Saudi Journal of Biological Sciences (2014) 21, 605–609

King Saud University

Saudi Journal of Biological Sciences

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Synthesis of silver nanoparticles using Sacha

inchi (Plukenetia volubilis L.) leaf extracts
Brajesh Kumar *, Kumari Smita, Luis Cumbal *, Alexis Debut

Centro de Nanociencia y Nanotecnologia, Universidad de las Fuerzas Armadas – ESPE, Av. Gral. Rumiñahui s/n, Sangolqui,
P.O. Box 171-5-231B, Ecuador

Received 27 April 2014; revised 9 July 2014; accepted 9 July 2014

Available online 24 July 2014

KEYWORDS Abstract Silver nanoparticles (AgNPs) are fabricated using Sacha inchi (SI) or (Plukenetia volubi-
Silver nanoparticles; lis L.) leaf extract as non-toxic reducing agent with particle size ranging from 4 to 25 nm. Optical,
TEM; structural and morphological properties of the synthesized nanoparticles have been characterized
Plukenetia volubilis L.; by using Visual, UV–Vis spectrophotometer, transmission electron microscopy (TEM) and dynamic
Antioxidant; light scattering (DLS) analysis. Selected area electron diffraction (SAED) confirmed the formation
DPPH of metallic Ag. Infrared spectrum measurement was carried out to hypothesize the possible phyto-
chemicals responsible for stabilization and capping of the AgNPs. It shows the significant antiox-
idant efficacy in comparison with SI leaf extracts against 1,1-diphenyl-2-picrylhydrazyl. From the
results obtained it is suggested that green AgNPs could be used effectively in future engineering
and medical concerns.
ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction attention to develop environmentally sociable technologies in

Nanotechnology is an emerging field of interdisciplinary
research on particles of size 1–100 nm (Sun and Xia, 2002)
and dedicated to the creation, improvement, and utility of
nanoscale structures for advanced studies. For the last two
decades, the biosynthesis of noble metal nanoparticles (silver,
gold, platinum, and palladium) has received considerable
* Corresponding authors. Tel.: +593 2 3989492.
E-mail addresses: krmbraj@gmail.com (B. Kumar), lhcumbal@espe.
edu.ec (L. Cumbal).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
material synthesis with different chemical compositions, sizes,
shapes, and controlled dispersities (Akhtar et al., 2013). The
nanoparticles are of great interest due to their extremely small
size and large surface to volume ratio, and they exhibited
utterly novel characteristics compared to the large particles
of bulk material. Silver nanoparticles (AgNPs) have been
employed against diseases causing bacteria, fungi, and viruses
(Sharma et al., 2009), high sensitivities in the biomolecular
detection and diagnostic (Schultz et al., 2000), therapeutic
(Eckhardt et al., 2013), catalysis (Kumar et al., 2014a),
micro-electronics fields (Gittins et al., 2000), Surface enhanced
Raman spectroscopy, SERS (Solano-Ruiz et al., 2010) and
many other engineering, biomedical applications.
Ecofriendly methods for nanoparticle synthesis using
plants or plant extracts (Huang et al., 2011), fruits (Kumar
et al., 2014b), agricultural wastes (Kumar et al., 2014c),

http://dx.doi.org/10.1016/j.sjbs.2014.07.004
1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.
606 B. Kumar et al.

microorganisms (Bansal et al., 2012), enzymes (Willner et al., program). The particle size distributions of nanoparticles were
2006), fungi (Vigneshwaran et al., 2007), have been suggested determined using the HORIBA, Dynamic Light Scattering
as possible alternatives to chemical and physical methods. Bio- (DLS) Version LB-550 program. Size and selective area elec-
logical approaches to the synthesis of nanoparticles are tron diffraction (SAED) pattern of nanoparticles are studied
attracting research attention (Kaushik et al., 2010). Biological on transmission electron microscopy, TEM (FEI, TECNAI,
molecules can undergo highly controlled, hierarchical assem- G2 spirit twin, Holland). The FTIR-ATR spectra were col-
bly (Dipankar and Murugan, 2012), which makes them suit- lected in the transmission mode (4000–650 cm1) using a
able for the development of reliable and ecofriendly Perkin Elmer spectrophotometer (Frontier FT-IR).
processes for metal nanoparticle synthesis (Upendra et al.,
2009). Furthermore, these methods are cheap, rapid and effi- 2.3. Evaluation of antioxidant activity
ciently produce single atoms or molecules with a wide variety
of shapes (spheres, prisms, or plates) in the nano scale level. The scavenging activity of the AgNPs was measured by using
Sacha inchi (SI) or Plukenetia volubilis L. is an important DPPH as a free radical model and a method adapted from
climbing shrub plant from the Euphorbiaceae that grows Magalhaes et al. (2006) with slight modification. An aliquot
mostly in the Amazon region of Ecuador, Peru, Brazil (1.0–0.2 mL) of AgNPs or control and (1.0–1.8 mL) of H2O
(Hamaker et al., 1992). Some studies have shown that seeds was mixed with 2.0 mL of 0.2 mM (DPPH) in absolute meth-
from this plant are an excellent source of oil (omega 3 and anol. The mixture was vortexed vigorously and allowed to
omega 6 fatty acids, 35–60%) and protein (27–33%) stand at room temperature for 30 min in the dark. Absorbance
(Hamaker et al., 1992; Follegatti-Romero et al., 2009). Leaves of the mixture was measured spectrophotometrically at
of SI are reported to contain terpenoids, saponins, phenolic
compounds (flavonoids) and other components responsible
for the antioxidant and antiproliferative activities (Nasci
mento et al., 2013). Some studies have been done using SI
seeds and leaves in order to reduce the lipid profile of patients
with postprandial lipemia and hyper cholesterolemia
(Garmendia et al., 2011). Its dried shell biomass (Kumar
et al., 2014c) and oil (Kumar et al., 2014d) are also used to
fabricate silver nanocatalyst to remove different synthetic
dyes. However, there is no scientific report about the use of
SI leaves for the synthesis of AgNPs. The aim of the work pre-
sented here was to synthesize AgNPs using an ecofriendly
route and evaluate the antioxidant effect.

2. Experimental

2.1. Synthesis of AgNPs using the SI leaf extract

All chemicals used were of analytical grade and used without

any purification. Silver nitrate (AgNO3, 99.0%) was purchased
from Spectrum (USA) and Sacha inchi (SI) leaves were col-
lected from a farm in Quito, Ecuador. 1,1-Diphenyl-2-pic- Figure 1 Colorimetric visual pattern of (a) 1 mM AgNO3
rylhydrazyl (DPPH, >99.5%) was purchased from Sigma solution, (b) SI leaf extract, and (c–f) is AgNPs after 2, 22, 96,
Aldrich, USA. Milli-Q water was used in all experiments. Col- 168 h.
lected leaves were washed thoroughly with double distilled
water and dried under shade for one week. Dried and crushed
SI leaf (800 mg) was transferred to a 100 mL flask containing 4,0 (b)Sacha Inchi Leaf Extract (aq)
Absorbance (a.u.)

50 mL of deionized water, mixed well, and heat for 30 min at 3,5

(c1)AgNPs (1 Hr Heating)
(c2)AgNPs (1 Hr Heating, after 2 Hr)
60–70 C. The extract obtained was red colored and filtered (d)AgNPs (1 Hr Heating, after 22 Hr)
3,0
through Whatman No. 1 filter paper. The filtrate was collected (e)AgNPs (1 Hr Heating, after 96 Hr)
2,5 (f) AgNPs (1 Hr Heating, after 168 Hr)
in 50 mL Erlenmeyer flask and stored at 4 C for further use.
To synthesize AgNPs, 1 mL of SI leaf extract was mixed with 2,0
10 mL of an aqueous solution of AgNO3 (1 mM) and stirred 1,5
for 60 min at 60–70 C. Reduction occurs rapidly as indicated 1,0
by the appearance of yellowish color after 60 min of heating 0,5
and studied the formation of the AgNPs for 168 h.
0,0

2.2. Characterization -0,5

200 300 400 500 600 700 800
Wavelength (nm)
UV–vis spectra was measured using a spectrophotometer
(Thermo Spectronic, GENESYS 8, England, Quartz Cell, Figure 2 UV–vis pattern of (b) SI leaf extracts and (c–f) is
path length 10 mm and graph plotted on the Origin 6.1 AgNPs after 2, 22, 96 and 168 h.
Synthesis of silver nanoparticles using Sacha inchi (Plukenetia volubilis L.) leaf extracts 607

Figure 3 DLS size distribution pattern of synthesized AgNPs.

Figure 4 TEM pictures of spherical silver nanostructures at different magnifications (a) 200 nm, (b) 100 nm, (c) 20 nm and (d) SAED
pattern.

517 nm, and the free radical scavenging activity was calculated 3. Results and discussion
using
Scavenging effect ð%Þ ¼ ½1  fabsorbance of sample=absorbance of controlg 3.1. Visual and UV–vis study
 100
ð1Þ To study the formation of AgNPs, Visual and UV–Vis spec-
trum of the reaction solution were carried out. Fig. 1a–f shows
The scavenging percentage of all samples was plotted. The the visual calorimetric pattern of samples before and after the
final result was expressed as % of DPPH free radical scaveng- reaction at different time intervals. It clearly shows that
ing activity (mL). AgNO3 appears colorless and the SI leaf extract is pink before
608 B. Kumar et al.

the reaction. The colorless aqueous AgNO3 solution became (a)AgNPs , 168 Hr
24 (b) SI Leaf extract (aq)
yellow within 1 h when treated with the SI leaf extract at 60– 22
70 C and it is a preliminary indication of the formation of 20

Scavenging (%)
AgNPs. The intensity of color changes from yellow to wine 18
red with time. The presence of AgNPs in the medium was con- 16
firmed by the surface plasmon resonance band (SPR) (Kelly 14
et al., 2003) observed by UV–Vis absorption spectroscopy. 12
10
Fig. 2b–f shows the UV–vis spectra recorded for AgNPs. It
8
is observed that, there is an appearance of absorbance peak 6
around 430 nm and steadily increased in intensity with time, 4
which, respectively, corresponds to the transverse and longitu- 2
dinal plasmon vibration in the AgNPs. For spherical AgNPs, 0
0,2 0,4 0,6 0,8 1,0
the characteristic surface plasmon resonance (SPR) absorption
band of AgNPs is observed in the range of 400–475 nm Volume (mL)
(Kumar et al., 2014a; Stamplecoskie and Scaiano, 2010).
Figure 6 Antioxidant activities of (a) AgNPs and (b) SI leaf
3.2. DLS study extract.

stretching groups of macromolecular association (cellulose,

The DLS histogram of AgNPs is shown in Fig. 3. The mean
diameter and S.D. in the size was found 28. 7 nm and
12.9 nm. The size measured by DLS was slightly larger than
that measured by TEM.
3.3. TEM-SAED study
The TEM micrograph of the synthesized AgNPs at 200, 100
and 20 nm scale is shown in Fig. 4a–c. It was found that
AgNPs were spherical in shape with maximum particles in
the size range of 4–25 nm. The SAED pattern is shown in
Fig. 4d and confirmed the presence of elemental AgNPs. It
was also observed that AgNPs were monodispersed with low
polydispersity index (PDI).
3.4. FTIR analysis
FTIR spectroscopy was used to characterize the presence of
different functional groups/phytochemical constituents in the
SI leaf extract, responsible for the stabilization and capping
of the bio-reduced AgNPs. Fig. 5 represents the compared
FTIR spectra of leaf extract and AgNPs. The broad peak
observed at 3286 cm1 and 3267 cm1 in the spectra of leaf
extract and AgNPs corresponds to –NH2 and O–H bond
pectin, polyphenols, etc.). The band observed at 2919 cm1
and 2850 cm1 could be assigned to the C–H stretching vibra-
tions of methyl, methylene and methoxy groups (Kumar et al.,
2014d). The IR spectrum of SI leaf extract exhibit a strong
band at 1624 cm1 corresponding to the C‚O (amide I)
stretching mode and this peak shifted to 1634 cm1 suggesting
the possible involvement of the aforementioned groups in
AgNPs synthesis. This amide I band shows that proteins can
bind to Ag+ through free amine groups or carboxylate ions.
The strong band at 1029 cm1 can be attributed to the glyco-
side/C–OH bonds in the polysaccharide/protein structure of
the SI leaf. The phytochemicals (Nascimento et al., 2013) pres-
ent in the leaf extract are powerful reducing and stabilizing
agents, which may be responsible for the reduction of Ag+.
Most possibly, the phytochemicals might have formed a layer
on AgNPs which also prevents agglomeration of the particles,
and thus the nanoparticles are stabilized in the medium.
3.5. Evaluation of antioxidant activity
In the present study, the percentage of free radical scavenging
activity at different volumes ranging from 0.2 to 1.0 mL for
AgNPs and SI leaf extracts were evaluated as shown in Fig. 6.
It was found that, the antioxidant efficacy of AgNPs was higher

Figure 5 FTIR spectrum of (a) SI leaf extract and (b) synthesized AgNPs.
Synthesis of silver nanoparticles using Sacha inchi (Plukenetia volubilis L.) leaf extracts
than SI leaf extracts against the DPPH assay. The DPPH activ-
ity of the AgNPs and SI leaf was found to increase in a dose
dependent manner. The DPPH radical scavenging activities
were found to be maximum of 22.5% in 0.6 mL for AgNPs
whereas 19% in 1.0 mL for SI leaf extracts. The AgNPs are
spherical in shape with larger surface area and SI leaf phyto-
chemical compounds might get adsorbed onto the active surface
of the AgNPs. The highest antioxidant efficacy against DPPH
is probably derived, through the electrostatic attraction
between negatively charged bioactive compounds (COO,
O) and neutral or positively charged nanoparticles. AgNPs
bound to the phytochemicals and their bioactivity increases
by synergistically. The effect of activity depends on the site of
attachment of the metals and its consequent impact on the activ-
ity of the antioxidant agent (Magalhaes et al., 2006).
4. Conclusion
We have demonstrated a green approach to the synthesis of Ag
nanosphere with precisely controlled sizes using the Sacha
inchi leaf extract. The present protocol could be used to pre-
pare Ag nanosphere with size in the range of 4–25 nm under
heating conditions. We have also demonstrated that spherical
AgNPs shows radical scavenging activity when DPPH was
added into the reaction solution. In addition, the as-obtained
spherical nanostructures are used as promising candidates
for various applications related to optics and biomedicine.
Acknowledgments
This scientific work has been funded by the Prometeo Project
of the National Secretariat for Higher Education, Science,
Technology and Innovation (SENESCYT), Ecuador. We
thank Mr. Abraham Rodolfo Sanchez Piñuela for providing
Sacha inchi leaf for performing experiments.
References
Akhtar, M.S., Panwar, J., Yun, Y.-S., 2013. Biogenic synthesis of
metallic nanoparticles by plant extracts. ACS Sustain. Chem. Eng.
1, 591–602.
Bansal, V., Bharde, A., Ramanathan, R., Bhargava, S.K., 2012.
Inorganic materials using ‘unusual’ microorganisms. Adv. Colloid
Interface Sci. 179–182, 150–168.
Dipankar, C., Murugan, S., 2012. The green synthesis, characteriza-
tion and evaluation of the biological activities of silver nanopar-
ticles synthesized from Iresine herbstii leaf aqueous extracts.
Colloids Surf., B 98, 112–119.
Eckhardt, S., Brunetto, P.S., Gagnon, J., Priebe, M., Giese, B.,
Fromm, K.M., 2013. Nanobio silver: its interactions with peptides
and bacteria, and its uses in medicine. Chem. Rev. 113, 4708–4754.
Follegatti-Romero, L.A., Piantino, C.R., Grimaldi, R., Cabral, F.,
2009. Supercritical CO2 extraction of omega-3 rich oil from Sacha
inchi (Plukenetia volubilis L.) seeds. J. Supercrit. Fluids 49, 323–329.
Garmendia, F., Pando, R.R., Ronceros, G., 2011. Effect of Sacha
inchi oil (Plukenetia volubilis L.) on the lipid profile of patients with
Hyperlipoproteinemia. Rev. Peruana Med. Exp. Salud Publ. 28,
628–632.
609
Gittins, D.I., Bethell, D., Nichols, R.J., Schiffrin, D.J., 2000. Diode-
like electron transfer across nanostructured films containing a
redox ligand. J. Mater. Chem. 10, 79–83.
Hamaker, B.R., Valles, C., Gilman, R., et al, 1992. Amino acid fatty
acid profiles of the Inca peanut (Plukenetia volubilis). Cereal Chem.
69 (1), 461–463.
Huang, J., Zhan, G., Zheng, B., Sun, D., Lu, F., Lin, Y., Chen, H.,
Zheng, Z., Zheng, Y., Li, Q., 2011. Biogenic silver nanoparticles by
Cacumen platycladi extract: synthesis, formation mechanism, and
antibacterial activity. Ind. Eng. Chem. Res. 50, 9095–9106.
Kaushik, N.T., Snehit, S.M., Rasesh, Y.P., 2010. Biological synthesis
of metallic nanoparticles. Nanomed.: Nanotechnol. Biol. Med. 6,
257–262.
Kelly, K.L., Coronado, E., Zhao, L.L., Schatz, G.C., 2003. The optical
properties of metal nanoparticles: the influence of size, shape, and
dielectric environment. J. Phys. Chem. B 107, 668–677.
Kumar, B., Smita, K., Cumbal, L., Debut, A., Pathak, R.N., 2014a.
Sonochemical synthesis of silver nanoparticles using starch: a
comparison. Bioinorg. Chem. Appl.. Article ID 784268, 8 pages.
Kumar, B., Smita, K., Cumbal, L., Debut, A., 2014b. Biogenic
synthesis of iron oxide nanoparticles for 2-arylbenzimidazoles
fabrication. J. Saudi Chem. Soc., in press. http://dx.doi.org/
10.1016/j.jscs.2014.01.003.
Kumar, B., Smita, K., Cumbal, L., Debut, A., 2014c. Sacha inchi
(Plukenetia volubilis L.) shell biomass for synthesis of silver
nanocatalyst. J. Saudi Chem. Soc., in press. http://dx.doi.org/
10.1016/j.jscs.2014.03.005.
Kumar, B., Smita, K., Cumbal, L., Debut, A., 2014d. Sacha inchi
(Plukenetia volubilis L.) oil for one pot synthesis of silver
nanocatalyst: an ecofriendly approach. Ind. Crops Prod. 58, 238–
243.
Magalhaes, L.M., Segundo, M.A., Reis, S., Lima, J.L.F.C., 2006.
Automatic method for determination of total antioxidant capacity
using 2,2-diphenyl-1-picrylhydrazyl assay. Anal. Chim. Acta 558,
310–318.
Nascimento, A.K.L., Silveira, R.F.M., Santos, N.D., Fernandes, J.M.,
Zucolotto, S.M., Rocha, H.A.O., Scortecci, K.C., 2013. Antioxi-
dant and antiproliferative activities of leaf extracts from Plukenetia
volubilis Linneo (Euphorbiaceae). Evid-Based Complemt. Alternat.
Med.. Article ID 950272, 10 pages.
Schultz, S., Smith, D.R., Mock, J.J., Schultz, D.A., 2000. Single-target
molecule detection with nonbleaching multicolor optical immu-
nolabels. PNAS 97, 996–1001.
Sharma, V.K., Yngard, R.A., Lin, Y., 2009. Silver nanoparticles: green
synthesis and their antimicrobial activities. Adv. Colloid Interface
Sci. 145, 83–96.
Solano-Ruiz, E., Sato Berrú, R., Ocotlán-Flores, J., Saniger, J.M.,
2010. Synthesis of silver nanoparticles by sonochemical induced
reduction application in SERS. J. Nano Res. 9, 77–81.
Stamplecoskie, K.G., Scaiano, J.C., 2010. Light emitting diode
irradiation can control the morphology and optical properties of
silver nanoparticles. J. Am. Chem. Soc. 132, 1825–1827.
Sun, Y., Xia, Y., 2002. Shape-controlled synthesis of gold and silver
nanoparticles. Science 298, 2176–2179.
Upendra, K.P., Preeti, S.S., Anchal, S., 2009. Bioinspired synthesis of
silver nanoparticles. Dig. J. Nanomater. Biostruct. 4, 159–166.
Vigneshwaran, N., Ashtaputre, N.M., Varadarajan, P.V., Nachane,
R.P., Paralikar, K.M., Balasubramanya, R.H., 2007. Biological
synthesis of silver nanoparticles using the fungus Aspergillus flavus.
Mater. Lett. 61, 1413–1418.
Willner, I., Baron, R., Willner, B., 2006. Growing metal nanoparticles
by enzymes. Adv. Mater. 18, 1109–1120.