Applied Science and Engineering Progress, Vol. 15, No.

3, 2022, 5511 1

Research Article

Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous
Fruit Extracts and Its Characterization

Marilen M. Martin and Rodolfo E. Sumayao Jr.*

Department of Chemistry, De La Salle University, Manila, Philippines

* Corresponding author. E-mail: [Link]@[Link] DOI: 10.14416/[Link].2021.10.011

Received: 7 June 2021; Revised: 16 July 2021; Accepted: 3 August 2021; Published online: 27 October 2021
© 2022 King Mongkut’s University of Technology North Bangkok. All Rights Reserved.

Abstract
Rubus rosifolius Linn is a wild raspberry found in the Philippines. The fruit extract is found to be rich
in flavonoids, terpenoids, tannins, and polyphenols. It has been shown to contain interesting profiles of
antioxidants and it is not previously explored as a reducing agent, specifically in the field of bio-nano research.
Some of these antioxidants have been documented to have anti-cancer potential. In this study, the fruit extracts
of sampinit were used as a reducing agent for the synthesis of silver nanoparticles (AgNPs) via a ‘one-pot’
facile approach. Sampinit aqueous fruit extract-AgNPs (SAFE-AgNPs) were synthesized by reducing silver
ions using sampinit extract. Optical, chemical, and morphological properties of the synthesized SAFE-AgNPs
were characterized. Maximal absorption of SAFE-AgNPs was observed at 415 nm which is associated with
the characteristic surface plasmon resonance profile of AgNPs. SAFE-AgNPs displayed highly stable and
homogeneous nanoparticles with hydrodynamic size of 85.6 ± 0.98 nm and a zeta potential of –29.5 ± 0.96 mV.
HR-TEM analysis of a single crystalline image of SAFE-AgNPs corresponds to hexagonal diffraction
pattern and the AgNPs were coated with soft carbon-rich materials. EDX and XRF analysis showed that
SAFE-AgNPs are chiefly composed of silver (Ag) and carbon (C), where C was largely localized on the
surface of AgNPs. FTIR analysis showed that SAFE-AgNPs contains key chemical functional groups
associated with sampinit-derived phytoconstituents. Overall, the biosynthesized SAFE-AgNPs produced
small size, spherical shape, and monodisperse population which can be a candidate therapeutic agent for the
treatment of various diseases such as cancer.

Keywords: Green Synthesis, Rubus rosifolius Linn, SEM, Silver Nanoparticles, TEM

1 Introduction Generally, AgNPs can be prepared by chemical

and physical methods. The traditional chemical method
The unique properties of metallic nanoparticles (NPs) for the synthesis of AgNPs involves chemical reduction
have attracted keen interest of researchers and have which requires a metal precursor (usually silver
prompted vast research on their synthesis, characterization, nitrate), a reducing agent, and/or stabilizing agents
and application. Recent developments in NP synthesis [4]. This synthetic method has been hampered by the
have opened tremendous opportunities for the application inconsistent yield of NPs that are homogeneous in size
of NP technology in the field of biomedical sciences and shape, as well as its associated toxicity to humans
and engineering. Silver NPs (AgNPs) have taken the and the environment [4], [5]. Physical methods, on the
spotlight due to their unique physical and chemical other hand, do not require toxic and highly reactive
properties. AgNPs have been widely recognized for chemicals and generally have rapid processing time.
their excellent antimicrobial activities against a wide However, physical methods have high energy input
range of microorganisms which include bacteria [1], and costly downstream processing [6]–[8]. To enhance
fungi [2], and parasites [3]. AgNP functionality and biocompatibility, researchers

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
2 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511

have been exploring green synthetic approaches, in

which utilize biological agents such as bacteria, fungi,
yeast, plant and algal extracts [4], [9]. The biological
capacity to produce NPs under standard conditions
requires lower capital and operating expenses and can
also be implemented in nearly any setting at any scales
[4], [9]. Since many biological agents can also act as
surfactants and/or capping agents, biogenic NPs have
the advantage of enhanced stability, biocompatibility,
and reduced toxicity, which effectively mitigates its
adverse health and environmental effects [9].
Recently, extracts from botanical materials such
as leaves, barks, roots, and fruits were used for the
biogenic synthesis of AgNPs [4]. Plants and their Figure 1: Rubus rosifolius Linn, a wild raspberry
various anatomical parts contain proteins, pigments, locally known as sampinit. It is a spiny shrub that has
secondary metabolites, and other phytoconstituents prickly stem belonging to the rose family. Its fruit is
which act as reducing agents to produce nanoparticles conically elongated in shape, has hairy receptacle and
from metal salts without producing toxic products. red in color.
Some of these phytoconstituents may also serve
as biosurfactants and/or stabilizing agents which observed only to some specific plants such as sampinit.
promote long-term stability to metallic nanoparticles. To our knowledge sampinit has not been systematically
Phytomediated synthesis of AgNPs yields nanoparticles reported as a reducing agent for the green synthesis of
which differed in size, morphology, stability, and bio- silver nanoparticles.
activity [10]. However, due to variety of biomolecules In the present study, sampinit aqueous fruit
present in plant extracts, it is impossible to differentiate extracts (SAFE) were used as a reducing agent for
and observe the effect of each molecule during the synthesis of AgNPs using a ‘one-pot’ approach.
the reduction, nucleation, and growth steps in the The SAFE-AgNPs were extensively characterized for
nanoparticle synthesis [11]. optical properties, morphology, stability, and chemical
Rubus rosifolius Linn (Figure 1), locally known composition. The use of SAFE-AgNPs produces
as sampinit, is a shrub that belongs to the rose family. desirable optical, biophysical, and structural properties.
It can grow to a height of six feet and normally
thrives in higher altitudes about 1000 to 2000 feet 2 Experimental
above sea level and can be found in the southeast
Asia [12]. Its fruits are red in color when fully ripe, 2.1 Sample collection
have hairy receptacle and are conically elongated in
shape with roughly 1.5–2 cm across in length [6], [13]. Rubus rosifolius Linn was harvested from Dolores,
Phytochemical analysis of sampinit confirmed the Quezon Province, Philippines with the following
presence of different kinds of flavonoids, phenolic coordinates, 14°02’45.8” N 121°26’13.7”E. The
acids, and other bioactive compounds [14], [15]. collected fruits were authenticated and verified by the
Previous studies also showed that sampinit fruit Bureau of Plant Industry, Crop Research and Production
extracts exhibit antimicrobial [16], antioxidant [17]– Support Division, San Andres St. Malate, Manila.
[19], anti-inflammatory [17] and anti-cancer properties
[20], [21]. 2.2 SAFE preparation
The need for biocompatible nanomaterials
requires new nanotechnology solutions for green Ripe fruits were selected for aqueous extract preparation.
synthesis of AgNPs with biocompatible surfaces. The fruits were washed thoroughly with distilled
Hence, surface functionalization is emphasized in water. The fruits were cut into small pieces and snap-
this study since this may constitute a unique property frozen in liquid nitrogen. Subsequently, the fruits were

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511 3

lyophilized using a freeze dryer (Heto PowerDry, 2.5 Characterization of SAFE-AgNPs

ThermoFisher Scientific, Waltham, MA USA). The
freeze-dried samples were ground into fine powder 2.5.1 UV-Vis spectrophotometry
using a motorized blender. Powdered sample (2.0 g)
was placed in an Erlenmeyer and distilled deionized Optical properties of SAFE-AgNPs were assessed by
water was added to a final concentration of 0.02 g/mL. UV-Vis spectrophometry. A fresh suspension of SAFE-
The mixture was subjected to constant stirring for 30 AgNPs was subjected to wavelength scanning between
min at room temperature (RT), protected from light. 300–800 nm with a resolution of 1 nm using a UV-Vis
The extract was filtered using Whatmann filter no. 1 spectrophotometer (Hitachi U-2900, Tokyo, Japan).
and the filtrate was stored at –80 °C until further DDI H2O was used as a blank. The characteristic
analysis. strong absorption at 415 nm was considered as a
All chemicals were purchased from Sigma- confirmation of the presence of AgNPs. The stability
Aldrich (St. Louis, MO USA) unless otherwise stated. of SAFE-AgNPs was assessed by performing the same
spectral analysis every week for 6 weeks.
2.3 Reducing power
2.5.2 Fourier Transform Infrared (FTIR) spectroscopy
The reducing power of SAFE was determined using
a method as described with slight modifications [22]. FTIR analysis was performed for both SAFE and
To 1.0 mL of 25 mg/mL SAFE, 2.5 mL of phosphate SAFE-AgNPs. Lyophilized samples were mixed
buffer (pH 6.6) and 2.5 mL of 1.0% potassium ferricyanide with the potassium bromide with a ratio of 1 : 100 and
were added. The solution was incubated at 50 °C for pelletized. The spectrum of the sample was collected
20 min. Then, 2.5 mL of 10% trichloroacetic acid was using an FTIR spectrophotometer (ThermoFisher
added to the solution. The solution was centrifuged Scientific Nicolet 6700, Waltham, MA, USA) at a
at 3000 rpm for 10 min at RT. The supernatant was resolution of 4 cm−1 and 64 interferogram scans in the
transferred to a glass test tube and added with 2.5 mL range of 400–4000 cm−1. The various functional groups
distilled water and 0.5 mL 0.01% ferric chloride. The were automatically assigned using the Omnic software
absorbance of the solution was measured at 700 nm using (ThermoFisher Scientific, Waltham, MA, USA).
a UV-Vis spectrophotometer (Hitachi U-2900, Tokyo,
Japan). Ascorbic acid (5 mg/mL was used as a positive 2.5.3 Scanning Electron Microscopy (SEM)
control. The absorbance and concentration of the
sample are directly related to its reducing power [23]. Powdered SAFE-AgNP (2.0 mg) was coated with thin
layer of gold and was placed on a copper grid plate. The
2.4 SAFE-AgNP synthesis structural morphology of SAFE-AgNPs was analysed
using a scanning electron microscope (Phenom XL,
Forty mL of 1.0 mM AgNO3 was mixed with 3.0 mL Waltham, MA, USA) SEM images were captured at
of 2.0 mg/mL SAFE. The solution was subjected 26 kV and 50,000X magnification and were analysed
to constant stirring at 60 °C for 60 min, protected using the ProSuite software (ThermoFisher Scientific,
from light. The pH of solution was adjusted to 6.83 Waltham, MA, USA).
using 0.1 M ammonia (NH3). The appearance of the
characteristic yellowish brown color associated with 2.5.4 High Resolution-Transmission Electron Microscopy
AgNP formation was monitored. The suspension was (HR-TEM) with Energy Dispersive X-ray Spectroscopy
allowed to cool to RT. The AgNP suspension was (EDX)
centrifuged at 13,000 rpm for 15 min at RT to allow
nanoparticle sedimentation. The supernatant was High resolution morphological analysis of SAFE-AgNPs
carefully aspirated and discarded while the pellet was performed using high resolution transmission
was washed twice with DDI H2O. The AgNPs were electron microscope (HR-TEM). In brief, ~5 µL of
lyophilized for 24 h and were stored at RT, protected aqueous suspension of SAFE-AgNPs was deposited
from light, until further analysis. on 200 mesh holey carbon film supported on a copper

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
4 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511

grid and was allowed to dry at RT. The images of the

SAFE-AgNPs were acquired using the JEM-2100 Plus
HR-TEM (Jeol, Tokyo, Japan) with the field emission
gun operating at an accelerating voltage of 200 kV.
Elemental profiling and mapping of SAFE-
AgNPs were performed through locally resolved EDX
microanalysis at different points HR-TEM lamellae.

2.5.5 Dynamic Light Scattering (DLS) analysis

The average hydrodynamic diameter, polydispersity

index (PDI) and zeta potential of the SAFE-AgNPs
were measured by DLS. An aqueous suspension of Figure 2: Reducing power (RP) of SAFE. Data are
SAFE-AgNPs was sonicated (Soner 220H, Rocker expressed as mean ± SD of four replicates per group.
Products, New Taipei City, Taiwan) at 53 KHz at 25 °C *** denotes statistically significant differences (α =
for 20 min and loaded into 10 mm polystyrene cuvettes. 0.001). AA represents ascorbic acid.
For zeta potential measurements, the samples were
loaded into a folded zeta capillary cell. Measurements methanol, ethyl acetate, and n-hexane leaf extracts
were performed at 25 °C using a fixed angle of 173° of R. rosifolius gave the FRAP capacities equal to
using the Zetasizer Nano ZS (Malvern Panalytical, 96.50%, 82.83%, and 73.51%, respectively indicating
Malvern, United Kingdom) operated at 150V. Data its antioxidant potential.
were analysed using the Zetasizer Ultra-Pro ZS
Xplorer software v1.00 (Malvern Panalytical). 3.2 SAFE-AgNP synthesis

3 Results and Discussion The appearance of a yellow-brownish color upon

the addition of a reducing agent to AgNO3 has been
3.1 Reducing power of SAFE (Ferric Reducing routinely used as an indicator of AgNP formation [20],
Antioxidant Potential-FRAP) [26]. The addition of SAFE to AgNO3 solution resulted
in a change in color (yellow brown). This color change
The reducing power is an estimate of the antioxidant is associated with the surface plasmon excitation of
potential of the compound [22]. Antioxidant potential AgNPs, which is the primary and most notable indication
is due to the reduction-oxidation (REDOX) properties of AgNP formation [27], [28]. The yellow brown color
of the sample. SAFE was determined to assess its intensified in the presence of NH3 [Figure 3(c)]. Aside
ability to reduce silver ion (Ag+) to metallic silver from pH adjustment, NH3 can catalyse formation of
(Ago). As shown in Figure 2, SAFE displayed higher AgNPs [29], promote nucleation and size-controlled
Fe3+-Fe2+ complex absorbance than ascorbic acid (1.17 synthesis of AgNPs [30]. Similar results for the effect
± 0.026 and 0.81 ± 0.089, p < 0.05), which indicates of NH3 with or without chemicals that control the size
that SAFE has a higher reducing power compared to and catalyse formation of AgNPs were presented in the
the potent reducing agent, ascorbic acid. The high synthesis of AgNPs using skim rubber latex [31] and
reducing potential of SAFE is due to the presence of sodium citrate [32].
highly reducing phytochemicals in sampinit such as In this study, modification of the previously
flavonoids and phenolics as reported in several studies published protocols for the biosynthesis of AgNPs was
[6], [15], [24]. Hence, SAFE may be considered as performed by optimization of the parameter conditions
an effective reducing agent for AgNP synthesis. This such as the mass ratio of the SAFE to AgNO3, pH,
result is consistent with the ability of Rubus rosifolius temperature, and reaction time. It was observed
leaf extract to reduce Fe3+ complex ion to Fe2+ [25]. that the higher the concentration of the fruit extract
This is correlated with the antioxidant potential with the right concentration of the AgNO3 used in
involving single electron transfer mechanism. The the synthesis of AgNPs, the smaller the size of the

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511 5

(a) (b) (c)

(d)

(e) (f)

Figure 3: Monitoring of the biosynthesis of SAFE-AgNPs: (a) SAFE (20 mg/mL) added with 100 mL deionized
water; (b) SAFE (20 mg/mL) added with 40 mL 1.0 mM AgNO3; (c) SAFE (20 mg/mL) added with 40 mL
1.0 mM AgNO3 and 0.1 mM NH3.; (d) UV-Vis spectrum of SAFE-AgNPs at 415 nm absorbance.; (e) Weekly
absorbance spectra of SAFE-AgNPs showing no shift in peaks.

nanoparticles produced. The reason is that the more This leads to a controllable growth of the nanoparticle
extract molecules that cap the surface of the AgNPs [36].
the more the growth could be controlled [33]. In this
study, optimized condition was achieved at slight 3.3 Characterization of SAFE-AgNPs
acidic pH 6.83. Synthesis of the small and uniform
sized AgNPs could be achieved by altering the pH of UV-Vis spectroscopy is an important characterization
the reaction mixture depending on the nature of the technique to monitor the formation and stability of
plant extract [34]. Slightly acidic to basic condition AgNPs in aqueous solutions [37]. AgNPs exhibit
facilitates fast nucleation process leading to formation strong absorption peak between 350 and 450 nm due
of smaller sized AgNPs [35]. Similar result was reported to the collective oscillation of free conduction band
to have reached a more benign AgNPs at pH 6 [36]. The electrons of particles also known as surface plasmon
optimized temperature achieved in the biosynthesis resonance (SPR) phenomenon [38], [39]. UV-Vis
of SAFE-AgNPs is 60 °C for an hour. As the temperature spectral analysis of SAFE-AgNPs yielded a strong
is increased the yellow brown colored solution of the absorption peak at 415 nm [Figure 3(d)]. The intensity
AgNPs was intensified. This is due to the decomposition of the absorbance of SAFE-AgNPs at 415 nm remained
of the precursor salt in the reaction mixture [36]. strong over a period of 6 weeks [Figure 3(e)]. The
Generally, nucleation takes place rapidly at high periodic observation of SAFE-AgNPs during the storage
temperature leading to the synthesis of smaller sized period of 6 weeks showed almost consistent absorption
AgNPs [36]. As for the reaction time, the longer the spectrum without shift in the peak wavelength which
aging process the more extract molecules that adhere indicate the stability of SAFE-AgNPs. The results agree
on the surface of the SAFE-AgNPs were formed [36]. with the previous study where the proanthocyanidins-

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
6 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511

functionalized AgNPs revealed stability during the

storage of longer than a month since no obvious
(a)
changes in the UV absorption spectrum were observed
[28]. Similar results were reported with the preparation
of AgNPs from the inflorescence of Cocos nucifera
[12] and from the peel of Punica grantum [17] with
longer period of storage, 2 and 4 months, respectively
where no variation on the spectral characteristics on
the absorption spectra was observed.

3.4 Structural and morphological properties of

SAFE-AgNPs
(b)
Dynamic Light Scattering (DLS) analysis was performed
to determine the size distribution profile, as well as
the polydispersity index (PDI), of SAFE-AgNPs.
There were 2 peaks, peak 1 and peak 2, detected in
the particle size distribution analysis where peak 2
represents larger particle size SAFE-AgNPs. Majority
of the size was represented by peak 2. This might be
due to the intensity of the light scattered by the larger Figure 4: (a) Particle size distribution profile and
particle sized SAFE-AgNPs which totally conceals (b) Zeta Potential distribution profile of SAFE-AgNPs.
the signal from smaller ones [40]. Hence, the average
hydrodynamic size of SAFE-AgNPs was 85.6 ± 0.98 distribution [19]. The average PDI obtained for
nm [Figure 4(a)]. SAFE-AgNPs was 0.266 ± 0.0097, which indicates
The zeta potential is an important parameter used that SAFE-AgNPs are relatively homogeneous [40].
to evaluate the surface charge of micro- or nanoparticles Overall, the results were in good agreement with the
in colloidal dispersions, which is associated with their biogenically synthesized AgNPs from green tea and
stability. The average zeta potential value obtained for pomegranate peel extracts where PDI values obtained
SAFE-AgNPs was –29.5 ± 0.96 mV [Figure 4(b)]. Zeta were 0.28 and 0.25, respectively indicating monodisperse
potential values above +25 mV and below –25 mV population for both AgNPs [21], [42].
have high degree of stability [41]. Therefore, SAFE- SEM was performed to establish the surface
AgNPs are considered highly stable. Several reports morphology, size, and shape of the SAFE-AgNPs.
demonstrated similar zeta potential values below The SEM images of SAFE-AgNPs revealed clusters of
–25 mV for AgNPs. Fumaria parviflora extract mediated quasi-spherical nanoparticles with sizes ranging from
biogenic synthesis of AgNPs obtained a zeta potential 33.3 to 59.2 nm [Figure 5(a) and (b)]. SEM images
value of –29.6 mV, which caused greater stability and revealed sizes of nanoparticles which are quite smaller
prevent agglomeration for the synthesized AgNPs [36]. than the sizes obtained in DLS. This might be due to
A study of the green tea extract mediated biogenic the aggregation of several nanoparticles which is often
synthesis of silver nanoparticles obtained a negatively detected as single particles by DLS [43]. In addition, the
charged zetapotential value, –39.6 mV, which suggested increased size of the synthesized AgNPs as determined
not only high stability of the biosynthesized AgNPs but by DLS may be due to the hydrated capping agents
also the presence of the active organic phytoconstituent, [44] and solvation effects. The SAFE-AgNPs used in
polyphenol, present in green tea [21]. DLS technique is in colloidal solution. The size of the
The PDI was measured to determine the size SAFE-AgNPs is not only determined by the metallic
population distribution of SAFE-AgNPs. A PDI value core but also by the other substances that adhered on
equal to 0 represents a monodisperse distribution and the surface of the AgNPs [40]. In addition, the thickness
a PDI value equal to 1 represents a polydispersed of electrical double layer might also add up to the size

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511 7

(a) (b)

(a) (b)
Figure 5: SEM micrograph of SAFE-AgNPs showing
clusters of quasi-spherical shapes. (a) x35k magnification
and (b) x50k magnification.

of the SAFE-AgNPs. The thickness of the electrical

double layer depends on the substances present in the
colloid and the biomolecules that capped the AgNPs
[40]. Similar results were obtained in the biosynthesized
AgNPs using Matricaria chamomilla flower extract (c)
where the sizes of AgNP-1 measured using SEM and
DLS, were 70 nm and 125 nm, respectively [45]. For Figure 6: High resolution transmission electron
AgNP-2, the sizes measured using SEM and DLS were microscopy bright field (HRTEM – BF) image of
52 nm and 105 nm, respectively [45]. SAFE-AgNPs at (a) x100k magnification and (b) x150
A more detailed structural analysis of SAFE- magnification. (c) A single crystallized AgNP covered
AgNPs was performed using HR-TEM. Consistent with organic phytoconstituents present in SAFE, and
with SEM analysis, HR image of SAFE-AgNP crystal the inset shows the diffraction pattern corresponding to
structure revealed a uniformly spherical-shaped the hexagonal crystallographic planes of SAFE-AgNPs.
nanoparticles with an approximate size of <50 nm
[Figure 6(a) and (b)]. The atomic planes of a single of a single crystalline AgNP were indeed sampinit-
crystalline silver are visible from the HR lattice derived organic phytoconstituents. Consistent with
image of an individual SAFE-AgNP [Figure 6(c)]. these findings, the EDX atomic emission spectrum
The diffraction patterns in the image correspond to also revealed strong signals from Ag and C at ~3.0
a hexagonal structure which was previously reported keV and ~0.3 keV [Figure 7(d)], respectively, which
for AgNPs [46], [47]. Interestingly, an HR image of further supports the sampinit-mediated capping of
a single crytalline AgNP revealed that it is uniformly AgNPs, which may have protomoted the long-term
coated with a soft material, which could possibly be stabilization of SAFE-AgNPs.
the phytoconstituents of sampinit acting as capping
agents to stabilize the SAFE-AgNPs. 3.5 Chemical composition of SAFE-AgNPs
EDX spectroscopy was performed along with
HR-TEM to confirm the elemental distribution in X-ray fluorescence (XRF) spectroscopy analysis was
SAFE-AgNPs [Figure 7(d)]. EDX point mapping of performed for complementary elemental analysis of
SAFE-AgNPs revealed a strong signal for Ag and SAFE-AgNPs. The XRF emission spectra of SAFE-
was mainly localized in the core domain of AgNPs AgNPs revealed two strong signals at ~3.0 and 21 keV
(green) [Figure 7(b)]. Interestingly, carbon (C) formed (blue and yellow peaks, respectively) (Figure 8), which
a huge component of the SAFE-AgNPs and was is indicative of metallic silver. Ag metal accounts
largely diffused across the surface of the AgNPs (red) for 42.5% of SAFE-AgNPs (data not shown). The
[Figure 7(c)]. The abundance of C on the surface of amount of silver obtained in SEM-EDX is higher
SAFE-AgNPs indicates that the soft materials that coat compared to XRF since it is the the dried purified form
the surface of AgNPs observed in the HR-TEM image of AgNP that was measured in the analysis whereas, in

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
8 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511

(a) (b) (c)

(d)

Figure 7: Scanning transmission electron microscopy (STEM)/Energy dispersive X-ray (EDX) Mapping
of SAFE-AgNPs. (a) The figure displays a bright field (BF) image of dark SAFE-AgNPs on a light
background. (b) The figure shows the energy dispersive mapping of silver characteristic x-ray which affirms its
distribution in SAFE-AgNPs. (c) The image shows large component of carbon (in SAFE–AgNPs) surrounding
the nanoparticles. (d) EDX spectrum of SAFE-AgNPs.

Figure 8: Elemental profile of SAFE-AgNPs determined by X-ray Fluorescence (XRF) spectroscopy.

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511 9

Figure 9: Overlaid FTIR spectra of SAFE and SAFE-AgNP.

XRF it is the AgNP suspension that was used. Possibly, [50], [51]. The bands observed observed at 1387.72
presence of some other bioconstituents might still be and 1256.28 cm–1 in SAFE-AgNPs correspond to the
involved in the anaysis. Overall, these results corroborates CH2 stretching bend, whereas the peak at 1071.23
the reduction capability of sampinit-derived cm–1 is attributed to –C=O stretching vibration of
phytoconstitutents in the formation of high-yield AgNPs. ester groups. Lastly, the band that appeared at 744.332 cm–1
Fourier-transform infrared (FTIR) spectroscopy is associated with the aliphatic carboxylic acid.
was performed to determine the chemical functional A slight shift in the absorption band at 3398.85 cm–1
gropus present in SAFE-AgNPs. The FTIR spectrum in SAFE-AgNPs may be attributed to the multiple
of SAFE-AgNPs strongly matched the spectrum of hydrogen bonding between the various functional
SAFE, which indicates that most of the SAFE-derived groups in SAFE which, in turn, could have facilitated
chemical compounds were successfully incorporated the stabilization of SAFE-AgNPs [16]. Furthermore,
into the AgNPs (Figure 9). The strong broad band the appearance of the absorption band at 1161.73 cm–1
obtained between 3300–3500 cm–1 is associated with and 1105. 48 cm–1 in SAFE-AgNPs may be associated
the hydroxyl and amine groups. The distinctive bands with the shift of the deformation vibration of –OH
at 3396.85 cm–1 and 3374.06 cm–1 are characteristic of groups, which suggests involvement of –OH groups
O-H and N-H stretching vibrations of hydroxyl and in the bioreduction process of Ag+ to Ago [48].
amines groups, respectively, where N-H is chracterized Overall, the various functional groups detected
by lower wave numbers than that of the O–H [48], by FTIR revealed the chemical diversity of sampinit-
[49]. These –OH groups may be associated with the derived extract which may have aided the successful
polyphenolic compounds, while the –NH groups may bioreduction of Ag+ to Ago. The presence of active
be associated with the primary and secondary amines functional groups such as alcohol, phenol, secondary
in amino acids and peptides present in SAFE [48]. The amines, amides, and carbonyl groups in SAFE-AgNPs
absorbance bands at 2926.61, 1627.96 and 1596.46 cm–1 may be attributed to the various sampinit-derived
are attributed to the symmetric and asymmetric aliphatic metabolites such as polyphenols, flavonoids, tannins, and
C–H and N–H stretching vibrations, to C=O or amide proteins/peptides [23], [50]. These organic constituents
groups and to C–C stretch in aromatic rings of the in sampinit may have also adhered on the surface of
flavonoids, polyphenols and terpenoids, respectively AgNPs, serving as a capping agent, which accounts

M. M. Martin and R. E. Sumayao Jr., “Facile Green Synthesis of Silver Nanoparticles Using Rubus rosifolius Linn Aqueous Fruit Extracts
and Its Characterization.”
10 Applied Science and Engineering Progress, Vol. 15, No. 3, 2022, 5511

for the long term stabilization of SAFE-AgNPs and

prevention of their agglomeration. This is supported by
a plethora of studies that utilized various plant materials
or fruit extracts as reducing agents for the biogenic
synthesis of AgNPs [51]–[53].
There are some studies reported on the successful
synthesis of AgNPs using different Rubus species. Most
of them reported the preparation of extract by washing,
grinding, heating the samples either by regular heating
[54] or via microwave [55]. Finally, heated samples are
allowed to cool and filtered. In this study, same steps
were performed but with the addition of snap-freezing
the samples with liquid nitrogen prior to lyophilization. Figure 10: Proposed mechanism for the formation of
These additional steps were done to avoid dehydration SAFE-AgNPs. The reduction of Ag+ to Ago involves
and to preserve the samples. For the synthesis of REDOX reaction.
SAFE-AgNPs, after the extraction of SAFE, it was
again subjected to lyophilization to get the exact 4 Conclusions
concentration of SAFE to be added to AgNO3. Then,
AgNO3 was added drop by drop to the extract until In this study, the conjugation of AgNPs with sampinit
the formation of AgNPs. This part of the synthesis aqueous fruit extract (SAFE) was demonstrated
is crucial to the formation of the desirable properties through a simple one pot synthesis. The developed
of SAFE-AgNPs. Several studies had sucessfully green process can be used as an alternative approach to
synthesized AgNPs from Rubus species [55], [56] but conventional methods in synthesizing biogenic silver
none had reported to the best of our knowledge the nanoparticles. The active phytoconstituents present
modifications made in this study. Furthermore, the use in SAFE as detected by FTIR may have aided the
of any Rubus species was able to reduce Ag+ to Ago but successful bioreduction of Ag+ to Ago and the long
only Rubus rosifolius linn gave a stable SAFE-AgNPs term stabilization of SAFE-AgNPs. Its ability to
with no shift in peak wavelengths within the storage effectively reduce Ag+ to AgNP is complemented by its
period of 6 weeks. high reducing power. Overall, the synthesized SAFE-
With all the several characterization techniques AgNPs having spherical particles of <50 nm in size, a
performed, significant results were obtained. Hence, PDI value of 0.266 ± 0.0097, and a zeta potential value
mechanism for the formation of stable SAFE-AgNPs of –29.5 mV ± 0.96 are remarkable characteristics and
is proposed. Reduction-oxidation, REDOX, reaction considered good attributes for effective nanocarriers.
involves the reduction of Ag+ to Ago. An electron is gained
by silver from the –OH ion present in flavonoids and Acknowledgements
polyphenols, which are the active phytoconstituents
present in SAFE as confirmed by the FTIR results. The authors would like to extend their gratitude to
These phytochemicals react with silver nitrate to Dr. Maria Carmen Tan (DLSU Chemistry Department
form the metallic silver. The –OH in polyphenols Instrument Laboratory) and Mr. Mark Mangogtong
reacts with Ag+ reducing it to AgNPs (Figure 10). (DKSH Philippines) for their technical assistance and
The biosynthesized SAFE-AgNPs was stabilized by to the DLSU Research Fellowship Program for the
capping the surface of the SAFE-AgNPs with the financial support.
SAFE secondary metabolites as corroborated by
the HR-TEM results. The capping of the secondary References
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