Appl Biochem Biotechnol

DOI 10.1007/s12010-013-0385-x

Ficus carica Latex-Mediated Synthesis of Silver

Nanoparticles and Its Application
as a Chemophotoprotective Agent

Hemant P. Borase & Chandrashekhar D. Patil &

Rahul K. Suryawanshi & Satish V. Patil

Received: 2 May 2013 / Accepted: 1 July 2013

# Springer Science+Business Media New York 2013

Abstract The present work provides scientific support on the use of latex of Ficus carica to
synthesize stable silver nanoparticles (AgNPs). AgNPs synthesized immediately after the
addition of latex to silver nitrate solution at room temperature. Synthesized nanoparticles
were of spherical shape with average size of 163.7 nm. Fourier transform infrared spectros-
copy analysis revealed capping of proteins and phenolic compound on AgNPs, while X-ray
diffraction analysis confirmed the fcc nature of AgNPs. Particles formed were stable for a
long time (6 months). It was found that incorporation of AgNPs with 2 and 4 % concentra-
tion exhibits synergistic increase in sun protection factor of commercial sunscreen and
natural extracts ranging from 01 to 12,175 % than control. Further characterization of latex
and AgNPs revealed total phenolic content of 98.75 and 94.88 μg/ml. The ferric ion
reduction potentials of latex and AgNPs were 79.69 and 18.79 %. Reduction potential of
ascorbic acid was synergistically increased after cumulative preparation of ascorbic acid
with latex and AgNPs and found to be 106.76 and 101.50 % for ascorbic acid + latex and
ascorbic acid + AgNPs, respectively.

Keywords Ficus carica . Nanoparticles . Sun protection factor . UV protection . Antioxidant

Introduction

Nanotechnology influences every aspect of human life. Much attention is now paid on the
synthesis of robust, stable, and tailor-made nanoparticles using biological sources due to
limitations associated with chemical and physical methods of nanoparticle synthesis [1–4].
Several biological agents are widely exploited for the synthesis of nanoparticles [5–12].
Biosynthesized nanoparticles are now known for their use in biosensors [13], water purifi-
cation process [14], catalyst [15], antibacterial [10], antiviral [16], and anti-insect- like [9]

Electronic supplementary material The online version of this article (doi:10.1007/s12010-013-0385-x)

contains supplementary material, which is available to authorized users.
H. P. Borase : C. D. Patil : R. K. Suryawanshi : S. V. Patil (*)
School of Life Sciences, North Maharashtra University, Post Box 80, Jalgaon 425001 Maharashtra, India
e-mail: satish.patil7@gmail.com
 Appl Biochem Biotechnol

applications. Plant as a source for nanoparticle synthesis has several advantages over other
methods [12, 17]. Numbers of plants were successfully shown to produce different metal
nanoparticles like silver, gold, copper, and others [18].
Sun burning cases are observed around the globe especially in a region of temperate
and tropical zone. Sax [19] estimated that each year, around one million of people suffered
from skin cancer. Several other complications also arise due to overexposure of skin to UV
rays of sun like photosensitivity, skin aging, phototoxicity, cutaneous degradation,
photocarcinogenesis, inflammation, erythema, pigmentation, and hyperplasia [20–22].
Natural protection against harmful effect of UV radiation was given by the melanin
pigment synthesized in plants, animals, and microorganisms [23]. Melanin deficiency,
sunlight exposure, and its intensity caused complication which arises from UV radiation.
UV radiation is categorized into three regions: UV A (from 400 to 320 nm), UV B (320 to
290 nm), and UV C (290 to 200 nm). UV C is filtered before reaching to the earth by
atmosphere; it is the UV B which responsible for imparting skin cancer and other harmful
effect. UV A also penetrates in epidermis and dermis causing skin aging. To protect the
skin from the UV radiation, the use of sunscreen is a common practice. Nowadays, the
addition of sunscreen compounds in commonly used personal care products like shampoo,
moisturizers, creams, and lotions are observed [24]. Several compounds are used as UV
radiation protectant in commercial products like benzophenone-3, octyl methoxycin-
namate, octyl salicylate, butylated hydroxytoluene, titanium oxide [25], and zinc oxide
[26]. Bioactive compounds from plants are also used for protection against UV rays like
Crocus sativus L. (Saffron) [27], melanin from the berry of Cinnamomum burmannii and
Osmanthus fragrans [28], sesame (Sesamum indicum L. Pedaliaceae) [29], and many
more.
Chemical compounds have several disadvantages like side effects upon frequent use,
environmental contamination through hazardous entities used in production process, and
growing consumer awareness towards the use of natural preparations. Thus, demand of
natural compounds tends to increase in sunscreen preparations. Bioorganic compounds
from plants contain diverse array of flavonoids, polyphenols, and phenolic acid which
are well documented for prevention against harmful effect of UV radiation [30]
Sun protection factor (SPF) is an index used to determine the quality of sunscreen to
protect the skin from UV radiation and remain adhered on the skin for prolonged time
after sweating. The measurement of SPF occupies the use of two methods in vivo and
in vitro. The in vivo method of COLIPA involves exposing the skin of a volunteer
animal applied with compound under study and without compound as control to UV
radiation and observing effect on skin called minimal erythema dose. This method raised
several ethical concerns [31] and now largely replaced by the in vitro method of Mansur
et al. [32], having advantages over in vivo method such as simple analysis, less
expensive, more accurate, and without any ethical concerns.
Free radicals like O2−, H2O2, OH, NO2, and NO cause damage to important biomolecules
like DNA, protein, and lipid leading to a pathological condition. Antioxidants are a class of
compound which relieves oxidative stress induced by photodamage and free radical [34].
There are various reports on the use of plant extracts as potent antioxidant like Shorea
roxburghii [35], Camellia sinensis [36], Daphne gnidium [37], Hypericum perforatum L.
[38], and Boerhavia diffusa [39].
The genus Ficus constitutes about 750 species found in tropical and subtropical regions
[35]. Ficus carica (Fig. 1), commonly called “fig” plant, is known to harbor diverse
Appl Biochem Biotechnol

chemical compounds with proven medicinal importance [40]. Different parts of the fig
plant contain flavonoids, polyphenols [41], triterpenoids, β-sitosterol, psoralen, and
taraxasterol [42, 43]. Latex contains resin, albumin, sugars, proteolytic enzymes, lipase,
diastase, catalase, peroxidase, and caoutchouc (2.4 %) [44]. Several researchers demon-
strate the medicinal importance of fig plant as an antioxidant [45, 46], antidiabetic [47],
hepatoprotective [48], antipyretic [49], and antimicrobial [50]. Latex of fig suppresses
cancer cell proliferation and has an antiviral potential [51, 52].
As per our knowledge, F. carica plant latex is used for the first time in nanoparticle
synthesis.
In order to explore the application of AgNPs in cosmetic industry, the change in SPF
was determined after the addition of AgNPs in commercial sunscreen preparations and
natural extract like cucumber (Cucumis sativus) and Aloe vera. The activity of latex and
latex-synthesized silver nanoparticles was also evaluated to check its beneficial role as
an antioxidant.

Materials and Methods

Chemicals

Silver nitrate (AgNO3), ascorbic acid (vitamin C), Folin–Ciocalteu reagent (phenol reagent),
trichloroacetic acid, gallic acid, potassium ferricyanide, ferric chloride, sodium carbonate,
and potassium bromide were procured from Merck, India. All chemicals were used without
any treatment and solutions were prepared with Millipore water.

Plant Latex Collection

Latex was collected from healthy plants of F. carica (Fig. 1) growing in the campus of
North Maharashtra University, Jalgaon, India. The method of latex collection was de-
scribed in our previous reports [8–10]. In short, a small cut near youngest leaves or fruit of
F. carica causes extrusion of milky latex. Latex was collected in sterile test tubes and
stored at 4 °C to avoid coagulation. This was used further for nanoparticle synthesis,

Fig. 1 Fruits of F. carica used to obtained latex

 Appl Biochem Biotechnol

characterization, SPF analysis, and antioxidant assay. Metabolites in latex were deter-
mined according to report of Kokate et al. [53].

Synthesis of Silver Nanoparticles

One milliliter of latex was added into 100 ml of 100 ppm silver nitrate solution. Silver nitrate
without latex was also kept as control. Both flasks were kept at 28 °C in an incubator without
stirring.

Characterization of AgNPs

Visual Observations

Test and control flasks were periodically observed for any kind of color change.

UV–Vis Spectroscopy

Silver nanoparticle formation with the latex of F. carica was monitored periodically by
withdrawing a sample from reaction mixture containing silver nitrate and latex. Samples
were centrifuged at 5,000 rpm for 10 min and the pellets thus obtained were suspended in the
Millipore water. This procedure was repeated three times to purify AgNPs. Final solution
was used in UV–Vis spectrophotometer at a resolution of 1 nm between 200 and 800 nm
(Shimadzu, Model 1601, Japan).

Fourier Transform Infrared Spectroscopy Analysis (FT-IR)

In a typical FT-IR (Shimadzu, model IR Prestige 21, Japan) analysis, approximately 3 mg of

lyophilized (Lyophilizer, Christ, model ALPHA 1-2 LD plus, Germany) latex was mixed
with 300 mg of dried KBr. Similar procedure was followed for AgNPs. The spectrum
obtained in the range of 400–4,000 cm−1 was then analyzed for presence, absence, appear-
ance, and disappearance of peaks denoting different functional group of biomolecules in
latex and those associated with AgNPs.

Environment Scanning Electron Microscopy (E-SEM) Analysis

Lyophilized AgNPs were analyzed for surface morphology (shape, size) using environment
scanning electron microscopy (FEI Quanta-200, USA) operating at 40 kV. Samples were
spotted on a double-sided adhesive carbon tape on copper grid as thin layer, and micrographs
were taken along with energy-dispersive spectroscopy (EDS) to confirm the presence of
elemental silver.

X-ray Diffraction (XRD) Analysis

The phases of prepared AgNPs were determined using an X-ray diffractometer (Brucker D8
Advance, Germany). Thin film of AgNPs was deposited on a glass slide. The diffracted
intensities were measured from 20 to 80 2θ angles.
Appl Biochem Biotechnol

Particle Size Analysis and Zeta Potential Analysis

Particle size analyzer system (Zetasizer, Malvern Instruments Ltd., USA) was used to
investigate the percent distribution of particles of different size and zeta potential. Clear
disposable cell was rinsed with ethanol and deionized water was used as dispersant. Sample
was put in clear disposable zeta cell. Twelve scans were made at 25 °C.

In Vitro SPF Determination

UV spectrophotometric method developed by Mansur et al. [32] was used for in vitro SPF
analysis and can be calculated using the following equation:

290 EE ðλÞ  I ðλÞ  AbsðλÞ

SPF ¼ CF  ∑320
where EE (λ) is the erythemal effect spectrum, I solar intensity spectrum, Abs absorbance of
sunscreen product, and CF correlation factor (10).
Value of EE (λ)×I is constant and is determined by Sayre et al. [33] (Table S1).
Three commercially available sunscreens labeled with SPF of 15, 30, and 45 and
cucumber (C. sativus L.) were purchased from a local market of Jalgaon, India, while A.
vera was obtained from the campus of North Maharashtra University, Jalgaon, India.
Extracts of A. vera and cucumber were prepared by washing the upper layer with
distilled water to remove soil and other dirt. Ten grams of both was taken separately
in mortar and pestle and ground to make a viscous extract. The extract thus obtained
was filtered through a thin fabric to get a clear extract. This extract was then used for
SPF analysis.
For SPF determination, 1 g of each commercial formulation and 1 ml of herbal extracts of
cucumber and A. vera were added separately in a 100-ml volumetric flask. Ethanol was used
to make up volume. To these flasks, AgNPs were added at 2 and 4 % of concentration. A
separate control was set for each test without addition of AgNPs. Test and control mixtures
were ultrasonicated for 5 min. The resulted solution was filtered. Initial 10 ml filtrate was
discarded; subsequent 5 ml of the filtrate was then added to 25 ml volumetric flak and
diluted with ethanol. This solution was immediately used to measure absorbance in wave-
length range from 290 to 320 nm at every 5 nm interval. Experiments were performed in
triplicate and values obtained were used to calculate SPF.

Determination of Antioxidant Potential

Total Phenolic Content Analysis

Total phenolic contents were estimated by the method of Singletone and Rossi [54]. One
milliliter of (1 mg/ml) latex of F. carica was dissolved in 5 ml distilled water. One milliliter
of Folin–Ciocalteau reagent was added, and mixture was allowed to incubate for 3 min.
Then, 3 ml of saturated sodium carbonate solution was added to the mixture and allowed to
incubate for 90 min in dark. Absorbance of blue color developed was noted at 725 nm by
UV–Vis spectrophotometer. Reading of three replicates was taken and concentration of
phenolic content in latex was expressed as microgram of gallic acid equivalent.
 Appl Biochem Biotechnol

Ferric Reducing Potential

In this assay, 5 mg of ascorbic acid as standard and 5 mg of latex, AgNPs, and combination
of ascorbic acid with latex and AgNPs (5:1 mg) were taken. To each tube, 2.5 ml of
phosphate buffer (pH 6.6) was added followed by the addition of 2.5 ml of 1 % potassium
ferricyanide solution. The mixture is allowed to incubate for 20 min and then 2.5 ml of
10 % trichloroacetic acid was added. Whole mixture was centrifuged at 3,000 rpm for
10 min. Of upper layer, 2.5 ml was added in 2.5 ml of distilled water, and in this mixture,
0.5 ml freshly prepared ferric chloride (0.1 %) solution was added. Volume was made up to
10 ml with distilled water. One milliliter from this mixture was again diluted to 10 ml with
distilled water, and finally, absorbance was recorded at 700 nm using the UV–Vis
spectrophotometer.

Result and Discussion

Visual Observation

Colorless solution turns reddish brown within a few minutes from the addition of latex into
silver nitrate solution. The intensity of color increases steadily as incubation period in-
creases, and within 1 h, solution appeared complete brown as compared to the control
showing no color change.

UV–Vis Spectroscopy

The absorbance of reaction mixture was taken at intervals of 5, 10, 15, and 20 min after latex
addition into AgNO3 solution (Fig. 2a). Control spectra of AgNO3 and latex were also noted.
Silver nitrate solution showed maximum absorbance at 297 nm, while latex shows peak at
318 nm. Surface plasmon resonance (SPR) peak is observed at 430 nm, clearly showing
AgNP formation. The absorbance of SPR peak steadily increases with an increasing
incubation time. After 40 min of latex addition, a very slow increase in the SPR absorbance
indicates completion of reaction (Fig. 2b) suggesting the role of latex as a catalyst and
biocompatible matrix for nanoparticle formation. It was found that nanoparticles are stable
up to 6 months with slight shift in SPR peak (Fig. 2c).

FT-IR Analysis

FT-IR analysis of latex-mediated AgNPs showed major peaks at 1,091.75, 1,384.94, 1,595,
and 3,263.66 cm−1. Peak at 1,091.75 cm−1 assigns to primary alcohols, 1,384.94 cm−1 arises
due to NO3− and may be due to C–N stretching vibrations of aliphatic and aromatic amines,
and 1,595 cm−1 peak was due to presence of nitro compounds, while peak at 3,263.66 cm−1
attributed to OH stretching in alcohol and phenolic compounds (Fig. 3). FT-IR results
showed the presence of latex metabolites in solution of silver nanoparticles.

Environment Scanning Electron Microscopy Analysis

Several researchers discussed the shape and size of nanoparticles using scanning and
transmission electron microscopy [6, 7, 10]. SEM image revealed that AgNPs produced
from F. carica were predominantly spherical in shape with no sign of agglomeration. Size
Appl Biochem Biotechnol

Fig. 2 a UV–Vis spectra of AgNPs taken at different time interval. b Plot of absorbance at λ max against time
showing an increase in SPR peak absorbance with an increase in incubation time. c UV–Vis spectra of AgNPs
after 3 and 6 months. d UV–Vis spectra of percent transmission of commercial sunscreen and sunscreen
incorporated with AgNPs

range of AgNPs was found at around 50 to 200 nm (Fig. 4a). Observation through E-SEM
matched closely with the results obtained from particle size analyzer system. Interestingly, it
was observed that upon addition of AgNPs to the solution of commercial creams and natural

Fig. 3 FT-IR spectrum of silver nanoparticles synthesized using latex of F. carica

 Appl Biochem Biotechnol

Fig. 4 a SEM of AgNPs synthesized from F. carica and b agglomeration of AgNPs after its addition in cream
solution

extract, the AgNPs tend to become agglomerated to form large clumps of nanoparticles
(Fig. 4b). EDS analysis confirmed the presence of silver in test samples (Fig. 5).

XRD Analysis

X-ray diffraction pattern of phytosynthesized AgNPs showed different peaks at 2θ value at

37.719°, 63.713°, and 76.471° corresponding to 111, 220, and 311 Bragg's reflection,
respectively (JCPDS card file no. 03-0921). Apart from these peaks, several other peaks
were also observed which may be due to the coating of latex metabolites on the surface of
nanoparticles (Fig. 6).

Particle Size Determination (PSD) and Zeta Potential Analysis

The average diameter of AgNPs on PSD system was 163.7 nm and 50 % of particles in
sample showed size range around 116 nm (Fig. 7). Zeta potential value was used for proof of
nanoparticle stability in colloidal solution. Zeta potential values were also very good (−19.3)
pointing towards stability of nanoparticles (Fig. 8) [8, 9].

Fig. 5 EDS of AgNPs

Appl Biochem Biotechnol

Fig. 6 XRD of silver nanoparticles

In Vitro SPF Determination

As discussed earlier, SPF determination is a universally recognized method to evaluate the

effectiveness of sunscreen. SPF of commercial sunscreen was made and it was found that
analyzed SPF closely matched with labeled SPF. The claimed SPF of three creams was 15,
30, and 45, and the SPF value found after analysis was 15.59±0.17, 29.80±0.44, and 44.71
±0.38, respectively (Table 1). Extract of A. vera and cucumber has an SPF value of 0.08
±0.006 and 0.17±0.008, respectively. Upon addition of 2 and 4 % AgNPs in the solution of
three commercial sunscreens and two natural extracts of A. vera and cucumber, a percent
increase of 18, 5, 1, 4,888, 22 and 23, 11, 03, 12,175, and 53 was obtained (Table 1). From
the spectrophotometric analysis, it was proved that there was decreased transmission of
sunscreen incorporated with AgNPs as compared to sunscreen alone showing higher trans-
mission (Fig. 2d).

Fig. 7 Particle size histograph of AgNPs

 Appl Biochem Biotechnol

Fig. 8 Zeta potential of AgNPs

Antioxidant Activity

Total Phenolic Content Analysis

The total phenolic content of latex of F. carica and latex-synthesized AgNPs was 98.75 and
94.88 μg/ml, respectively (Table S2).

Ferric Reducing Potential

The ferric reducing potential of latex, AgNPs, and combination with standard antioxidant
was done (Table 2). Latex has significantly higher antioxidant property of 79.69 % than the
latex-mediated AgNPs with only18.79 %. It was also found that cumulative activity of
ascorbic acid and latex showed increased activity up to 106.76 %. Similarly, ascorbic acid
and AgNP combination also showed higher antioxidant potential, i.e., 101.50 % (Table 2).

Table 1 SPF of commercial sunscreen preparations and natural extract before and after addition of AgNPs
synthesized from the latex of F. carica

Sr. Commercial Active ingredients Label Found SPF after AgNP addition (v/v)
no. product/natural SPF SPFa (% increase from labeled SPF)*
extract
2% 4%
(% increase) (% increase)

1 Sunscreen 1 Methylene 15 15.59±0.17 18.42±0.21 (18) 19.12±0.07

bis-benzotriazolyl, (23)
octyl methoxycinnamate,
Helianthus annuus extract
2 Sunscreen 2 Sandalwood, Aloe vera, 30 29.80±0.44 31.30±0.11 (5) 33.14±0.10
chamomile, liquorice (11)
3 Sunscreen 3 Methyl paraben, cetyl 45 44.71±0.38 45.36±0.07 (1) 46.04±0.10
alcohol, glyceryl (3)
stearate, xanthan gum
4 Aloe vera Alkaloid, tannin, saponins, NA 0.08±0.006 3.91±0.02 (4,888) 9.74±0.01
steroids, etc. (12,175)
5 Cucumber L (+) lactic acid, NA 0.17±0.008 0.22±0.00 (29) 0.26±0.00
(Cucumis Z-6-nonenol, (53)
sativus) E-2-nonenal, etc.
a
Analysis was performed in our laboratory. Results of triplicates were denoted by mean±standard error
Appl Biochem Biotechnol

Discussion

Use of latex for AgNP synthesis has several advantages over plant extract and microbial
method of nanoparticle synthesis. It does not require any kind of pretreatment like drying of
leaf and stem, solvent extraction, use of costly media, and other equipment. All these steps
can be circumvented with the use of latex.
Color change is a preliminary indication of nanoparticle formation [8, 9, 12, 17].
Different metals in their nanoform show peculiar change in color with respect to their bulk
color, viz. gold chloride solution is initially yellow but it transformed into pink after
conversion into gold nanoparticles [55, 56]. The formation of reddish brown color is due
to surface plasmon resonance property exhibited by nanoform of silver [57]. Other peaks in
spectroscopic analysis may arise due to biomolecules capped on AgNPs [57]. FT-IR analysis
revealed the presence of latex metabolites in solution of silver nanoparticles. These metab-
olites may contribute to metal reduction and can layer around particles to prevent agglom-
eration [58, 59].
Phenolic compounds are known for their role as antioxidant. Phytochemical analysis
revealed the presence of phenolic compounds in latex (Table S3). These phenolics with other
metabolites may be responsible for showing antioxidant activity [30, 35]. Synthesized
AgNPs also show the presence of phenolics which indicate the role of phenolics in reduction
and capping process. Phenolic content is one of most important components reported for
enhancing antioxidant property of any antioxidant preparation. This would be useful in the
sunscreen preparation by working as a UV radiation protectant as well as by removing
oxidative radical produced on skin.
Similarly, latex showed good ferric reduction potential than the standard ascorbic acid
and synthesized AgNPs. Also, individual combination of AgNPs and latex with ascorbic
acid showed synergistic effect on reducing potential of ascorbic acid. Thus, latex contents
might influence the reduction capacity of AgNPs. In E-SEM images, we found aggregation
of AgNPs after the addition of nanoparticle solution in commercial creams and natural
extract. We believe that this aggregation of AgNPs can reflect UV radiation and thus
contribute to an increase in SPF of test compounds as compared to the control without
AgNPs. Similar results were reported by Lin et al. [60]. Many organic and inorganic UV
filters are commercially used in sunscreen formulations. Commonly used inorganic metal
oxides as UV protectant include ZnO [61] and titanium dioxide [26, 60]. Various reports
explained non-harmful nature of nanoparticles on the skin when it is incorporated in
sunscreen, and very few reports focus on the use of nanoform of metal in sunscreen
[62–64]. Thus, more research work is required in this unexplored application of
nanotechnology.

Table 2 Antioxidant activity of ascorbic acid, latex of F. carica, AgNPs, and combination of ascorbic acid
with latex of F. carica AgNPs

Sr. no. Aliquots Absorbance at 700 nm Antioxidant activity (%)

1 Ascorbic acid 0.133±0.002 100

2 Latex 0.106±0.003 79.69
3 AgNPs 0.025±0.001 18.79
4 Ascorbic acid + latex (5:1 mg w/w) 0.142±0.003 106.76
5 Ascorbic acid + AgNPs (5:1 mg w/w) 0.135±0.001 101.50
 Appl Biochem Biotechnol

Conclusion

Simple, efficient, and stable silver nanoparticles were prepared using latex of F. carica. The
use of AgNPs in commercial sunscreen preparations to enhance SPF and to protect the skin
from UV radiation is a new area with potential of future development in nano preparations.

Acknowledgments Hemant P. Borase is a DST-INSPIRE fellow (grant file no. DST/INSPIRE Fellowship/
2011[149]).

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