues, indicates that HSA can act as a biocompatible carrier or transporter for zinc nanoparticles, potentially

facilitating their circulation in the bloodstream. The involvement of residues like HIS105 and ARG196, which

are frequently implicated in metal coordination and polar interactions, further supports the possibility of transient

but functionally relevant nanoparticle binding.[41], [42], [45], [59], [60]

Overall, the docking study provides meaningful insight into the molecular-level interaction mechanism between

ZnNPs and HSA. The combination of blind docking for exploratory binding site identification and site-specific

docking for precise residue-level interaction analysis proves to be a robust approach in nanoparticle–protein

interaction studies. These findings underscore the importance of surface characteristics, size compatibility, and

electrostatic properties in governing the docking efficiency and biological compatibility of metal

nanoparticles[43], [60]

CONCLUSION

The present study demonstrated that the antimicrobial activity of the synthesized nanoparticles is both

concentration-dependent and time-dependent [3], [4]. Higher concentrations and prolonged exposure times

significantly enhanced the inhibition of microbial growth, suggesting that the nanoparticles possess strong and

sustained antimicrobial potential. These findings emphasize the importance of optimizing both dosage and

duration of treatment for effective microbial control. The results also support the suitability of green-synthesized

nanoparticles as promising candidates for antimicrobial applications in medical, pharmaceutical, and

environmental fields.[47], [61] Further in-depth studies, including mechanistic and in vivo analyses, are

recommended to validate and expand upon these findings for potential real-world applications [5], [8], [48].

Conflict of Interest: Authors declare no conflict of Interest.

Ethics declaration: Ethics declaration not applicable.

Funding Declaration: This research did not receive any funding support.

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Tables

Table-1 : Dose-

Dependent antimicrobial Study of Nanoparticles

Table 2: Time-Dependent Antimicrobial Study of Nanoparticles Figures