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Energy Procedia 89 (2016) 239 – 247

CoE on Sustainable Energy System (Thai-Japan), Faculty of Engineering, Rajamangala University

of Technology Thanyaburi (RMUTT), Thailand

Environmental-friendly method for synthesis of silver nanoparticles

from dragon fruit peel extract and their antibacterial activities
Siriporn Phongtongpasuka*, Sarinya Poadanga, Niti Yongvanichb
a
Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakornpathom, 73000, Thailand
b
Department of Materials Science and Engineering, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakornpathom,
73000 Thailand

Abstract

A facile, green and eco-friendly approach was introduced for the synthesis of silver nanoparticles using dragon fruit peel extract
(DPE) as reducing as well as capping agent. The effect of pH on the formation of silver nanoparticles was studied utilizing
various characterization techniques including UV-Vis spectroscopy, FTIR, XRD, EDX, TEM and zeta potential. The formation
of silver nanoparticles was confirmed by UV-Vis spectroscopy with the presence of surface plasmon resonance band between
430 and 460 nm. TEM images demonstrated that the size of synthesized silver nanoparticles was reduced with an increase of pH.
The silver nanoparticles was spherical in shape with a negative charge on the surface of the nanoparticles. The size of the
nanoparticles was in the range of 25-26 nm. XRD and EDX analysis indicated that the biosynthesized nanoparticles were
crystallized in face centered cubic symmetry with the existence of Ag2O. A possible organic compound in DPE involving in the
reduction and stabilization of nanoparticles was revealed by FTIR. Moreover, the synthesized nanoparticles exhibited effective
antibacterial action against representative pathogenic bacteria.

©
© 2016
2016TheTheAuthors. Published
Authors. by Elsevier
Published Ltd. Ltd.
by Elsevier This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of the 12th EMSES 2015.
Peer-review under responsibility of the organizing committee of the 12th EMSES 2015
Keywords:Antibacterial activity;Dragon fruit; Peel extract; Silver nanoparticles

* Corresponding author. Tel.: +6-634-219-360 ; fax: +6-634-219-360.

E-mail address: [email protected]

1876-6102 © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of the 12th EMSES 2015
doi:10.1016/j.egypro.2016.05.031
240 Siriporn Phongtongpasuk et al. / Energy Procedia 89 (2016) 239 – 247

1. Introduction

Silver nanoparticles are clusters of silver atoms in the size range of 1-100 nm. They have gained momentum due
to distinctive electrical, optical, and biological properties [1]. The latter property has drawn much attention due to a
broad-spectrum antimicrobial activity [2]. Therefore, silver nanoparticles have been popularly incorporated into
several products including wound dressing, household antiseptic sprays, textiles and antimicrobial coating for
medical devices that sterilize air and surfaces [3].
Generally, silver nanoparticles are mainly synthesized by physical and chemical approaches, which are
economically expensive and involve in chemical toxic [4]. In addition, silver nanoparticles derived from chemical
synthesis could have a toxic chemical residue being absorbed on the surface of nanoparticle resulting in adverse
effect for biomedical applications. Therefore, biogenic synthesis using plant extract has been recently emerged as an
alternative method for the synthesis of silver nanoparticles. The plant extract is commonly composed of different
types of phytochemical compound which participate in the reduction of Ag + to Ag0 and stabilization of silver
nanoparticle by acting as a capping agent. The biological synthetic approach for silver nanoparticles is advantageous
over physicochemical method because it is simple, cost effective, environmental-friendly, and easy to scale up for
mass production [5]. Furthermore, the biogenically synthesized silver nanoparticle could be safely utilized for many
therapeutic applications as it is biocompatible. However, there are only few studies on the synthesis of silver
nanoparticle using agricultural wastes.
Dragon fruit (Hylocereus undatus) or red pitaya is a tropical fruit which has a red skin with green fins on the fruit.
It belongs to the cactus family, Cactaceae. The pulp of dragon fruit contains high quantity of vitamin C and water-
soluble fiber. Usually, dragon fruit is consumed directly or being processed into juice for functional drink.
Consequently, it has resulted in the accumulation of the fruit peels as waste materials. The key chemical
compositions in dragon fruit peel are betanin, phyllocactin, hylocerenin, betacyanin, pectin, triterpenoids, and
steroids [6,7]. These compounds have many pharmacological activities such as antitumor, anti-inflammatory, and
antioxidant properties [8,9]. The latter action is interesting due to the availability of electrons to neutralize free
radicals. It indirectly implies that the organic compounds in the dragon fruit peel could have the ability for the
reduction of metal salt ion in the synthesis of metal nanoparticle. Therefore, the objective of this study was to
synthesize silver nanoparticles by a green approach using a dragon fruit peel extract at different pH levels and to
characterize the synthesized silver nanoparticles utilizing UV-Visible spectroscopy, EDX, TEM, XRD, FTIR, and
zeta potential analysis. Besides, their antibacterial activity against representative of human pathogenic bacteria was
also investigated.

Nomenclature

BSA Bovine serum albumin

DPE Dragon fruit peel extract
FRAP Ferric reducing antioxidant power
GAE Gallic acid equivalent
RE Rutin equivalent
TFC Total flavonoid content
TPC Total phenolic content

2. Materials and methods

2.1. Materials

Dragon fruit were obtained from Pathommongkol market (Nakornpathom, Thailand). All chemicals used are
analytical grade. All solutions were prepared with deionized water. Silver nitrate (AgNO3) and Folin-Ciocalteu
reagent were purchased from Merck (Darmstadt, Germany). All other chemicals used in the experiments were
obtained from Sigma Aldrich (St. Louise, USA).
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2.2. Preparation of the peel extract

Dragon fruit peel extract was obtained by extracting fresh peel with water (ratio 1:20 w/v) at 100 ºC for 15 min.
Then, the filtration was applied to obtain the extract. The filtrate was collected and stored at 4 ºC for further use.

2.3. Synthesis of silver nanoparticles

A reaction mixture contained 220 ml of dragon fruit extract in 110 ml of 10 mM silver nitrate (AgNO 3) solution.
The solution was stirred for 24 h on a magnetic stirrer at room temperature. Silver nanopaticles were separated from
the reaction mixture by centrifugation at 12,000 rpm for 15 min at 4 ºC. The pellet was redispersed in deionized
water and centrifuged again at the same condition as mentioned above to gain the purity of nanosilver. Later,
lyophilization was performed to obtain silver nanoparticle powder. The effect of pH was studied by adjusting the pH
of DPE to 3.35, 4.35 and 5.35 prior to reacting with AgNO3.

2.4. Characterization of silver nanoparticles

To examine the absorption pattern of biogenic silver nanoparticles, the spectrum of sample was recorded by UV-
Vis spectrophotometer (Biochrom Libra S22, UK) between 250-800 nm. The size and morphology of silver
nanoparticles were examined using TEM (Tecnai G2 20 S-Twin, USA). The samples were prepared by placing a
drop of silver nanoparticles on a carbon coated copper grid. The elemental analysis of the silver nanoparticles was
carried out using energy dispersive X-ray spectroscopy (EDX) incorporated to TEM. X-ray diffraction (XRD)
patterns were obtained using a Rigaku RINT2000 model (CuKD). FTIR (Nicolet 6700, Thermo Fisher Scientific,
USA) scanning at the wavenumber range of 4000-400 cm-1 was employed to analyze the functional groups capping
on silver nanoparticles. The surface charge and stability of silver nanoparticles were achieved by zeta potential
measurement (ZetaPlus, BrookHaven Instrument Corp., USA).

2.5. Phytochemical content in fresh dragon fruit peel extract

2.5.1. Total phenolic content (TPC)

Folin-Ciocalteu method was applied for the determination of total phenolic content with a slight modification
[10]. Briefly, 0.25 ml of the peel extract was well mixed with 1.25 ml of Folin-Ciocalteu reagent (10x dilution) for 5
min. Then, 1 ml of 7.5% Na 2CO3 solution was added. The reaction mixture was incubated at 40 qC for 30 min.
Absorbance of blue complex solution was measured at 760 nm using UV-Vis spectrophotometer. Distilled water
was used as a blank. A standard curve was prepared using a various concentrations of gallic acid. TPC was
expressed as milligram of gallic acid equivalent per gram sample (mg GAE/g sample).

2.5.2. Total flavonoid content (TFC)

The colourimetric method was introduced to the determination of total flavonoid content with some
modifications [10]. First, 0.5 ml of dragon fruit peel extract was mixed with 2.25 ml of distilled water. Then, 0.3 ml
of 5% NaNO2 solution was added and the reaction mixture was incubated. After 6 min, 0.3 ml of 10% AlCl 3.6H2O
was added and left to stand for 5 min. To the reaction mixture, 1 ml of 1M NaOH was added and mixed. The
absorbance of the reaction mixture was recorded at 510 nm using spectrophotometer. An array of rutin
concentrations were prepared for a calibration curve. TFC was presented in milligram rutin equivalent per gram
sample (mg RE/g sample).

2.5.3. Protein assay

Protein measurement in the peel extract was determined using a Bradford assay [11]. To 10 Pl of the peel extract
in eppendrof, 200 Pl of Bradford reagent was added. The reaction mixture was well mixed. After 5 min, the
242 Siriporn Phongtongpasuk et al. / Energy Procedia 89 (2016) 239 – 247

absorbance was measured at 595 nm using microplate reader. A standard curve was prepared with different
concentrations of bovin serum albumin (BSA). Result was expressed in term of mg BSA/g sample. Samples were
analyzed in triplicate.

2.5.4. Reducing sugar assay

The determination of reducing sugar was carried out using DNS method [12]. A reaction mixture contained 1 ml
of the peel extract and 3 ml of DNS reagent. The solution was heated at 95 ºC for 10 min and cooled down to room
temperature. The absorbance was measured at 550 nm using spectrophotometer. The quantification was carried out
using a calibration curve with different concentrations of glucose in the range of 0.2-1.2 mg/ml. The results were
displayed in mg glucose/g sample.

2.5.5. Ferric reducing power assay

Reducing ability was determined using the FRAP assay [13]. The peel extract (200 Pl) was reacted with 1.8 ml of
the FRAP solution (300 mM acetate buffer pH 3.6, 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl 3.6H2O).
After 4 min, the absorbance was monitored at 593 nm using a spectrophotometer. An array of known concentrations
of FeSO4 were prepared for a standard curve. The results were expressed as Pmol FeSO4/g sample.

2.6. Antibacterial assay

The antibacterial screening of silver nanoparticles was carried out against pathogenic bacteria including
Escherichia coli and Pseudomonas aeruginosa (gram negative), and Staphylococus aureus (gram positive) using
disc diffusion method. Briefly, 0.1 ml each of the seeded broth containing 105 CFU/ml of the test organisms was
swabbed uniformly on nutrient agar using sterile cotton swabs. Then, 20 Pl of 0.5 mg/ml of peel extract and 0.5
mg/ml of biosynthesized silver nanoparticles were dropped on the center of paper disc. A commercial paper disc
with 10 Pg of gentamicin was used as positive control. All discs were placed on the nutrient agar plates. After
incubation at 37qC for 24 h, the diameter of inhibition zone was measured in millimeter. Three independent
experiments were performed with each strain.

3. Results and discussion

3.1. Characterization of biosynthesized silver nanoparticles

The effect of pH on the biosynthesis of silver nanoparticles was investigated by varying the pH of DPE to 3.35,
4.35 or 5.35. The formation of silver nanoparticles was monitored by visual observation and UV-Visible
spectroscopy. The color of the reaction mixture started to change from pink to yellowish brown within 15 min,
indicating the generation of silver nanoparticle due to the bioreduction of silver metal ion (Ag +) into silver
nanoparticle (Ag0) by the organic molecules present in the DPE. The color of silver nanoparticle suspension is
attributed to the excitation of surface plasmon resonance (SPR) depending on the particle size, shape, aggregation
and the surrounding dielectric medium [14]. As shown in Fig. 1., a well-defined SPR band for silver nanoparticle
was observed at about 430-460 nm. The color of the reaction mixture was pH dependent. The silver nanoparticles
synthesized at pH of 3.35, 4.35 and 5.35 demonstrated absorption peaks at 457, 449 and 431 nm, respectively.
Interestingly, the absorption peaks shifted to shorter wavelength and became narrower with the rise of pH value. It is
possibly due to the size reduction or anisotropic degree of the silver nanoparticles [15].
 Siriporn Phongtongpasuk et al. / Energy Procedia 89 (2016) 239 – 247 243

Fig. 1 Visual observation and UV-vis absorption spectra of biosynthesized silver nanoparticles at pH 3.35, 4.35 and 5.35 compared to DPE.

The morphology and size of the synthesized silver nanoparticles prepared at different pH values were analyzed
by TEM as shown in Fig. 2. All prepared silver nanoparticles were spherical in shape and polydisperse. An
aggregation of the biogenic silver nanoparticle was obviously monitored when the biogenic silver nanoparticle
prepared under lower pH: 3.35 and 4.35. The average diameter of the particle prepared at pH 3.35, 4.35 and 5.35
were 26.2±8.2, 25.7±8.7 and 25.3±7.9 nm, respectively. This result corresponds to hypsochromic shift of the
absorption bands in the UV-Visible spectra in Fig. 1. In addtion, silver nanoparticles were surrounded by a thin layer
which could be an organic constituent in the fruit peel extract.

Fig. 2 TEM images (A-C) and particle size distribution histograms (D-F) of silver nanoparticles synthesized at pH 3.35, 4.35 and 5.35,
respectively.

The XRD pettern of the silver nanoparticles compared with DPE is depicted in Fig. 3. The XRD peaks at 2T of
38.1 and 77.4 correspond to (111) and (311) crystalline planes of the face-centered cubic structure of metallic silver.
However, a few additional and yet unassigned peaks were also noticed in the XRD pattern of silver. This
unindentified peak was apparently not originated from organic compounds found in DPE because no peak was
observed in the XRD spectrum of DPE. In addition, the most intense peak at 32.5 possibly indicated a presence of
Ag2O in the synthesized silver nanoparticles.
244 Siriporn Phongtongpasuk et al. / Energy Procedia 89 (2016) 239 – 247

Fig. 3 XRD spectra pattern of synthesized silver nanoparticles using dragon fruit peel extract at different pH values including 3.35, 4.35 and 5.35.
The “X” symbol represents Ag2O peak while the dark rectangle depicts Ag peak.

EDX analysis (Fig. 4) reveals a strong signal of the metallic silver region at 3 keV and confirms the formation of
silver nanoparticle using DPE. A weak signal for oxygen was monitored in the EXD profile. This result would
correspond to the existence of Ag2O from XRD analysis. Two moderate signals of copper and carbon were detected
due to the carbon coated copper grid for sample preparation.

Fig. 4 EDX profile of biogenic silver nanoparticles.

FTIR spectroscopy was employed to identify the functional groups responsible for the biosynthesis of silver
nanoparticles. FTIR spectra of DPE (before reaction without AgNO 3) and synthesized silver nanoparticles (after
reaction with AgNO3) are illustrated in Fig.5. It can be seen that both FTIR spectra showed a shift in the following
peaks: 3420-3433 (due to O-H stretching of phenol or carboxylic acids), 2934-2922 (due to C-H stretching of
alkane), 1600-1627 and 1409-1428 (due to C=C stretching of alkene), 1103-1022 (due to C-OH stretching of
carboxylic acid. In addition, DPE showed peak at 1741 cm -1 corresponding to C=O stretching of carbonyl group
however this peak was absent in the synthesized silver nanoparticle. This reveals that carbonyl group of the DPE is
involved in the reduction of silver ions. The FTIR analysis indicates that the functional groups (-OH, -COOH and
C=O) may involve the reduction and stabilization of biosynthesized silver nanoparticle. Dragon fruit peel mainly
contain pectin, betanin, phyllocactin, hylocerenin, and betacyanin [7]. These compositions contain several functional
groups which possibly mediate the reduction of Ag+ to Ag0.
 Siriporn Phongtongpasuk et al. / Energy Procedia 89 (2016) 239 – 247 245

Fig. 5 FTIR spectra of DPE and AgNPs synthesized from the peel extract.

Zeta potential values of silver nanoparticles in water synthesized at pH 3.35, 4.35 and 5.35 were -28.16±0.38, -
16.61±0.26 and -32.36±1.08 mV, respectively. Overall, it reveals that the capping molecules present on the surface
of biogenic silver nanoparticles are principally composed of negatively charged functional groups. Generally, a
suspension that shows a zeta potential higher than ±20 mV confirms a great long term stability of the suspension due
to the repulsion among the particles preventing agglomeration [17]. In present study, the absolute zeta potential of
silver nanoparticle synthesized at pH 3.35 and 5.35 were higher than 20 mV proving that they could be well
dispersed and stable in the medium while pH 4.35 condition might resulted in moderate stability.

3.2. Phytochemical content in the peel extract

Quantitative phytochemical contents was examined by colormetric method as illustrated in Table1. The result
exhibits that dragon fruit peel extract consists of several phytochemical contents including TPC, TFC, reducing
sugar, and protein at 4.21±0.036 mg GAE/g sample, 1.35±0.113 mg RE/g sample, 23.64±0.003 mg glucose/ g
sample, and 1.26±0.013 mg BSA/g sample, respectively. This indicates that these biomolecules in the peel extract
contribute in the bioreduction and stabilization of the silver nanoparticle synthesis. Furthermore, FRAP assay was
performed to imitate the power of phytochemical constituent in the peel extract for the bioreduction of Ag + to Ag0.
The FRAP value (32.63±0.019 Pmol FeSO4/g sample) had a relatively moderate reducing ability when compared
with a standard chemical such as ascorbic acid. This implies that organic molecules in DPE were involved with the
reduction of Ag+ to Ag0 for the nanoparticle formation.

Table 1. Phytochemical contents in dragon fruit peel extract.

TPC TFC Reducing sugar FRAP Protein

(mg GAE/g sample) (mg RE/ g sample) (mg glucose/ g sample) (Pmol FeSO4/g sample) (mg BSA/ g sample)

DPE 4.21±0.036 1.35±0.113 23.64±0.003 32.63±0.019 1.26±0.013

3.3. Antibacterial activity of silver nanoparticles

The antibacterial activity of silver nanoparticles against selected human pathogenic bacteria was elucidated by
disc diffusion assay. Bacterial growth inhibition around the paper disc is due to the release of diffusible inhibitory
compounds from tested material. As shown in Table 2, gentamicin, used as positive control, demonstrated the
highest inhibition zone of diameter at 15 mm, 13 mm, and 14 mm for E. coli, P. aeruginosa, and S. aureus,
respectively. Moreover, all silver nanoparticles mediated by DPE at pH 3.35, 4.35, and 5.35 similarly exhibited an
246 Siriporn Phongtongpasuk et al. / Energy Procedia 89 (2016) 239 – 247

antibacterial activity with inhibition zone of diameter of 8.0 mm for E. coli, 7.0-7.3 mm for P. aeruginosa and 9.0-
9.3 mm for S. aureus. The result suggests that the synthesized nanoparticles display a good antibacterial activity
against both gram negative and gram positive bacteria but they demonstrate an effective stronger antibacterial
activity against S. aureus (gram positive) than P. aeruginosa and E. coli (gram negative). In addition, an
antibacterial activity in this study could be from both silver nanoparticle and Ag2O [18].

Table 2. Antibacterial activity against pathogenic bacteria.

Bacteria Diameter of inhibition zone (mm)

Gentamicin DPE pH 3.35 pH 4.35 pH 5.35

(10 Pg) (0.5 mg/ml) (0.5 mg/ml) (0.5 mg/ml) (0.5 mg/ml)

E. coli 15 ND 8.0 8.0 8.0

P. aeruginosa 13 ND 7.3 7.0 7.0

S. aureus 14 ND 9.2 9.3 9.0

4. Conclusion

The biogenic silver nanoparticles using dragon fruit peel extract were synthesized. The reduction and
stabilization of silver nanoparticles were attributed to the phytochemical compositions in DPE. The characterization
of synthesized silver nanoparticle was fully elucidated by several microscopic and spectroscopic techniques
including UV-Vis spectroscopy, TEM, EDX, XRD, and zeta potential confirming the formation of biosynthesized
silver nanoparticles. Furthermore, Ag2O was also present in the final product as shown in XRD and EDX analysis.
The effect of pH directly resulted in the morphology, size, stability, and antibacterial efficacy of the silver
nanoparticles. The proposed pH for the nanoparticle synthesis using DPE was pH 5.35 as given the smallest particle
size and good dispersion in the medium leading to a great stability of suspension. All biosynthesized nanoparticles
exhibited a great antibacterial activity against tested pathogenic bacteria. This biosynthesized nanoparticle could be
a potent antibacterial agent for the treatment of infection diseases caused by pathogenic bacteria.

Acknowledgements

The financial support provided by the Higher Education Research Promotion (HERP) and National Research
University project of Thailand (NRU), Office of the Higher Education Commission (SURDI-HERP 570112).

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