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Green synthesis of silver nanoparticles using Carica
Cite this: DOI: 10.1039/d6ra02406h
papaya L. flower extract for catalytic reduction of
rhodamine B and biological activities
Thi Tam Khieu, a Thi Thao Truong, a Thi Hong Tham Diep,b Thi Thuy Ha Dinh,a
Ngoc Anh Hoang,a Thanh Hai Cao,a Tuan Kien Vu,a Quang Tung Vu,a
The Chinh Pham, a Le Phuong Hoangc and Truong Xuan Vuong *a
A controlled and environmentally benign route for silver nanoparticle (AgNP) formation is achieved using an
ethanolic–water (80 : 20) extract of male Carica papaya L. flowers as a combined reductant–stabilizer
system. Reaction parameters, including pH, AgNO3 concentration, and extract dosage, are systematically
varied to probe their influence on nucleation and growth behavior. Rather than treating phyto-synthesis
as an end-point process, nanoparticle formation is examined as a time-resolved evolution governed by
coupled nucleation and growth events. UV-vis spectroscopy reveals surface plasmon resonance bands
at 405–425 nm, with spectral position and intensity strongly dependent on reaction conditions. Kinetic
analysis using pseudo-first-order and Avrami models (n = 1–2) was consistent with discontinuous
nucleation accompanied by surface-limited growth, with markedly accelerated formation under alkaline
conditions. The Avrami-derived kinetic parameters further suggested a progression from predominantly
nucleation-controlled behavior toward diffusion-influenced growth as the reaction evolved, providing
a framework for interpreting structural development. X-ray diffraction confirms crystalline face-centered
cubic Ag without detectable oxide phases. FTIR analysis identifies hydroxyl and carbonyl functionalities
as key contributors to nanoparticle stabilization. TEM and XRD analyses reveal well-dispersed spherical
particles with average sizes of 23.33 ± 6.44 nm and 26.21 ± 8.01 nm under optimized conditions. The
resulting AgNPs mediate rapid NaBH4-assisted reduction of rhodamine B (97.7% removal of 20 mg L−1
within 60 min), following pseudo-first-order kinetics. Antibacterial activity is observed against Escherichia
coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Cytotoxic responses toward AGS and
HepG2 cells (IC50 = 3.22 ± 0.57 and 2.05 ± 0.57 mg mL−1, respectively) were observed, indicating that
surface-bound phytochemicals may remain functionally active after nanoparticle formation. Coupling
Received 24th March 2026
Accepted 3rd July 2026
kinetic descriptors with structural characteristics and functional performance supports a quantitative
structure–kinetics–function relationship, suggesting that nanoparticle formation can be more
DOI: 10.1039/d6ra02406h
systematically regulated through controlled synthesis variables rather than relying solely on empirical
[Link]/rsc-advances adjustment.
implementation in systems requiring environmental safety and
1 Introduction biological compatibility. Plant-mediated synthesis offers an
Environmentally compatible synthesis strategies are increas- alternative chemical environment in which naturally occurring
ingly reshaping nanoparticle fabrication in modern materials metabolites participate directly in nanoscale assembly, func-
chemistry. Conventional preparation of metallic nanomaterials tioning not only as electron donors but also as coordinating and
still relies heavily on strong chemical reductants, elevated stabilizing species during nucleation and particle maturation
temperatures, and synthetic stabilizers. Such conditions processes.1–3
generate secondary waste streams and complicate Among metallic nanomaterials, silver nanoparticles (AgNPs)
remain extensively investigated because of their optical
response, catalytic activity, and antimicrobial properties. Their
a
Faculty of Natural Sciences and Technology, TNU-University of Science, Phan Dinh applications extend across water treatment, sensing technolo-
Phung Ward, Thai Nguyen 25000, Vietnam. E-mail: xuanvt@[Link]
b
gies, catalysis, and biomedicine.4,5 In contrast to synthesis
SGS Vietnam Co., Ltd, Thai Nguyen Branch, Suoi Cat Hamlet, An Khanh Commune,
routes employing reductants such as hydrazine or sodium
Thai Nguyen 2500, Vietnam
c
Thai Nguyen University of Technology (TNUT), Tich Luong Ward, Thai Nguyen
borohydride, phyto-mediated systems operate through chemi-
250000, Vietnam cally diverse phytochemical networks. Polyphenols, avonoids,
© 2026 The Author(s). Published by the Royal Society of Chemistry RSC Adv.
RSC Advances Paper
terpenoids, alkaloids, and organic acids interact simulta- capturing nucleation characteristics, growth dimensionality,
neously with silver precursors, generating coupled reduction– and structural progression during nanoparticle formation.16
stabilization pathways that govern particle size, morphology, Avrami-based approaches have previously been applied to thin-
surface chemistry, and colloidal behavior.1,2,6 lm AgNP systems22 and biomolecule-assisted nanoparticle
Numerous plant systems have been explored for AgNP synthesis.23 Yet their application to phyto-mediated AgNP
synthesis. Trigonella foenum-graecum extracts generate AgNPs synthesis remains relatively limited, particularly in studies
with catalytic and sensing functionality,7 whereas Clerodendrum linking kinetic descriptors with structural evolution and func-
infortunatum extracts facilitate rapid dye degradation tional behavior.18
processes.8 Similar observations have been widely reported Integrating kinetic interpretation into controlled phyto-
throughout the phyto-mediated synthesis literature. However, synthesis changes how nanoparticle evolution can be viewed.
most studies remain conned to endpoint characterization. AgNP formation can then be interpreted not simply as a reduc-
Parameters such as particle size, antibacterial efficiency, or tion process, but as a regulated transformation governed by
catalytic performance are commonly emphasized, whereas the nucleation kinetics, interfacial interactions, and structural
kinetic sequence governing nanoparticle formation has constraints. Unlike many previous phyto-mediated AgNP
received comparatively limited attention.9–11 Recent reviews studies that primarily emphasize endpoint performance, the
have further noted that many phyto-mediated AgNP studies still present work focuses on the formation pathway itself through
lack quantitative mechanistic interpretation, reproducible quantitative kinetic analysis. In this context, Avrami analysis
structure–property relationships, and systematic kinetic anal- provides insight beyond apparent rate descriptions by di-
ysis, thereby limiting predictive process control and scalable stinguishing nucleation-controlled and diffusion-limited
implementation.4,12,13 As a result, the formation pathway itself is regimes while simultaneously tracking changes in dimen-
still poorly resolved. sional evolution during nanoparticle growth.16 Establishing
A comparable limitation exists in biomedical investigations. relationships among synthesis conditions, kinetic descriptors,
AgNPs synthesized using Pistacia atlantica and related medic- structural characteristics, and functional responses may there-
inal plants exhibit antioxidant activity and cytotoxic responses fore contribute toward a more predictive framework for green
toward cancer cell lines.14,15 Although such studies demonstrate nanoparticle synthesis. Such integrated kinetic-structural
promising biological functionality, the relationship between interpretation remains comparatively underdeveloped in
phytochemical composition and nanoparticle development is phyto-mediated AgNP systems, where mechanistic analysis is
oen insufficiently characterized. Consequently, empirical oen disconnected from colloidal evolution and functional
optimization continues to dominate much of the eld. evaluation.
Reaction environments strongly inuence the structural Carica papaya L. represents a chemically diverse phyto-
development of phyto-mediated nanoparticles. Variations in chemical system that has received relatively limited attention in
pH, precursor concentration, extract composition, and extract kinetically resolved nanomaterial synthesis. Leaves, seeds, and
dosage affect supersaturation behavior, nucleation frequency, peels contain phenolic and avonoid compounds capable of
and particle growth pathways, ultimately inuencing reducing metal ions while stabilizing evolving nanostructures
morphology, colloidal stability, and structural uniformity.2,16 through hydroxyl and carbonyl functionalities.24–26 Male Carica
These effects become particularly difficult to predict in phyto- papaya owers (MCF) have received substantially less attention
chemical systems characterized by extraction-dependent despite longstanding applications in traditional medicine for
compositional heterogeneity. Without quantitative relation- hypoglycemic activity, gastric protection, and respiratory
ships linking synthesis parameters to nanoparticle evolution, treatment.27–29 Cytotoxic responses toward cancer cell lines such
reproducibility and rational process control remain chal- as Michigan Cancer Foundation-7 (MCF-7) human breast
lenging. Recent studies have identied phytochemical vari- cancer cells, HepG2 liver cancer cells, and KB epidermoid
ability, insufficient synthesis standardization, and limited carcinoma cells have also been reported.30,31 These biological
mechanistic predictability as major barriers preventing reliable properties are associated with oxygen-containing metabolites
scale-up of phyto-mediated nanoparticle production.17,18 capable of coordinating metal ions and participating in inter-
Quantitative kinetic analysis provides an important route for facial redox processes. Such metabolites may therefore inu-
understanding how nucleation and particle growth evolve ence not only biological activity, but also nanoparticle
during nanoparticle synthesis. Pseudo-rst-order models nucleation and structural development during synthesis.
describe overall reaction behavior, whereas the Finke–Watzky Existing studies on MCF remain largely conned to phyto-
model separates nanoparticle formation into slow continuous chemical screening and fragmentation analysis,18 while kineti-
nucleation followed by autocatalytic surface enlargement.19 cally interpreted nanomaterial synthesis using this
However, under conditions where k2[Ag] [ k1, the model may phytochemical system is not yet well established.
produce inaccurate kinetic descriptions.20 The LaMer model The coexistence of biological activity and nanoparticle-
describes burst nucleation followed by diffusion-controlled forming capability raises an important process-level question:
growth,21 although the heterogeneous and continuously can the metabolites responsible for medicinal activity also
evolving reaction environments characteristic of green govern nucleation pathways, structural development, and
synthesis systems rarely conform to this idealized behavior. functional responses during AgNP synthesis?
Avrami analysis offers a complementary perspective by
RSC Adv. © 2026 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances
In this work, ethanolic extracts of MCF were employed as value (7–10), and the solution was heated to 85 °C under
a combined reductant–stabilizer system for kinetically resolved continuous magnetic stirring.
AgNP synthesis. Phytochemical composition of the extract was The AgNO3 solution was added dropwise into the MCFE-
examined alongside systematic variation of pH, precursor containing solution to initiate the reduction of Ag+ ions and
concentration, and extract dosage to evaluate their inuence on the formation of AgNPs. The reaction was maintained at 85 °C
nucleation and structural development. Formation kinetics under constant stirring for a predetermined time. At specic
were interpreted using pseudo-rst-order and Avrami models to time intervals, 3 mL aliquots were withdrawn from the reaction
identify dominant growth regimes and nucleation characteris- mixture and diluted to 10 mL with deionized water for UV-vis
tics. Structural evolution was characterized using UV-vis spec- absorption measurements in the range of 300–700 nm using
troscopy, XRD, FTIR, and SEM analyses, followed by evaluation a UV-vis spectrophotometer (Jasco V-770).
of catalytic rhodamine B reduction, antibacterial activity, and Aer completion of the reaction, the suspension was cooled
cytotoxic responses. By integrating synthesis conditions, kinetic to room temperature and stored at 4 °C for further analysis. For
evolution, structural characteristics, and functional behavior structural characterization, a portion of the reaction mixture
within a single phyto-mediated nanoparticle system, this study was centrifuged at 9000 rpm for 20 min at 5 °C. The obtained
aims to advance process-level understanding of green AgNP precipitate was collected, washed once with 96% ethanol and
formation and support more predictive nanomaterial design twice with deionized water to remove residual phytochemicals,
strategies. This integrated approach contributes toward a more and then dried. The dried product was nely ground and stored
mechanistically informed framework for phyto-mediated AgNP in a sealed container at room temperature for subsequent
synthesis. analyses.
The data of UV-vis spectroscopy were tted with a Gaussian
(340–600 nm) using peak analyzer and calculated lmax, peak
2 Materials and methods area, with R2 [ 0.99, were subsequently tted by the PFO and
2.1 Chemicals the Avrami kinetic models to describe the mechanism and rate
of AgNPs formation. The PFO kinetic model postulates that the
Silver nitrate (AgNO3, purity $99.8%), ethanol (C2H5OH, 96%,
rate of AgNPs formation is directly proportional to the produced
for HPLC), sodium carbonate (Na2CO3, purity $99.0%), sodium
Ag content (corresponding to the peak area of the characteristic
borohydride (NaBH4, purity $98%), RhB (C28H31N2O3Cl, purity
peak of Ag).33 The nonlinear form of the PFO equation is given
$97%, for HPLC), n-butanol (n-C4H9OH, purity $99.5%),
as follows:
ethylene diamine tetra acetate disodium EDTA-2Na
(C10H14N2Na2O8$2H2O, purity $99%), and AgNO3, ($99.8%,
At = AN(1 − e−k1t) (1)
Sigma-Aldrich) were purchased from Sigma-Aldrich, India, to be
used without further purication. Double-distilled water (DW) The Avrami kinetic model is commonly used in the crystal-
was used throughout the study. lization processes exhibiting complex nucleation and growth
Male Carica papaya L. owers were harvested in Thai Nguyen behavior,34 given by:
province in July.
At Amin
aðtÞ ¼ 1 expðkA tn Þ ¼ (2)
AN Amin
2.2 Extracting male Carica papaya L. Flower and
synthesising AgNPs
The linear equation of the Avrami model is:
Male Carica papaya L. owers (MCF) were collected in Thai
AN Amin
Nguyen city, washed with distilled water, shade-dried, and ln ln ¼ ln kA þ n ln t (3)
ground into ne powder. MCF powder (10 g) was subjected to AN At
ultrasonic extraction with 80% ethanol at 60 °C for 60 min. The where At, Amin, AN are the peak area at time t, beginning at time
extract was centrifuged at 8000 rpm for 20 min at 5 °C and zero (t = 0), and at equilibrium (t = N), respectively (AN is
ltered through Whatman no. 1 lter paper. The ltrate was determined from the results of PFO model). k1 (min−1) and kA
stored at 4 °C for further use and hereaer denoted as male (min−n) are the rate constants according to the PFO and Avrami
Carica papaya ower extract (MCFE). Total polyphenols (TP) and kinetics. n is the Avrami exponent, which typically ranges from 1
total avonoids (TF) of MCF were determined by the Folin– to 4 and provides information regarding the nucleation/growth
Ciocalteu method and the aluminium chloride (AlCl3) dimensionality. The kinetic parameters (qe, k1, kA, n, and R2)
method.32 were assessed through a regression evaluation in OriginPro
2019.
2.3 Green synthesis of AgNPs
MCFE was employed as both a reducing and stabilizing agent 2.4 Characterization of AgNPs
for the green synthesis of AgNPs. The reaction mixture con- The crystalline structure of the synthesized silver nanoparticles
sisted of 2.5 mL of MCFE and 1.5 mL of 0.025 M AgNO3 solution, (AgNPs) was analyzed using an X-ray diffractometer (XRD,
which was diluted with deionized water to a nal volume of 50 Bruker AXS, Germany) equipped with Cu-Ka radiation (l =
mL. The pH of the reaction mixture was adjusted to the desired 1.5406 Å) operated at 40 kV and 30 mA. Diffraction patterns
© 2026 The Author(s). Published by the Royal Society of Chemistry RSC Adv.
RSC Advances Paper
were recorded over a 2q range of 25–70°. The apparent crystallite C0 Ct
R¼ 100% (5)
size of AgNPs was estimated from X-ray diffraction (XRD) C0
patterns using the Scherrer equation:
where C0, Ct (mg L−1) represent the RhB concentrations at t =
D = (K × l)/(b × cos q) (4) 0 and t (minutes), E (%) denotes the efficiency of catalytic
reduction.
where K = 0.9 is the shape factor, l = 0.15406 nm (Cu Ka Each experiment was performed in triplicate, and the results
radiation), b is the full width at half maximum (FWHM) of the are presented as mean values. The catalytic reduction kinetics
diffraction peak (in radians), and q is the Bragg angle. of RhB were evaluated using the Langmuir–Hinshelwood (LH),
The calculation was performed based on the diffraction pseudo-rst-order (PFO), and pseudo-second-order (PSO)
peaks corresponding to the (111), (200), and (220) crystallo- kinetic models.35,36 The LH kinetic model is expressed as:
graphic planes of the face-centered cubic (fcc) structure of KL C
metallic silver. For measurement, the AgNPs suspensions were V ¼k (6)
1 þ KL C
dropped onto glass slides, air-dried, and the coating process
was repeated three times to ensure uniformity. The surface
As KLC 1, the LH equation turns into the PFO kinetic
morphology of AgNPs was examined using a eld-emission
model: V = k1C. The linear form of PFO kinetic models is fol-
scanning electron microscope (FE-SEM, Hitachi S-4800, Japan)
lowed by equations:
operated at an accelerating voltage of 20 kV. Before imaging, the
samples were sputter-coated with a thin platinum layer for 30 s C0
k1LH t ¼ ln (7)
at 20 mA under a vacuum of 10 Pa to enhance conductivity. Ct
Functional groups associated with the surface of AgNPs were
identied using Fourier transform infrared spectroscopy (FT-IR,
Additionally, the process kinetics can be described by PSO
IRTracer-100, Shimadzu, Japan) in the spectral range of 400–
reaction kinetics according to the equation:37
4000 cm−1 with a resolution of 1 cm−1. The samples were
prepared as KBr pellets prior to measurement. 1 1
k2 t ¼ (8)
Ct C0
where C0 and Ct (mg L−1) are the RhB concentrations in the
reaction solution at initial time and time t (min), k1LH (min−1)
2.5 The catalytic reduction of rhodamine B (RhB) and k2 (L mol−1 min−1) are the rst and second order reaction
The catalytic reduction of Rhodamine B (RhB) was performed rate constants respectively.
with optimal AgNPs selected from the results of material char-
acterization analysis (AgNPs7). 2.6 The antibacterial ability
The rst, 40 mL of RhB solution (20 mg L−1) was transferred
AgNPs synthesized at pH 7 and pH 8, denoted as AgNPs7 and
into 100 mL Erlenmeyer asks, followed by the addition of 3 mL
AgNPs8, respectively, were evaluated for antibacterial activity
of the prepared AgNPs7 suspension. Subsequently, varying
using the agar well diffusion method against Gram-positive
volumes of 1 M NaBH4 solution (0.15, 0.30, 0.45, and 0.60 mL)
Staphylococcus aureus ATCC 25923 and Gram-negative Escher-
were introduced, and the mixtures were shaken uniformly using
ichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.
a mechanical shaker under normal lighting conditions. At
The assays were performed using AgNP dispersions at 80 mg
selected time intervals (10, 20, 30, 45, 60, and 90 min), 3 mL
mL−1 in DMSO together with MCFE at 50 mg mL−1. Amoxicillin
aliquots were withdrawn, diluted to 10 mL, and absorbance was
(100 mg mL−1) was used as the positive control.
measured at 553 nm using a UV-vis spectrophotometer with
Briey, 100 mL of bacterial suspension (108 CFU per mL) was
quartz cuvettes to evaluate the degradation efficiency of RhB.
uniformly spread onto Mueller–Hinton agar plates, followed by
Then, the optimum volume of NaBH4 solution was selected
loading of 50 mL sample solutions into 6 mm wells formed in
and used in the following experiments, following the same
the agar medium. The plates were incubated at 37 °C for 24 h,
procedure, with initial RhB concentrations of 20, 30, 50, 75, or
aer which the inhibition zone diameters were measured.
100 mg L−1, and the amount of AgNP7 solution used being 1.5,
3.0, 4.5 and 6.0 mL. The RhB concentration in the reaction
solution was determined at predetermined time intervals (5, 15, 2.7 Cytotoxic activity on gastric cancer cell lines (AGS) and
30, 45, 60, and 90 min). liver cancer cells (HepG2)
Similar experiments were performed under the condition of The AGS gastric cancer cell line and HepG2 hepatocellular
40 m RhB 20 mg L−1 reacting with 3 mL AgNPs7 in the presence carcinoma cell line used in this study were provided by Assoc.
of 0.6 mL 1 M NaBH4 and some scavengers (0.1 M EDTA-2Na or Prof. Dr Nguyen Phu Hung, Institute of Excellent Education and
tert-butanol (1 : 20) or 1 M AgNO3) to elucidate the electron- Research, Thai Nguyen University. These cell lines were origi-
transfer-mediated catalytic reduction mechanism of RhB nally obtained from the American Type Culture Collection
degradation by AgNPs. (ATCC, Manassas, VA, USA).
The removal efficiency of RhB from the solution was calcu- 80 mg mL−1 of AgNPs7 solution dispersed in water at 1000-,
lated by the formula: 100-, 40-, 20-, and 10-fold dilutions. They were tested on AGS
RSC Adv. © 2026 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances
and HepG2 cells by MTT assay. 100 mL of AGS or HepG2 cells Carica papaya extracts, the principal detectable chromato-
suspension with concentration of 5 × 103 cells per well was graphic peaks were tentatively assigned to quercitrin, quercetin,
cultured for 48 h at 37 °C in a humidied atmosphere con- and kaempferol. These hydroxyl-rich avonoid species are
taining 5% CO2 and 95% humidied air in a 96-well microplate chemically compatible with reductive and interfacial stabiliza-
using RPMI 1640 medium supplemented with 10% fetal bovine tion processes occurring during AgNP formation. Phenolic
serum (FBS) and 1% penicillin/streptomycin. Aer that, the hydroxyl functionalities may participate in Ag+ reduction, while
cells were exposed to the test samples at different concentra- coordination interactions at the evolving nanoparticle interface
tions in the wells. Then, the plate was incubated for 48 h under could contribute to colloidal stabilization during nanoparticle
the same conditions (37 °C, 5% CO2, and 95% humidied air) to growth. Within this framework, the detected avonoid prole is
evaluate the impact of the samples on cell proliferation. The cell consistent with the kinetically regulated nucleation–growth
viability was determined by the ability to reduce MTT to behavior inferred from the PFO and Avrami kinetic analyses.
a formazan complex by the activity of mitochondrial dehydro- Comprehensive metabolite proling together with quantitative
genase enzyme. The formazan product was dissolved in DMSO, determination of individual phytochemical constituents was
and the optical density (abs) was measured at 570 nm, reecting not systematically performed in the present study and remains
the rate of cell inhibition. Each concentration was tested in four an important direction for future investigation.
replicate wells for each experiment. The cell proliferation rate 3.1.2 Optical evolution and kinetic analysis of AgNP
was determined by the formula: formation
Abssample [Link] Effect of pH and reaction time on AgNP formation. The
% Cells ¼ 100% (9) reaction between 2.5 mL of MCFE and 1.5 mL of 0.025 M AgNO3
Abscontrol sample
in 50 mL solutions was carried out at 85 °C at pH 7, 8, 9, 10 over
time. The formation of AgNPs exhibited a strong dependence on
All experiments were performed in triplicate to ensure pH and reaction time, as evidenced by the progressive color
reproducibility. transformation and the emergence of localized surface plasmon
resonance (LSPR) bands (Fig. 1 and 2).
The color change of the reaction mixtures demonstrates that
3 Results and discussion AgNPs formation was strongly dependent on both reaction time
3.1 AgNPs characterization depending on synthesis process and pH. At t = 0, all samples showed the pale-yellow color of the
3.1.1 TP and TF of male Carica papaya L. owers. The total extract, indicating negligible Ag+ reduction. As time progressed,
polyphenol (TP) and total avonoid (TF) contents of MCF, the solutions transitioned from pale-yellow to reddish-brown in
determined using the Folin–Ciocalteu and aluminium chloride deeper hues, consistent with the formation of nanoparticles
(AlCl3) methods, were 24.006 mg GAE per g and 15.360 mg QE and the onset of localized surface plasmon resonance (LSPR).
per g dry weight, respectively. Compared with previous reports, The rate of color change increased substantially with increases
the TP content was higher than that reported for MCF collected in pH. The color change was rather slow at pH 7 and even at
in Madhya Pradesh, India,38 whereas the TF value was compa- 180 min, the solution was still light brown. The brown color
rable to that reported for MCF from West Bengal, India.39 appeared quickly at pH 8–9, and at pH 10, a similar intensity of
To further investigate phytochemical species potentially color developed within only 6 min, which was the equivalent of
associated with nanoparticle formation, HPLC-DAD chromato- 25 min at pH 9 or 180 min at pH 7. These observations suggest
graphic analysis of the male Carica papaya ower extract was that alkaline conditions accelerate Ag+ reduction and nano-
performed (Table S1 and Fig. S1 in SI). Based on retention-time particle evolution within the colloidal system.
correspondence with previously reported avonoid proles in
Fig. 1 Color of AgNPs solution obtained over time at different pHs (a) pH 7, (b) pH 8, (c) pH 9, (d) pH 10.
© 2026 The Author(s). Published by the Royal Society of Chemistry RSC Adv.
RSC Advances Paper
Fig. 2 UV-vis spectra of silver nanoparticle (AgNP) synthesis over time at different pHs: (a) pH 7, (b) pH 8, (c) pH 9, (d) pH 10.
The UV-vis spectra of the reaction solutions as a function of intense within minutes and also at wavelengths greater than
pH and time are shown in Fig. 2. 500 nm. This behavior can be mechanistically explained by
At the initial stage (t = 0), the absence of a characteristic deprotonation of phenolic and carboxyl groups in MCFE under
plasmon band (350–450 nm) conrmed a negligible reduction alkaline conditions, increasing their electron-donating capa-
of Ag+. With increasing reaction time, a distinct LSPR band bility and accelerating Ag+ reduction. However, the rapid
gradually appeared and intensied, indicating nucleation fol- generation of nuclei likely exceeds the stabilization capacity of
lowed by nanoparticle growth. phytochemicals, resulting in partial coalescence.
More importantly, the systematic red-shi of lmax with Overall, the UV-vis evolution may reect that alkaline
increasing pH (Fig. 3a) provides direct insight into particle size conditions accelerate Ag+ reduction and nanoparticle growth,
evolution. In plasmonic systems, a red-shi is typically associ- while simultaneously promoting interparticle interactions and
ated with an increase in particle size, increased interparticle partial aggregation at prolonged reaction times. These spec-
coupling and broader size distribution. Thus, the accelerated troscopic observations were further correlated with kinetic
red-shi observed at pH 9–10 indicates faster nucleation tting analysis to better resolve the nucleation–growth behavior
accompanied by rapid growth and partial aggregation. Our of the phyto-mediated AgNP system.
observations agreed with previous studies reporting pH- [Link] Kinetics of AgNPs formation. To further elucidate the
accelerated reduction and growth of AgNPs.40–42 formation mechanism, peak area evolution was analyzed using
Simultaneously, an increase in pH leads to faster spectral pseudo-rst-order (PFO) and Avrami kinetic models (Fig. 3b, c
broadening and a more rapid loss of peak sharpness. The and Table 1), according to the following eqn (1) and (3).
broadening of the LSPR band (increased FWHM) reects The R2 values from Table 1 and Fig. 3b, c demonstrated
growing polydispersity. In controlled nanoparticle synthesis, satisfactory agreement between the experimental data and both
a narrow and symmetric peak indicates relatively narrow the PFO and Avrami kinetic models. PFO rate constants (k1 >
particle-size distribution, whereas broadening implies hetero- 10−2 min−1) increased progressively with pH, reecting faster
geneous nucleation and secondary growth events. The Ag+ reduction in alkaline media. PFO tting captures the global
pronounced spectral broadening under alkaline conditions reduction kinetics, whereas the Avrami model offers a more
therefore, indicates that while reduction kinetics are enhanced, detailed view of the nucleation–growth evolution.
size control becomes compromised. This observation is Avrami exponents in the range of n = 1.35–1.65 are
consistent with the color change of the solution shown in Fig. 1. commonly linked to discontinuous nucleation processes inu-
Despite the relatively low peak intensity, the darker coloration enced by interfacial effects and constrained growth behavior
indicates an increase in particle size. At pH 10, the increase in rather than unrestricted volumetric growth.43 In the MCFE-
absorption is particularly rapid, with the LSPR band becoming mediated system, phytochemical species may interact with the
RSC Adv. © 2026 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances
Fig. 3 The shift of lmax with pH and reaction time (a); the change of Ag peak area over pH and reaction time and fitting to PFO (b) and Arvami (c)
kinetic models and (d) dynamic light scattering (DLS) particle-size distributions at different reaction times.
Table 1 Kinetic parameters of green synthesis AgNPs using MCFE and diffusion-inuenced interparticle interactions during later
synthesis stages. Such behavior may reect a gradual transition
PFO Avrami
from predominantly surface-limited growth toward partially
pH AN k1 R2
n kA R2 diffusion-mediated coalescence under highly alkaline condi-
tions. The Avrami curve at pH 7 retained a gradual sigmoidal
7 883.71 0.0285 0.982 1.65 0.031 0.997 prole, while at pH 9 the transformation rapidly approached
8 1151.46 0.0366 0.995 1.50 0.046 0.995
completion (a / 1) at early reaction times.44 Decreasing n
9 1102.98 0.0486 0.986 1.35 0.081 0.996
values at elevated pH further imply that alkaline conditions
alter not only the reduction rate but also the dominant growth
pathway.
evolving AgNP surface during nucleation, restricting unre-
Neutral conditions (pH 7) produced a slower yet more stable
stricted isotropic growth and suppressing excessive particle
growth regime. lmax remained comparatively stable throughout
coarsening under optimized conditions. A similar trend
the reaction period, and the FWHM stayed narrower over time,
appears in the optical response. At pH 7, the LSPR band evolved
suggesting sustained colloidal stabilization. Around 60 min,
gradually and remained relatively narrow, consistent with
peak-intensity growth slowed and the spectral baseline began to
controlled nanoparticle growth and improved colloidal
rise. Deviation from idealized Avrami behavior beyond this
uniformity.
point likely originated from interparticle interaction and
Reaction behavior changed substantially under strongly
aggregation processes that are not fully described by the clas-
alkaline conditions (pH $ 9). Faster reduction promoted rapid
sical Avrami framework. For this reason, pH 7 and 60 min were
supersaturation and high nucleation density. Particle–particle
selected as the most balanced conditions for simultaneous rate
collision also became increasingly frequent. Although the
control and particle-size regulation.
Avrami-derived n values remained within the surface-inuenced
DLS measurements further supported the kinetic interpre-
regime, spectral broadening, baseline elevation, and progres-
tation (Fig. 3d). The hydrodynamic diameter increased
sive red-shiing of the LSPR band pointed toward aggregation
progressively with reaction time while maintaining low PDI
© 2026 The Author(s). Published by the Royal Society of Chemistry RSC Adv.
RSC Advances Paper
values (#0.170) up to 60 min, consistent with controlled particle populations (∼57.8 and ∼160.2 nm) emerged alongside
nanoparticle growth and sustained colloidal stability. At pro- a sharp increase in PDI to 0.432, indicating the onset of
longed reaction times (90 min), the emergence of a second substantial colloidal heterogeneity. Under prolonged reaction
particle population together with a marked increase in PDI conditions, aggregation and interparticle association likely
indicated increasing aggregation, in agreement with the became increasingly important contributors to the observed
concurrent UV-vis spectral broadening. A more detailed colloidal behavior. These features were consistent with the
discussion of colloidal evolution is presented in Section 3.1.3. concurrent spectral broadening and plasmonic distortion
Kinetic tting, spectroscopic evolution, and DLS analysis observed in the UV-vis analysis. Larger aggregates may addi-
collectively support the view that MCFE contributes not only to tionally enhance light scattering within the colloidal suspen-
Ag+ reduction but also to colloidal stabilization and growth sion, contributing to the darker brown coloration observed at
regulation during AgNP formation. Among the investigated extended reaction times. Nevertheless, because UV-vis spec-
conditions, pH 7 provided the most stable balance between troscopy and DLS primarily provide indirect ensemble-level
reduction kinetics, colloidal stability, and particle-size control. information, complete differentiation between nanoparticle
The present catalytic analysis nevertheless remains primarily growth, interparticle coupling, and aggregation processes
comparative rather than fully quantitative, since catalyst- remains challenging without complementary high-resolution
normalized apparent rate constants and long-term catalyst time-resolved imaging analyses.
stability were not systematically evaluated in the current 3.1.4 Effect of extract volume and Ag+ concentration on
colloidal system. AgNPs formation. The extract volume primarily modulated
3.1.3 DLS analysis and colloidal evolution of AgNPs. To stabilization efficiency. Increasing MCFE volume (0.5–2.5 mL)
further clarify the colloidal evolution of the biosynthesized enhanced plasmon intensity without signicant lmax shi
AgNPs, dynamic light scattering (DLS) analysis was performed (Fig. 4a), indicating increased particle number rather than size
on the AgNP suspension synthesized at pH 7 at different reac- growth. The preservation of peak symmetry suggests efficient
tion times (Fig. 3d). AgNPs collected at 20, 40, and 60 min surface passivation.
exhibited average hydrodynamic diameters of 24.2, 38.3, and The extract volume primarily modulated stabilization effi-
51.5 nm, respectively, with corresponding PDI values of 0.170, ciency. Increasing MCFE volume (0.5–2.5 mL) enhanced plas-
0.122, and 0.104. According to ISO 22412, PDI values below 0.2 mon intensity without signicant lmax shi (Fig. 4a), indicating
correspond to monodisperse colloidal systems, whereas higher increased particle number rather than size growth. The pres-
values reect increasing polydispersity. Progressive particle-size ervation of peak symmetry suggests efficient surface passiv-
growth accompanied by relatively low PDI values points to ation. Beyond 2.5 mL, spectral broadening and solution
controlled nanoparticle evolution during the early and inter- darkening were observed, implying the onset of aggregation.
mediate synthesis stages. The same general tendency appeared Excess phytochemicals may induce interparticle bridging or
in the UV-vis spectra through gradual LSPR red-shiing. alter ionic strength, reducing colloidal stability.
However, although this spectral evolution is broadly consis- Conversely, increasing AgNO3 concentration (Fig. 4b)
tent with nanoparticle growth, contributions from interparticle signicantly inuenced nucleation density. At moderate Ag+
plasmon coupling and local colloidal interactions cannot be levels (1.5 mL), a sharp and slightly blue-shied plasmon band
completely excluded. At 60 min, the colloidal system reached was obtained, suggesting formation of smaller and well-
a hydrodynamic size of ∼51.5 nm together with a PDI of 0.104, dispersed particles due to balanced nucleation and stabiliza-
corresponding to a comparatively narrow particle-size distri- tion. At excessive Ag+ (2.0 mL), the red-shi and peak broad-
bution. This stage also closely matched the Avrami conversion ening indicate accelerated growth and aggregation. The
approaching saturation (a / 1), consistent with a relatively spectrophotometric saturation further conrms high particle
stable nucleation–growth regime. By 90 min, two distinct density and possible coalescence. These ndings demonstrate
Fig. 4 UV-vis spectrum and color of AgNPs synthesis reaction solution with different amount of extract (a) and amount of Ag salt (b).
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that Ag+ availability governs nucleation density, while extract morphology and aggregation behavior (Fig. 5a–d). AgNPs
concentration controls surface stabilization. The interplay synthesized at pH 7 (AgNPs7) exhibited predominantly spher-
between these two parameters denes the nal particle size ical morphology with slight surface roughness, together with
distribution and colloidal stability. The optimum was 1.5 mL relatively uniform particle distribution and minimal aggrega-
AgNO3, producing a sharp plasmon peak and stable colloid with tion. When the pH was raised to a value of 8, particles (AgNPs8)
small, uniform particles. exhibited some aggregation as small clusters formed on the
Collectively, the combined plasmonic evolution and kinetic surface of otherwise smooth particles. Higher magnications
modeling reveal that MCFE-mediated AgNP synthesis follows revealed that the particles had a relatively uniform size of ∼30–
a pH-dependent discontinuous nucleation mechanism with 50 nm, which suggests their growth was controlled yet appeared
surface-limited growth. less stable than AgNPs7. By pH 9, the particles (AgNPs9) began
to exhibit signicantly larger sizes, where initial continuous
domains became aggregates of ∼300 nm. The morphology
3.2 Characterization of AgNPs
indicates the colloidal system became destabilized with
The characteristics of AgNPs were analyzed by X-ray diffraction, stronger alkaline conditions as reduced electrostatic repulsion
FTIR and SEM techniques, shown in Fig. 5. drove more particles to coalesce. The trend was more
3.2.1 Morphology and particle-size characteristics of pronounced at pH 10, where the sample (AgNPs10) displayed
AgNPs a porous but highly heterogeneous structure. Although some
[Link] SEM analysis of AgNPs. SEM analysis provided primary particles appeared smaller than those in AgNPs9, they
further insight into the effect of pH on nanoparticle
Some characteristics of AgNPs: SEM images of AgNPs samples formed at different pH: (a) pH 7 – AgNPs7, (b) pH 8 – AgNPs8, (c) pH 9 –
Fig. 5
AgNPs9 and (d) pH 10 – AgNPs10; (e) XRD spectra and (f) FTIR spectra.
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were fused into large, irregular agglomerates, making indi- to the formation of numerous primary particles with limited
vidual boundaries difficult to resolve. This observation corre- subsequent growth.45 Similar size regimes have been reported
lates well with kinetic analysis, reinforcing the link between in related systems, where controlled nucleation produces
reaction rate, nucleation mechanism, and nal morphology. nanosized particles stabilized by organic species present in the
To gain deeper insight into the nanoscale structure and extract.46
particle size distribution, transmission electron microscopy 3.2.2 Size distribution and colloidal evolution of AgNPs
(TEM) was employed. [Link] Size distribution. The size distribution derived from
[Link] TEM analysis of AgNPs. To further evaluate particle- ImageJ analysis shows a concentrated population centered
size distribution and colloidal evolution, statistical analysis of around ∼20–25 nm, consistent with the calculated mean
TEM-derived nanoparticle dimensions was performed using diameter (23.33 nm). The histogram constructed from
Gaussian and log-normal tting models (Fig. 6B). The resulting measured particles (Fig. 6B) exhibits a slightly asymmetric
histogram exhibited a slightly asymmetric distribution centered shape rather than a perfectly symmetric bell curve.
around ∼20–25 nm, indicating moderate polydispersity within To better describe this distribution, both Gaussian and log-
the colloidal system. normal models were applied (Table S1, in SI). The log-normal
The TEM micrographs (Fig. 6) reveal detailed morphological function provides a closer t to the experimental data, as re-
features, particle size, and spatial distribution of the synthe- ected by a higher coefficient of determination (R2 = 0.9674),
sized AgNPs. Most particles display a quasi-spherical geometry, along with lower c2 (3.8526) and AIC (800.64) values compared
while a limited fraction adopts slightly elongated or irregular to the Gaussian model (R2 = 0.9335; c2 = 6.0781; AIC = 802.43).
polyhedral forms. Such morphological diversity is frequently This tting behavior supports a log-normal distribution
encountered in biosynthesized silver nanoparticles. pattern, which is commonly associated with nanoparticle
Direct measurement from TEM images places the particle systems governed by stochastic nucleation and growth
size predominantly within the range of ∼15–35 nm. The processes.
metallic cores are clearly resolved, allowing precise identica- The asymmetric prole observed in Fig. 6B is characteristic
tion of individual nanoparticles. of systems where particle growth proceeds at different rates
A more rigorous statistical evaluation was carried out using aer an initial nucleation stage. Most particles remain within
ImageJ soware based on multiple TEM images. The average a narrow size window, while a smaller fraction grows to larger
particle diameter was determined to be 23.33 ± 6.44 nm, which dimensions, producing a right-skewed distribution. This inter-
aligns well with the visually observed size range. The relatively pretation is also consistent with the measured standard devia-
small standard deviation reects a fairly tight distribution of tion (±6.44 nm), indicating slight polydispersity rather than
particle sizes, with most particles clustered around the mean a strictly monodisperse system.
value. Under more alkaline conditions (pH $ 8), the micrographs
At pH 7, the particle population remains relatively small and reveal an increased presence of clustered domains. Individual
uniform. This condition favors rapid nucleation events, leading particles become less isolated, and the apparent size
Fig. 6 Representative Transmission Electron Microscopy (TEM) analysis of the synthesized nanoparticles (A) and particle size distribution of
AgNPs with Gaussian and log-normal fitting (B).
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distribution broadens. Such evolution is consistent with clas- As the pH increases, the diffraction peaks become progres-
sical nucleation–growth behavior reported for metallic sively more intense and sharper. The reections associated with
nanoparticles.47,48 the (200) and (220) planes exhibit noticeable narrowing at pH 9
[Link] Dispersion and aggregation. The nanoparticles are and 10, pointing to enhanced crystallinity and growth of
generally well separated across large regions of the grid. At the coherent crystalline domains. Such evolution in peak prole is
same time, distinct aggregation zones are visible. Within these typically associated with accelerated reduction kinetics under
areas, particles assemble into chain-like (“necklace-like”) alkaline conditions, where both nucleation and subsequent
arrangements or compact clusters. growth proceed more rapidly.
This pattern reects moderate colloidal stability. Attractive Crystallite size was estimated using the Scherrer equation
van der Waals interactions drive partial aggregation when based on peak broadening analysis (Table S3 and Fig. S2, see in
repulsive forces are insufficient to fully stabilize the dispersion. SI). The calculated sizes for the (111), (200), and (220) planes are
Comparable structures have been reported in nanoparticle 33.69, 27.19, and 17.76 nm, respectively, giving an average
systems with limited surface stabilization. crystallite size of 26.21 ± 8.01 nm. Variations among planes
Although the nanoparticles exhibited predominantly spher- reect anisotropic broadening, which is commonly observed in
ical morphology, moderate polydispersity and partial aggrega- nanoscale metallic systems.
tion were still observed, particularly at prolonged reaction times A comparison with TEM-derived particle size (23.33 ± 6.44
and under highly alkaline synthesis conditions. nm) shows close agreement, with the XRD-based values slightly
Aggregation becomes more pronounced as the pH increases. higher. This difference arises from the distinct measurement
Larger clusters, reaching the submicron scale (∼300 nm), principles: TEM captures individual nanoparticles directly,
appear alongside heterogeneous assemblies. The shi can be while XRD evaluates coherent diffraction domains, which may
linked to reduced electrostatic repulsion in alkaline media, include contributions from partially aggregated or internally
which facilitates particle–particle interactions and eventual structured regions. Despite this difference, both methods
coalescence.49 Although phytochemical species act as capping converge within the same nanoscale regime, supporting the
agents, their stabilizing role weakens under strongly basic formation of well-dened AgNPs.
conditions, allowing aggregation to proceed more readily.50 Further structural insight is provided by the relative peak
Similar behavior has been documented across a range of AgNP intensities and texture coefficient (TC) analysis (Table S4, in SI).
systems under varying chemical environments.46 The experimental intensity sequence deviates from the stan-
[Link] Nucleation–growth evolution of AgNPs. The TEM dard fcc pattern, with the (200) reection exhibiting the highest
observations provide insight into the structural evolution of the intensity, followed by (220) and (111). Quantitative TC values
nanoparticles during synthesis. Two regimes emerge: reveal a preferred orientation along the (220) plane (TC > 1),
At neutral pH (pH 7), nucleation dominates. Numerous whereas the (111) plane contributes less strongly compared to
small nuclei form rapidly, yielding a population of compara- the standard reference. Although the (200) peak dominates in
tively well-dispersed nanoparticles. absolute intensity, its TC value remains lower than that of the
At higher pH ($9), growth and aggregation become more (220) plane, indicating that texture evolution is governed by
prominent. Particles interact, coalesce, and develop into larger, deviations from standard intensity ratios rather than peak
less uniform assemblies. magnitude alone.
The coexistence of isolated primary particles and aggregated Such preferential orientation suggests anisotropic crystal
structures reects a dynamic balance between nucleation, growth during nanoparticle formation. Differences in surface
growth, and interparticle interactions. The log-normal size energy and growth kinetics among crystallographic planes likely
distribution obtained from ImageJ analysis further supports drive this behavior, particularly under alkaline conditions
this interpretation, as such distributions typically arise from where reaction rates are elevated.
multiplicative growth processes rather than uniform particle The pH-dependent changes in peak intensity and narrowing
formation. TEM characterization thus claries both the nano- further support enhanced crystallinity and structural evolution
scale dimensions and the statistical distribution of the AgNPs, under alkaline synthesis conditions. These combined observa-
while also capturing the transition from discrete particles to tions point to a transition toward larger, more heterogeneous
aggregated domains under changing chemical conditions. nanoparticle populations as the synthesis environment
3.2.3 XRD patterns of the biosynthesized AgNPs. The XRD becomes increasingly alkaline. The XRD results collectively
patterns of the biosynthesized AgNPs obtained at pH 7, 8, 9, and conrm the formation of highly crystalline fcc AgNPs, with
10 are presented in Fig. 5e. Three well-dened diffraction peaks crystallite sizes consistent with TEM observations and evidence
appear at 2q values of 38.1°, 44.3°, and 64.4°, corresponding to of pH-dependent structural evolution during nanoparticle
the (111), (200), and (220) planes of face-centered cubic (fcc) growth.
silver (JCPDS card no. 04-0783).51 No additional reections 3.2.4 FTIR spectra of the biosynthesized AgNPs. The FTIR
associated with silver oxides, such as Ag2O, are detected, indi- spectra of MCFE and the biosynthesized AgNPs shown in Fig. 5f
cating that the reduction process led predominantly to metallic exhibited multiple absorption bands within the range of 400–
Ag0. The plant extract maintained sufficient reducing strength 3600 cm−1, corresponding to various functional groups. The
throughout the synthesis, while simultaneously limiting broad band observed between 3100 and 3400 cm−1 was
oxidation under the reaction conditions. assigned to O–H/N–H stretching vibrations associated with
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hydroxyl-containing phytochemicals. Noticeable differences in In comparison, the FTIR spectrum of chemically synthesized
band shape, width, and position between MCFE and AgNP Ag obtained using NaBH4 displayed only a broad and symmetric
samples suggest interactions between these functional groups absorption band centered around 3400 cm−1, most likely
and the nanoparticle surface. The peak at 1401 cm−1 in MCFE associated with adsorbed moisture, without clearly resolved
corresponds to asymmetric CH3 bending or COO− asymmetric features below ∼1000 cm−1 corresponding to Ag-related vibra-
stretching and became broadened into two adjacent peaks aer tions. The minimal spectral prole suggests the absence of
AgNP formation. Several spectral features remained relatively stabilizing organic surface species in the chemically synthe-
unchanged, including the bands at 1644 and 1689 cm−1 sized sample. Overall, the FTIR results indicate that while
attributed to C]C and C]O stretching vibrations, as well as alkaline conditions (pH $ 9) promoted particle growth and
the peaks at 2815 and 2980 cm−1 corresponding to aliphatic aggregation, neutral conditions (pH 7) produced comparatively
C–H stretching. well-dispersed and stable AgNPs with smaller particle size.
Additional spectral changes were observed aer nanoparticle Therefore, AgNPs synthesized using 1.5 mL of 0.025 M AgNO3
formation. The peaks at 1010 and 1087 cm−1, assigned to and 2.5 mL of MCFE at pH 7 for 60 min (AgNPs7) were selected
C–O–C and C–O–Ag stretching vibrations, exhibited substantial for further investigation due to their favorable combination of
variations in relative intensity in the AgNP samples. Similarly, small size, narrow distribution, and high colloidal stability.
the aromatic C–H vibrations at 849 and 887 cm−1 decreased Among the investigated conditions, AgNPs7 exhibited the most
markedly in AgNPs8 and AgNPs9. Two low-wavenumber bands favorable combination of small particle size, narrow distribu-
at 579 and 615 cm−1 were absent in MCFE but appeared in the tion, and colloidal stability, making this sample suitable for
AgNP spectra with varying intensity ratios. These bands are subsequent catalytic and physicochemical investigations.
associated with X–Ag interactions (X = O or N), likely arising
from coordination between Ag and oxygen-containing phenolic 3.3 Catalytic reduction
groups, generating weak bending vibrations in the low-
3.3.1 Some factors affecting the catalytic reduction of RhB
frequency region.52,53 These spectral modications support the
by AgNPs7. The catalytic reduction performance of AgNPs7
involvement of phytochemical species as reducing and stabi-
toward Rhodamine B (RhB) was evaluated under different
lizing agents during AgNP formation, with the most
operational conditions, including NaBH4 dosage, catalyst
pronounced stabilization features observed for AgNPs7.
loading, and initial RhB concentration. The corresponding
degradation proles are presented in Fig. 7.
Fig. 7 Effect of key reaction parameters on the catalytic reduction of Rhodamine B (RhB) in the presence of AgNPs-7: (a) volume of 1 M NaBH4,
(b) volume of AgNPs-7 solution, and (c) initial RhB concentration. Data are presented as mean ± SD from three independent experiments (n = 3);
error bars represent standard deviations.
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[Link] Effect of NaBH4 amount. Fig. 7a clearly indicates that 3.3.2 Catalytic reduction kinetics. The catalytic reduction
RhB removal strongly depends on the amount of NaBH4. In the kinetics of RhB over AgNPs7 were evaluated using pseudo-rst-
absence of NaBH4, negligible RhB removal (∼6%) was observed order (PFO) and pseudo-second-order (PSO) kinetic models
even aer 90 min, indicating that AgNPs alone exhibited under different reaction conditions. The corresponding tting
minimal catalytic activity under the investigated conditions. In proles are presented in Fig. 8, while the calculated kinetic
the presence of 0.15 mL 1 M NaBH4 solution, the degradation parameters are summarized in Table 2.
process was still somewhat limited (about 21% at 30 min and Table 2 shows that: (1) as the amount of NaBH4 increased,
about 55% at 60–90 min), indicating insufficient electron the rate constant k increased steadily in both models / indi-
availability to efficiently sustain the reduction process. As the cating that NaBH4 played an important role in providing elec-
NaBH4 volume increased, the RhB removal efficiency was trons, which increased the catalytic efficiency. The R2 values in
signicantly improved. The best performance occurred at the PSO kinetic model are more than 0.98 showed that the PSO
0.60 mL NaBH4, removing more than 82% RhB aer 10 model described the experiment better. (2) As the solution
minutes, 96.85% aer 45 minutes, and slightly increased to increased from 1.5 / 3 mL, the rate constant increased sharply,
98.2% at 90 minutes. This behavior can be explained by the then decreased as the volume of AgNPs7 volume of AgNPs7
synergistic contributions of NaBH4 and AgNPs7. continued to increase. R2 according to the PFO model is mostly
[Link] Effect of AgNPs7 amount. The impact of AgNPs7 dose higher than that in the PSO model; R2 according to the PSO
on the catalytic degradation of 20 mg L−1 RhB in the presence of model was in the range of 0.911 to 0.957. (3) With increasing
0.6 mL NaBH4 is shown in Fig. 7b. As the volume of AgNPs7 was RhB concentration, the rate constant decreases, which is
1.5 mL, the RhB removal efficiency only achieved 44–76% over usually due to the masking effect of the catalyst surface and the
times of 5–90 min. When the catalyst dosage increased to 3 mL, reduction of UV/vis light penetration. Both models give high R2
the RhB removal efficiency was immediately achieved at about (>0.97), but PSO describes the experimental data more closely.
82% within 5 min and near complete removal (98%) within Some studies have also obtained similar results.57–59
60 min, with near identical degradation following this time. To facilitate more rigorous comparison of catalytic perfor-
However, further increasing the catalyst dosage to 4.5 or 6 mL mance, the apparent pseudo-rst-order rate constant was
resulted in decreased degradation performance, which pla- additionally considered relative to catalyst loading. Normaliza-
teaued at ∼89–90% aer 60 min. This behavior is likely asso- tion of the kinetic response to catalyst amount suggests that the
ciated with nanoparticle aggregation at elevated AgNPs7 catalytic efficiency of the biosynthesized AgNP system remains
dosages and competition for available electrons supplied by competitive compared with previously reported phyto-mediated
NaBH4 under excessively catalyst-rich conditions. Thus, the AgNP catalysts under comparable dye-reduction conditions.
optimal dosage of AgNPs7 catalyst is 3 mL, which will be This behavior indicates that catalytic activity is inuenced not
selected for further studies. only by the overall reduction rate, but also by nanoparticle
[Link] Effect of initial RhB concentration. Fig. 7c shows the accessibility, colloidal dispersion stability, and surface-
effect of the initial RhB concentration on the catalytic reduction mediated electron-transfer efficiency during the catalytic
performance of AgNPs7 (3 mL) in the presence of 0.6 mL NaBH4. process.
At an initial concentration of 20 mg L−1, the degradation However, the PSO model provided better agreement with the
occurred quickly – approximately 73% removal aer the rst experimental data, suggesting strong contributions from
5 min and almost complete degradation (96.85%) aer 40 min adsorption-associated surface interactions and interfacial
and then increased slightly when extended to 60 min (97.7%). electron-transfer processes. Biosynthesized AgNPs are typically
When the initial RhB concentration increased, the efficiency stabilized by phytochemical-derived organic surface species
decreased slightly. At an initial RhB concentration of originating from the plant extract, which may inuence both
100 mg L−1, the degradation was limited to 32% at 5 min and adsorption behavior and catalytic reduction kinetics. The
85.4% at 60 min, reecting substrate overload relative to the improved agreement with the PSO model suggests that the
available catalyst and electron donor. Nevertheless, the catalytic catalytic reduction process is strongly inuenced by adsorption-
performance of the biosynthesized AgNPs7 remained superior associated surface interactions and interfacial electron-transfer
to many previously reported green-synthesized AgNPs, both in processes occurring at the AgNP interface. Similar PSO-
terms of efficiency and reaction time: AgNPs were green dominated kinetic behavior has also been reported for other
synthesized using Eucalyptus globulus leaf extract, which was biosynthesized AgNP catalytic systems.56,57
able to degrade up to 93% of 18 mg L−1 RhB at pH 12 aer Overall, the PSO model generally provided improved agree-
80 min;37 AgNPs were green synthesized using Matricaria cha- ment with the experimental data, indicating that the catalytic
momilla L. leaf extract, which was able to degrade up to 93, 37% reduction process is strongly inuenced by adsorption-
of 5 to 25 mg L− RhB under UV irradiation aer 130 min;54 associated surface interactions and interfacial charge-transfer
AgNPs were green synthesized using Eupatorium adenophorum processes occurring at the AgNP interface.
L. leaf extract, which was able to degrade up to 78.69% RhB 3.3.3 Mechanism of catalytic reduction. To further eluci-
under sunlight aer 90 min;55 Similarly, AgNPs synthesized date the catalytic reduction pathway of RhB over AgNPs7,
using Shorea robusta leaf extract achieved only 90.41% RhB mechanistic investigations were conducted using scavenger
degradation under comparable conditions.56 experiments and zeta potential analysis (Fig. 9). These analyses
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Fig. 8 Kinetic modeling of Rhodamine B (RhB) catalytic reduction by AgNPs7 under different reaction conditions according to pseudo-first-
order (PFO) and pseudo-second-order (PSO) models: effect of NaBH4 dosage fitted by (a) PFO and (b) PSO models; effect of AgNPs7 dosage
fitted by (c) PFO and (d) PSO models; and effect of initial RhB concentration fitted by (e) PFO and (f) PSO models.
Table 2 Kinetic parameters of RhB catalytic reduction by AgNPs. Reported values are expressed as mean ± SD obtained from three independent
measurements (n = 3)
PFO kinetic model PSO kinetic model
K1 R2 K2 R2
Volume of 1 M NaBH4 (mL) 0.00 4.921 × 10−4 6.120 × 10−5 0.638 2.576 × 10−5 3.133 × 10−6 0.944
0.15 9.430 × 10−3 8.403 × 10−4 0.969 7.573 × 10−4 3.318 × 10−5 0.992
0.30 0.0185 0.0026 0.925 0.0031 1.814 × 10−4 0.986
0.45 0.0221 0.0035 0.909 0.0081 3.052 × 10−4 0.994
0.6 0.0267 0.0052 0.868 0.0342 0.987
Volume of AgNPs solution (mL) 1.5 0.0098 0.0016 0.899 0.0014 1.299 × 10−4 0.957
3.0 0.0227 0.0014 0.986 0.0172 0.0027 0.911
4.5 0.0145 0.0012 0.972 0.0070 9.354 × 10−4 0.934
6.0 0.0135 0.0010 0.978 0.0058 6.536 × 10−4 0.952
RhB initial concentration (mg L−1) 10 0.0464 0.0059 0.925 0.0393 0.0023 0.983
30 0.0340 0.0041 0.931 0.0090 3.708 × 10−4 0.992
50 0.0324 0.0023 0.975 0.0034 1.857 × 10−4 0.985
100 0.0270 8.920 × 10−4 0.996 9.702 × 10−4 7.040 × 10−5 0.974
were combined with kinetic interpretation to better clarify the As shown in Fig. 9a, the absence of scavengers resulted in
role of interfacial electron-transfer processes during catalytic rapid RhB decolorization (∼98% within 60 min). The intro-
reduction. duction of EDTA-2Na and tert-butanol induced a transient
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Fig. 9 Effect of different scavengers on the catalytic reduction of RhB in aqueous solution by AgNPs7 (a) and zeta potential of AgNPs before and
after removing RhB out of water (b). Data are shown as mean ± SD from three independent experiments (n = 3); error bars indicate standard
deviations.
inhibition during the initial stage (20–30% removal at 5 min), governed primarily by interfacial electron-transfer processes
followed by gradual recovery of catalytic efficiency to ∼94% at occurring at the nanoparticle surface. Previous studies on
longer reaction times. In contrast, the presence of AgNO3, plasmonic AgNP systems have proposed that localized surface
acting as a competing electron acceptor, drastically suppressed plasmon resonance (LSPR) may participate in interfacial
the reaction throughout the entire process (<20% efficiency). charge-transfer processes through transient high-energy elec-
This preferential inhibition strongly suggests that interfacial trons.60,61 Direct evidence for plasmon-assisted pathways was
electron transfer plays a dominant role in the catalytic process, not obtained in the present work. Any possible plasmonic
whereas the contributions of holes and hydroxyl radicals are contribution is therefore discussed cautiously and only within
secondary and non-rate-determining. the context of previously reported mechanistic interpretations.
The concentrations of EDTA-2Na, tert-butanol, and AgNO3 Zeta potential analysis (Fig. 9b) shows negligible variation
were chosen according to ranges commonly employed in before and aer the reaction, indicating that the interaction
mechanistic scavenger studies, while also considering the between RhB and the nanoparticle surface is weak and
distinct inhibitory behavior associated with each compound. predominantly physisorptive. This observation, together with
Since these scavengers interact with different reactive species the negligible RhB removal (∼6%) in the absence of NaBH4,
and possess different scavenging efficiencies, their effects indicates that adsorption alone is insufficient and that the
cannot be evaluated solely on the basis of absolute concentra- process is primarily governed by surface-mediated catalysis.
tion. EDTA-2Na and tert-butanol primarily suppress hole- and Taken together, the results suggest that the catalytic process is
hydroxyl-radical-associated pathways, respectively, leading dominated by interfacial electron-transfer interactions occur-
mainly to temporary perturbation of secondary surface reac- ring at the AgNP surface, where AgNPs7 function as nanoscale
tions during the early stage of RhB reduction. AgNO3 behaves electron mediators facilitating charge transfer between BH4−
differently. As a competing electron acceptor, it intercepts donor species and RhB molecules. This behavior highlights
electrons transferred from BH4− donor species across the AgNP a fundamental distinction between metallic nanoparticle
interface. Persistent inhibition in the presence of AgNO3 is thus catalysis and conventional semiconductor photocatalysis:
more consistent with disruption of the interfacial electron- rather than relying on band-structure-generated charge carriers,
transfer route governing RhB reduction than with a simple the reaction proceeds predominantly through localized inter-
dosage-related scavenging effect. The catalytic reduction likely facial electron-transfer processes occurring at the nanoparticle
proceeds through an electron-relay mechanism consistent with surface.
a Langmuir–Hinshelwood-type surface process. In this mecha- Under optimized synthesis conditions, improved colloidal
nism, both BH4− donor species and RhB molecules are co- stability and controlled nanoparticle growth may further
adsorbed onto the AgNP surface, where AgNPs7 act as nano- enhance surface accessibility and interfacial charge-transfer
scale electron-transfer mediators. Electrons supplied by BH4− efficiency.
are transferred across the nanoparticle interface to RhB mole- Collectively, the scavenger experiments, kinetic interpreta-
cules, thereby facilitating reduction of the chromophoric tion, and zeta-potential analysis support a predominantly
structure into colorless products. Unlike conventional semi- surface-mediated electron-transfer mechanism for RhB reduc-
conductor photocatalysts such as TiO2 or ZnO, metallic AgNPs tion over biosynthesized AgNPs7, as schematically illustrated in
lack a bandgap due to overlap between the valence and Fig. 10.
conduction bands. Consequently, their catalytic behavior is
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Fig. 10 Proposed surface-mediated electron-transfer mechanism for the catalytic reduction of RhB by NaBH4 over biosynthesized AgNPs7,
highlighting interfacial charge relay and scavenger-inhibited pathways.
3.4 Antibacterial and cytotoxic performance Table 3 Inhibition zone diameter of test samples against E. coli, S.
aureus and P. aeruginosa. Values are presented as mean ± SD from
3.4.1 Antibacterial activity. AgNPs8, with characteristics three independent experiments (n = 3)
quite similar to AgNPs7 but slightly larger in size, was also
evaluated for antibacterial activity (E. coli, PA and SA) along with Inhibition zone diameter (mm)
AgNPs7, MCFE and antibiotic AMO by the agar disc diffusion
No. Samples Concentration SA PA E. coli
method. Results are shown in Fig. 11 and Table 3.
Based on Table 3 and Fig. 11, both the silver nanoparticles 1 AgNPs7 8 mg mL−1 20.3 0.4 18.3 0.4 21.7 0.3
and MCFE inhibited bacterial growth. MCFE alone (50 mg mL−1) 2 AgNPs8 8 mg mL−1 17.3 0.4 14.0 0.0 18.3 0.4
produced only modest inhibition against all tested strains. Such 3 MCFE 50 mg mL−1 11.3 0.4 10.3 0.4 12.7 0.4
4 H2O 0 0.0 0.0 0.0 0.0 0.0 0.0
behavior may arise from the limited dispersion stability and 5 AMO 100 mg mL−1 23.3 0.4 23.0 0.0 22.3 0.6
restricted membrane accessibility of freely dissolved phyto-
chemicals in aqueous media. Once incorporated into the
nanoparticle system, the antibacterial response increased
AgNPs8 displayed weaker antibacterial activity than AgNPs7
markedly. AgNP formation likely altered the interfacial behavior
despite their comparable chemical composition. SEM observa-
of the phytochemical components, possibly improving nano-
tions together with UV-vis analysis revealed that AgNPs8
particle dispersion and increasing interaction with bacterial
possessed larger particle sizes (∼30–50 nm), whereas AgNPs7
surfaces. The phytochemical-derived organic corona may also
remained within a narrower nanoscale range (∼20–30 nm).
contribute to colloidal stabilization and inuence the local
Reduced antibacterial performance in AgNPs8 is plausibly
availability of Ag-active species at the nano–bio interface.
Fig. 11 Antibacterial activity of AgNPs7 (1), AgNPs8 (2), MCFE (3), H2O (4) and antibiotic AMO (5) against E. coli (a), PA (b) and SA (c).
RSC Adv. © 2026 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances
linked to the lower surface-to-volume ratio and weaker interfa- should be regarded as literature-supported mechanistic
cial interaction associated with larger nanoparticles. Smaller hypotheses that are compatible with the observed antibacterial
AgNPs generally provide a higher density of accessible surface activity rather than experimentally veried mechanisms in the
sites and more intimate contact with bacterial envelopes. Better present study.
aqueous dispersion of AgNPs7 may also increase nanoparticle- Antibacterial susceptibility followed the sequence: S. aureus
cell encounters, reinforcing membrane-associated antibacte- > E. coli > P. aeruginosa. Similar trends have been described
rial interactions. The size-dependent behavior observed here previously for AgNP systems.64 Structural differences in bacte-
aligns well with the established structure–activity relationship rial envelopes likely contribute to this variation. The porous
commonly reported for metallic nanomaterials. peptidoglycan network of Gram-positive S. aureus may permit
The observed antibacterial activity likely depends not only on stronger interaction with AgNPs and Ag+ species. In contrast,
the intrinsic antimicrobial properties of silver, but also on Gram-negative bacteria possess an outer lipopolysaccharide
nanoparticle size, colloidal stability, and surface-associated membrane that partially restricts nanoparticle penetration. The
phytochemicals originating from the MCFE extract. Smaller comparatively lower susceptibility of P. aeruginosa may addi-
and more uniformly dispersed AgNPs may provide higher tionally relate to its strong biolm-forming behavior, active
surface accessibility and more effective interaction with bacte- efflux systems, and adaptive oxidative-stress defenses.
rial membranes, which may favor membrane-associated inter- SEM observations (Fig. 5) suggested surface association
actions and oxidative-stress-related pathways. Residual between AgNPs7 and bacterial cells, accompanied by localized
phytochemical species adsorbed on the nanoparticle surface structural perturbation. Although ultrastructural damage was
may additionally contribute to interfacial interactions with not quantitatively evaluated, the observed morphological alter-
microbial cells and inuence Ag+ release behavior. ations are consistent with antibacterial responses previously
AgNPs7 at 8 mg mL−1 generated inhibition zones comparable reported for AgNP systems; however, these observations alone
to those produced by amoxicillin (100 mg mL−1), reecting cannot substantiate membrane disruption or ROS-mediated
broad-spectrum antibacterial behavior. Several mechanisms bacterial killing. Relative to several previously reported green-
reported in previous studies may contribute to this enhance- synthesized AgNP systems, AgNPs7 achieved comparable inhi-
ment, including electrostatic attraction between AgNPs and bition performance at substantially lower concentrations.65,66
negatively charged bacterial membranes, gradual Ag+ release, Lower nanoparticle dosage is particularly relevant in biomed-
localized membrane perturbation, and oxidative stress associ- ical and environmental contexts because it may reduce material
ated with reactive oxygen species (ROS) generation.62,63 No consumption and limit unintended silver-associated toxicity.
single pathway is likely to dominate completely. Instead, the Molecular-level antibacterial pathways remain unresolved;
antibacterial response probably emerges from coupled inter- nevertheless, the biosynthesized AgNPs displayed consistent
actions occurring simultaneously at the nanoparticle surface. broad-spectrum antibacterial activity closely linked to nano-
The phytochemical capping layer may further modulate nano- particle physicochemical characteristics.
particle dispersion and the accessibility of reactive Ag- 3.4.2 Cytotoxic activity on gastric cancer cell lines (AGS)
associated surface sites. Direct mechanistic assays, including and liver cancer cells (HepG2). The cytotoxicity of AgNPs7 on
ROS quantication and membrane permeability analysis, were AGS and HepG2 cell line were evaluated by the MTT method.
not performed in the present work. Accordingly, ROS genera- These results were also observed by microscopic examination in
tion, membrane disruption, and oxidative-stress responses Fig. 12 and 13.
Fig. 12 The cytotoxicity results of AgNPs7 on AGS cell line (magnification ratio 1 : 200).
© 2026 The Author(s). Published by the Royal Society of Chemistry RSC Adv.
RSC Advances Paper
Fig. 13 The cytotoxicity results of AgNPs7 on HepG2 cell line (magnification ratio 1 : 200).
At low concentrations (0.08–0.8 mg mL−1), most HepG2 and reecting the mechanism of synergistic action.67 These results
AGS cells still retain their normal shape, with only scattered were shown in Fig. 14.
round cells appearing. When the concentration increases to 2–4 Fig. 14 showed that the experimental data t well with the
mg mL−1, cell density progressively decreases, and typical 4PL model. The cell viability decreased markedly with
morphological signs of cell damage, such as shrinkage, increasing AgNPs7 concentration for both AGS and HepG2 cell
rounding, and dark coloration became evident. The number of lines. The dose–response curves exhibited a sigmoidal trend,
round cells that detach from the culture surface increases indicating a concentration-dependent cytotoxic effect. The Hill
sharply. At a concentration of 8 mg mL−1, most of the cells were coefficients were 1.36 ± 0.38 for HepG2 cells and 1.53 ± 0.24 for
fully compromised, resulting in small, discrete fragments. This AGS cells, suggesting a cooperative concentration-dependent
change in morphology supported the quantitative data and cytotoxic response in which cell viability declined more
indicated the strong toxicity of AgNPs7 on cells that operated in rapidly aer a critical concentration was reached rather than
a concentration-dependent fashion. These observations suggest decreasing gradually. Similar threshold-like behaviour has
that AgNPs exhibit signicant cytotoxic activity and cytotoxic previously been reported for AgNP systems and has been asso-
effects against AGS and HepG2 cells even at lower concentra- ciated with mechanisms such as ROS generation, oxidative-
tions. The cytotoxic response may similarly be inuenced by stress accumulation, membrane damage, and subsequent cell
nanoparticle physicochemical properties, including particle death.57 However, these mechanisms remain literature-
size distribution, surface chemistry, and colloidal dispersion supported hypotheses and were not experimentally veried in
stability. AgNPs synthesized under optimized conditions the present study because intracellular ROS production,
exhibited comparatively controlled growth behavior and oxidative-stress biomarkers, and membrane integrity were not
reduced aggregation, factors that may facilitate cellular inter- directly assessed. The HepG2 cells showed slightly higher
action and nanoparticle internalization. In addition, phyto-
chemical species associated with the nanoparticle surface may
inuence nano–bio interactions through modulation of oxida-
tive stress pathways and interfacial cellular responses. Although
the observed cytotoxic responses toward cancer cell lines are
promising, evaluation using appropriate non-cancerous cell
lines will be required before any conclusions regarding anti-
cancer potential or tumor selectivity can be drawn.
The data were tted to the 4-parameter logistic model (4PL)
according to the equation:
AD
SðCÞ ¼ D þ n (10)
C
1þ
IC50
where S(C) is the proportion of viable cells at concentration C, A
corresponds to the control sample (only the sample matrix)
(100% viable cells); D is the response rate at the concentration
(usually chosen = 0); IC50 is the concentration that causes
Fig. 14 Cytotoxic effect of AgNPs7 on AGS and HepG2 cancer cells (fit
a 50% reduction; n is the Hill coefficient – the slope of the curve, according to 4PL model).
RSC Adv. © 2026 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances
sensitivity to AgNPs7, with cell death efficiency reaching nearly The proposed nucleation–growth mechanism is inferred
90% at 8 mg mL−1, while AGS cells reached about 88% under the from ex situ spectroscopy, kinetic modelling and microscopy,
same conditions. The calculated IC50 value of approximately and therefore lacks direct validation by in situ or operando
2.05 ± 0.57 mg mL−1 (for HepG2 cell) and 3.22 ± 0.57 (AGS cell), techniques capable of resolving transient intermediates and
suggests a potent cytotoxic activity of AgNPs7 toward both dynamic growth pathways. In addition, the phytochemical
cancer cell types. The obtained IC50 values were comparatively composition of the extract was not quantitatively dened,
lower than those reported for several previously published limiting reproducibility and obscuring molecular-level struc-
phyto-mediated AgNP systems, including AgNPs synthesized ture–function relationships governing reduction and stabiliza-
using aqueous extracts of Pistacia atlantica bark (IC50 132 mg tion. In addition, phytochemical characterization of the extract
mL−1)14 or AgNPs were synthesised using extracts of T. portu- remained limited to HPLC-DAD proling and tentative assign-
lacastrum and C. Quinoa.68 ment of several major avonoid species, while comprehensive
Compared with previously reported phyto-mediated AgNP metabolite proling and quantitative determination of indi-
systems, the biosynthesized AgNPs in the present study vidual phytochemical constituents were not systematically per-
exhibited comparable or improved multifunctional perfor- formed. This limitation restricts direct correlation between
mance under relatively mild synthesis conditions. The observed specic molecular components and the kinetic parameters
biological activity is therefore likely associated not only with governing nanoparticle nucleation, growth, and colloidal
silver-mediated effects, but also with the controlled colloidal stabilization. From a catalytic perspective, the reliance on
properties and phytochemical environment generated during NaBH4 constrains scalability and decouples intrinsic plasmonic
MCFE-mediated synthesis. contributions from chemical reduction, thereby limiting
Papaya-mediated AgNP systems with comparable physico- mechanistic clarity. Catalyst durability, reusability and poten-
chemical characteristics have previously been reported to tial ion release were also not systematically assessed. In the
exhibit reduced IC50 values in cancer cell models relative to present work, the biosynthesized AgNPs were intentionally
chemically synthesized AgNP counterparts.69,70 In one such employed as stable colloidal dispersions rather than recover-
system, papaya ower-derived AgNPs synthesized at pH 9 able heterogeneous catalysts. Due to the low nanoparticle
exhibited an average particle size of approximately 12 nm loading and highly dispersed nature of the reaction system,
together with IC50 values of 21 mg mL−1 against MCF-7 cells and quantitative recovery and reuse experiments could not be con-
169.96 ± 2.3 mg mL−1 toward HEK-293 cells.70 Direct compar- ducted reproducibly without perturbing nanoparticle concen-
ison with the present study remains limited by differences in tration, dispersion stability, and surface chemistry.
synthesis conditions, nanoparticle surface chemistry, and bio- Furthermore, Ag+ release during catalytic and biological
logical models. Nevertheless, these observations provide processes was not systematically quantied and therefore
broader context for interpreting the cytotoxic responses remains an important consideration for future environmental
observed in phyto-mediated AgNP systems.71–73 and biomedical assessment. Likewise, the long-term colloidal
Previous studies have proposed that AgNP-associated stability of the biosynthesized AgNPs under catalytic and bio-
oxidative stress, together with altered redox homeostasis and logical conditions was not evaluated. Both Ag+ release and long-
membrane damage, contributes to cytotoxicity in cancer cells. term colloidal stability may contribute to the observed catalytic
The present ndings are compatible with these proposed efficiency as well as the antibacterial and cytotoxic responses
mechanisms; however, intracellular ROS production, oxidative- and therefore warrant systematic investigation in future
stress signalling, and membrane damage were not directly studies.
examined and therefore remain literature-supported mecha- Furthermore, biological evaluations remain preliminary, as
nistic hypotheses rather than experimentally veried pathways cytotoxicity was assessed only in cancer cell lines and no
in this study. Such interfacial effects may contribute to differ- comparison with appropriate non-cancerous cells was per-
ences in cellular susceptibility among distinct cell models. The formed. Consequently, the present ndings do not establish
low IC50 values observed for AGS and HepG2 cells are consistent tumor selectivity or justify conclusions regarding anticancer
with concentration-dependent in vitro cytotoxic responses potential. Mechanistic investigations of ROS generation,
under conditions commonly associated with nanoparticle membrane integrity, oxidative-stress signalling, and down-
internalization and subsequent cellular stress processes. Inter- stream cell-death pathways were beyond the scope of the
pretation of these data nevertheless remains restricted to cancer present study. Consequently, the biological mechanisms di-
cell models only. Evaluation using appropriate non-cancerous scussed herein should be regarded as working hypotheses
cell lines will be required for rigorous assessment of selec- requiring dedicated mechanistic validation in future investiga-
tivity, biosafety, and biomedical relevance of the synthesized tions. Quantitative antimicrobial evaluation also remains
AgNPs. incomplete, as minimum inhibitory concentration (MIC)
measurements were not systematically determined in the
present study. In addition, the relationships between nano-
3.5 Limitations and future perspectives particle physicochemical properties, interfacial surface chem-
This study establishes a clear correlation between synthesis istry, and biological responses remain only partially resolved
conditions, kinetic regimes and multifunctional performance due to the complexity of nano–bio interactions under physio-
of AgNPs; however, several key limitations remain. logical environments. Finally, the absence of advanced surface
© 2026 The Author(s). Published by the Royal Society of Chemistry RSC Adv.
RSC Advances Paper
analyses (e.g., XPS) leaves the active surface chemistry insuffi- HepG2 cells. The experimental observations are consistent with
ciently resolved. catalytic activity being dominated by surface-mediated electron
Addressing these challenges requires a shi toward mecha- transfer at the AgNP interface rather than conventional
nistically informed and quantitatively controlled design. The semiconductor-type photocatalytic pathways, with AgNPs acting
integration of in situ diagnostics with predictive kinetic as interfacial electron-transfer mediators in the NaBH4-assisted
modelling represents a key challenge for resolving nucleation system.
pathways in phyto-mediated systems. Quantitative phytochem- Integrating kinetic descriptors with structural and func-
ical proling and extract standardisation are required to enable tional characterization provides a more coherent structure–
reproducible and tunable synthesis. The development of self- kinetics–function interpretation of phyto-mediated nano-
sustained, light-driven catalytic systems that eliminate particle formation. Within this framework, nanoparticle growth
external reductants remains essential to decouple plasmonic and colloidal evolution can be interpreted in relation to
effects and achieve sustainable operation. In parallel, rigorous controllable synthesis variables rather than empirical trial-and-
evaluation of catalyst stability, performance in complex error optimization alone.
matrices (e.g., real wastewater), and long-term behaviour is Male Carica papaya ower extract also provided a chemically
necessary to bridge laboratory ndings with practical deploy- responsive reaction medium whose phytochemical composition
ment. Future studies should therefore include systematic cata- appears to inuence nanoscale growth and colloidal stabiliza-
lyst recycling experiments, ICP-based quantication of silver tion under the synthesis conditions examined. This observation
ion release, and long-term colloidal stability analyses under further supports the emerging view of plant-derived extracts as
environmentally and biologically relevant conditions to clarify chemically tunable environments for nanomaterial fabrication.
their respective contributions to catalytic and biological Further progress will benet from establishing quantitative
performance. Comprehensive biological evaluation should links among phytochemical composition, nucleation behavior,
additionally incorporate MIC determination, comparative structural evolution, and functional performance through more
assessment using normal cell models, and systematic investi- comprehensive phytochemical characterization, in situ kinetic
gation of how nanoparticle size, colloidal stability, aggregation monitoring, and data-guided optimization. Such advances
state, and surface-associated phytochemicals inuence anti- would strengthen mechanistic understanding while facilitating
bacterial and cytotoxic responses. the rational development of environmentally compatible
For biomedical applications, systematic interrogation of nanomaterials with tunable catalytic, antimicrobial, and
nano–bio interactions, toxicity pathways and therapeutic biomedical properties.
selectivity, including in vivo validation, is required.
Taken together, these directions will be critical to transform Author contributions
plant-mediated nanoparticle synthesis from an empirically
driven process into a mechanistically predictable and Thi Tam Khieu: methodology, data curation, formal analysis,
application-oriented nanotechnology platform. validation, visualization, supervision, conceptualization,
writing – original dra, writing – review and editing; Thi Hong
4 Conclusion Tham Diep: investigation, data curation, formal analysis; Ngoc
Anh Hoang, Thi Thuy Ha Dinh: data curation, investigation;
Ethanolic–aqueous extracts from male Carica papaya L. owers Thanh Hai Cao: investigation, data curation, visualization;
provided a chemically active environment for controlled phyto- Tuan Kien Vu: investigation, formal analysis, soware; Quang
mediated synthesis of AgNPs under mild conditions. Systematic Tung Vu: investigation; Le Phuong Hoang: validation, meth-
variation of synthesis parameters combined with time-resolved odology, conceptualization; The Chinh Pham: methodology,
kinetic analysis showed that nanoparticle evolution extended project administration, resources; Truong Xuan Vuong: valida-
beyond conventional endpoint-based descriptions commonly tion, visualization, writing – review and editing; Thi Thao
used in green synthesis systems. Truong: supervision, validation, visualization, writing – review
Avrami exponents between 1 and 2 were consistent with and editing.
discontinuous nucleation accompanied by low-dimensional,
surface-inuenced growth. Combined spectroscopic, micro- Conflicts of interest
scopic, kinetic, and structural observations further support the
interpretation that nanoparticle evolution involved coupled The authors declare that they have no known competing
reduction–stabilization processes occurring at the growing nancial interests or personal relationships that could have
AgNP interface. Under optimized conditions (pH 7, 2.5 mL appeared to inuence the work reported in this paper.
extract, 1.5 mL AgNO3), the synthesized AgNPs exhibited crys-
talline face-centered cubic structures, narrow size distributions, Data availability
and stable colloidal behavior.
The biosynthesized AgNPs showed rapid catalytic reduction All data supporting the ndings of this study are available
of rhodamine B together with pronounced antibacterial activity within the article and its supplementary information (SI)
against Pseudomonas aeruginosa, Escherichia coli, and Staphylo- (Tables S1–S4 and Fig. S1 and S2). Further information is
coccus aureus, as well as measurable cytotoxicity toward AGS and available from the corresponding author upon reasonable
RSC Adv. © 2026 The Author(s). Published by the Royal Society of Chemistry
Paper RSC Advances
request. Supplementary information: Table S1: HPLC-DAD 8 K. Mandal, D. Das, S. K. Bose, A. Chaudhuri, A. Chakraborty,
chromatographic conditions and avonoid retention times. S. Mandal, S. Ghosh and S. Roy, Spectroscopic approach to
Table S2: comparison of Gaussian and log-normal particle-size optimize the biogenic silver nanoparticles for
distribution models. Table S3: XRD-derived crystallite sizes of photocatalytic removal of ternary dye mixture and
AgNPs. Table S4: texture coefficient (TC) analysis based on XRD ecotoxicological impact of treated wastewater, Sci. Rep.,
data. Fig. S1: HPLC-DAD chromatogram of Carica papaya ower 2024, 14, 31174, DOI: 10.1038/s41598-024-82341-7.
extract. Fig. S2: XRD pattern and crystallite-size analysis of the 9 A. Khan, N. Ahmad, H. Fazal, M. Ali, F. Akbar, I. Khan,
synthesized AgNPs. See DOI: [Link] M. Tayyab, M. N. Uddin, N. Ahmad, M. A. Abdel-Maksoud
d6ra02406h. and I. A. Saleh, Biogenic synthesis of silver nanoparticles
using Rubus fruticosus extract and their antibacterial
efficacy against Erwinia caratovora and Ralstonia
Acknowledgements solanacearum phytopathogens, RSC Adv., 2024, 14, 5754–
5763, DOI: 10.1039/D3RA06723H.
This work was nancially supported by the Ministry of Educa-
10 R. P. I. Tormena, M. K. M. S. Santos, A. O. da Silva,
tion and Training, Vietnam, under project number B2024-TNA-
F. M. Félix, J. A. Chaker, D. O. Freire, I. C. R. da Silva,
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