IET Nanobiotechnology

Research Article

Efficacy of biogenic silver nanoparticles ISSN 1751-8741

Received on 18th January 2017
Revised 13th April 2017
against clinical isolates of fungi causing Accepted on 18th May 2017
doi: 10.1049/iet-nbt.2017.0003
mycotic keratitis in humans www.ietdl.org

Monika Lachmapure1, Priti Paralikar1, Manikandan Palanisamy2,3, Monica Alves4, Mahendra Rai1
1Nanobiotechnology Lab., Department of Biotechnology, Sant Gadge Baba Amravati University, Maharashtra, India
2Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore 641 014, Tamil Nadu, India
3Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah University, Majmaah 11952, Saudi Arabia
4Department of Ophthalmology, University of Campinas, Brazil

E-mail: mahendrarai@sgbau.ac.in

Abstract: Mycotic keratitis is mainly responsible for vision loss caused by various fungi. Sometimes, proper treatment of such
infection is not possible due to unavailability of effective antifungal agents and development of resistance of such fungi to
antimycotic drugs. Hence, it is necessary to search for potential antifungal agents, which can effectively eradicate fungal
infection of eyes. Nanoparticles-based antifungal drugs overcome this problem by increasing permeability and properties of drug
molecules. In the present study, silver nanoparticles were synthesised by using Helminthosporium sp. and Chaetomium sp.
following sequential reduction technique. The synthesised silver nanoparticles were detected primarily by UV-visible
spectrophotometer showing absorption spectra at 424 and 433 nm, respectively. Nanoparticles tracking analysis confirmed the
mean particle size of silver nanoparticles as 45 and 55 nm. The synthesised AgNPs showed significant antifungal activity
against fungi causing mycotic keratitis, when used alone and in combination with ketoconazole and amphotericin B in the range
of 30–70 microgram per millilitre of minimum inhibitory concentration. Thus, the synthesised AgNPs can be used to enhance the
activities of ketoconazole and amphotericin B.

1 Introduction synthesis is carried out by physical and chemical methods [20].

Keratitis is an inflammation of cornea, which may be superficial,
chronic or deep. Mycotic keratitis is a potentially blinding fungal
infection, with ulceration and suppurative infection. It is the most
common infectious disease occurring in different parts of the world
[1, 2]. The risk factor with mycotic keratitis are use of contact
lenses, insufficient use of antibiotics, injuries to cornea and
sometimes ocular surgery [3–5]. Different species of Fusarium,
Aspergillus, Curvularia, Paecilomyces and Scedosporium
apiospermum are the principal causes of filamentous fungal
keratitis [6–10]. Various antifungal agents have been employed in
therapy such as natamycin, amphotericin B, miconazole,
ketoconazole, itraconazole, fluconazole and voriconazole [11, 12].
Although, the conventional eye drops are most commonly
prescribed for the treatment of ocular infections and drug delivery.
They have low bioavailability due to pre-coronal retention, which
leads to tear turn over that cause drug loss. The most frequent use
of eye drops may induce coronal dryness, cause toxicity etc., [13–
15]. Nanoparticles due to their unique bioactive properties can help
to improve therapeutic strategy for targeted drug delivery on the
site of infection [16]. Further, the nanoparticles can provide new
therapeutic/prophylactic options for the treatment of ocular
infections [17].
Nanotechnology is an interesting field of research and can be
applied to various fields ranging from medicine, agriculture,
environment and consumer goods because of their higher surface
area to the volume ratio. All the nanotechnology approaches for
ocular drug delivery have been found to be efficacious and may
have significant opportunities for development of novel
therapeutics, which can be used for efficient treatment of these
diseases [18]. The application of nanotechnology in
ophthalmology, however, enabled to improve drug delivery
systems, medicine solubility and short half-life in biological
systems, controlled release, targeted delivery, bioavailability,
diffusion limitations and biocompatibility [19].
Generally, the silver nanoparticles can be synthesised by non-
biological and biological methods. In non-biological methods, the
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However, there are disadvantages of using these methods being
expensive and may involve hazardous chemicals [21]. This is the
reason why many researchers have been attracted towards the
biological method of synthesis. The silver nanoparticles can be
synthesised extracellularly or intracellularly [22]. Many bacteria,
fungi, plant extracts, yeasts and so on have been employed for
nanoparticles synthesis [23, 24]. Endophytic fungi have been
emerged as promising microbes for nanoparticles synthesis [25,
26], which contain a variety of bioactive compounds with novel
bioactivities. They are easy to handle and largely cultivated [27].
Biological approach using an endophytic fungi is a novel way
towards the development of safe, environmental friendly and
economically viable method for the synthesis of silver
nanoparticles.
Further, the metabolites isolated from medicinal plants and their
endophytes have also been reported to be a potential source of
natural antioxidants [28–30]. The endophytic fungi have been
emerged as ‘green’ alternative to the chemical method of synthesis.
Sunkar and Nachiyar [31] reported efficient extracellular synthesis
of silver nanoparticles from endophytic fungus occurring in
Garcinia xanthochyumus and found to show significant
antimicrobial activity. However, a few studies have been carried
out on exploitation of endophytic fungi for the synthesis of silver
nanoparticles [32, 33].
Our aim in the present study was to synthesise silver
nanoparticles by using aqueous extract of Helminthosporium sp.
and Chaetomium sp., which were isolated from the healthy leaves
of Gymnema sylvestre and Terminalia arjuna, respectively.
Moreover, antifungal activity of AgNPs was also evaluated against
fungi causing mycotitic keratitis.
2 Materials and methods
2.1 Fungi causing mycotic keratitis
The fungal isolates of Aspergillus flavus, A. fumigatus, A. niger,
Curvularia sp. and Bipolaris sp. causing mycotic keratitis in
humans were procured from Arvind Eye Hospital, Coimbatore,
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Tamil Nadu, India. The isolates of fungi were maintained on
sabouraud dextrose agar slants at 4°C.
2.1.1 Helminthosporium sp. and Chaetomium sp.: The
endophytic fungi Helminthosporium sp. and Chaetomium sp. were
isolated from leaves of Gymnema sylvestre and Terminalia arjuna
plants respectively, on potato dextrose agar medium (HiMedia,
Mumbai, India). The inoculated agar plates were incubated at 27°C
for 7 days. The fungi were then suspended in to potato dextrose
broth (HiMedia) and incubated at 27°C. The fungal biomass was
obtained after seven days in the form of a fungal mat.
2.2 Synthesis of silver nanoparticles using endophytic fungi
The fungal biomass was filtered and resuspended in sterilised
double-distilled water in conical flasks separately for each fungus.
The biomass was harvested by filtration through Whatman filter
paper No. 1 and rinsed with sterile distilled water to remove excess
medium contents. The biomass obtained from both fungi was
transferred in100 ml sterile distilled water separately for 48h at
27°C. After incubation, the suspension was filtered through
Whatman filter paper No. 1 and then centrifuged at 4000 rpm for
25 min to get clear filtrate. The resultant respective filtrates were
challenged with silver nitrate salt solution (final concentration 1  3 Results
mM) to obtain silver nanoparticles (AgNPs) suspension. The
AgNPs suspension was then centrifuged at 42,933 g for 20 min to
obtain pellet of silver nanoparticles.
2.3 Detection and characterisation of silver nanoparticles
2.3.1 Visual observation and UV-visible (UV-Vis)
spectrophotometer analysis: The primary detection was carried
out by observing change in colour of fungal filtrate after treatment
with silver nitrate to dark-brown colour, which indicates formation
of silver nanoparticles. The resultant reaction mixtures were then
subjected to optical analysis using UV-vis spectrophotometer
(Shimadzu UV-1700, Tokyo, Japan).The spectrum was scanned at
the resolution of 1 nm from 200–800 nm for each sample.
2.4 Nanoparticles tracking analysis
The nanoparticles solution was diluted with distilled water and 0.5  concentration of 0.27 × 108 particles/ml as shown in Fig. 2.
mL of each diluted sample was injected into the sample chamber
and observed using a Nanosight LM 20 device (NanoSight Ltd.,
Amesbury, UK). The size of the synthesised nanoparticles and
tracking motion of the nanoparticles was observed through the
camera fixed with the instrument.
2.5 Zeta potential analysis
The zeta potential was measured using a Zetasizer (3000 HS;
Malvern Instruments, Malvern, UK) with a zeta dip cell. Sample
preparation for zeta analysis involved 1 : 10 dilution of the
mycosynthesised AgNPs, the total volume of the sample (1 ml)
was kept into a clear disposable zeta cell for measurement of zeta
potential.
2.6 Fourier transform infrared spectroscopy (FTIR) analysis
FTIR analysis was made to analyse surface chemistry of
mycosynthesised silver nanoparticles. The suspensions of
synthesised silver nanoparticles were analysed by FTIR (Perkin-
Elmer FTIR-1600 USA) in the range 450–4000 cm−1 at resolution
of 4 cm−1.
2.7 In vitro evaluation of antifungal activity of silver
nanoparticles
The in vitro activity of silver nanoparticles synthesised by using
Helminthosporium sp. and Chaetomium sp. was evaluated against
A. flavus, A. fumigatus, A. niger, Curvularia sp., Bipolaris sp. and
Fusarium sp. singly and in combination with ketoconazole and
amphotericin-B (Hi-Media Pvt Ltd Mumbai, Maharashtra, India)
by impregnating 20 μl of nanoparticles solution into antibiotics
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disc using Kierby-Bauer disc diffusion method. The plates were
incubated at 25°C for 48 h. On completion of incubation period,
the zones of inhibition were measured with zone measurement
scale.
2.8 Determination of minimum inhibitory concentration of
silver nanoparticles
Minimum inhibitory concentration of silver nanoparticles was
determined by broth dilution method against four fungi responsible
for causing mycotic keratitis. The spore count of fungal cultures
was first adjusted to 10 5 CFU/ml. The stock solution of AgNPs and
standard antibiotic amphotericin-B was prepared with
concentration of 1 mg/ml. 10 dilutions of silver nanoparticles
solution and amphotericin-B were prepared (10–100 μl) by diluting
in (990–900 μl) RPMI (Roswell Park Memorial Institute medium)
medium. 200 μl of each grade of nanoparticles solution and
amphotericin-B was added in two different columns of microtitre
wells. 20 μl of fungal culture was added to both nanoparticles
solution and amphotericin loaded wells of microtiter plate. For the
visual observations, the microtitre plate was kept for incubation at
27°C for 24h in order to observe colour change.
Fungal filtrates of Helminthosporium sp. and Chaetomium sp. were
found to be most promising for the synthesis of silver nanoparticles
because after addition of AgNO3 (1 mM), the colour of fungal
filtrate changes rapidly from colourless to brown (Fig. 1). The UV-
vis spectra of colloidal silver nanoparticles from Helminthosporium
sp. and Chaetomium sp. were recorded after 24 h of incubation.
The spectral analysis showed a typical plasmon resonance in the
form of absorbance peak at about 424 nm and 433 nm, respectively
(Fig. 1). Nanoparticle tracking analysis (NTA) was made for the
detection of size of nanoparticles on their Brownian motion of
particles in suspension using (NanoSight LM-20). NTA showed
polydispersed population of synthesised AgNPs. The mean size of
AgNPs synthesised using Helminthosporium sp. was 49 ± 34 nm
and concentration was 0.12 × 108 particles/ml; while in case of
Chaetomium sp. The particles size was found to be 55 ± 71 nm in a
In addition to this, stability and charge achieved by the
synthesised AgNPs were characterised by Zeta potential analysis.
The Zeta potential value of silver nanoparticles from
Helminthosporium sp. and Chaetomium sp. was found to be −15.1 
mV and −15.1 mV, respectively, which indicates that the
synthesised nanoparticles were moderately stable (Fig. 3). Later,
FTIR analysis of AgNPs was carried out to identify the possible
biomolecules responsible for the synthesis and stabilisation of
AgNPs. In comparison with fungal filtrate, silver nanoparticles
synthesised from Helminthosporium sp. showed peaks at ∼ 3340 
cm−1, ∼2104 cm−1, ∼1636 cm−1, ∼608 cm−1, ∼544 cm−1 and
∼563 cm−1 (Fig. 4); while FTIR analysis for AgNPs obtained from
Chaetomium sp. demonstrated peaks at ∼3297 cm−1, ∼2236 cm−1,
∼2112 cm−1, ∼1636 cm−1, ∼1237 cm−1, ∼613 cm−1, ∼574 cm-1,
∼533 cm−1 (Fig. 5).
In vitro antifungal activity of AgNPs synthesised from
Helminthosporium sp. and Chaetomium sp. was evaluated against
fungi causing mycotic keratitis namely, A. flavus, A. fumigatus, A.
niger, Curvularia sp., Bipolaris sp., and Fusarium sp. Synthesised
AgNPs showed growth inhibitory effect in terms of zone of
inhibition observed for AgNPs alone and in combination with
antimycotic drugs ketoconazole and amphotericin B against all test
fungal pathogens (Tables 1 and 2). MIC was recorded as the lowest
concentration at which no visible growth of test pathogens was
observed. Silver nanoparticles synthesised from Helminthosporium
sp. and Chaetomium sp. showed significant antifungal activity
against A. flavus, A. fumigatus, A. niger, Curvularia sp., Bipolaris
sp., and Fusarium sp.; whereas, for A. flavus and A. fumigatus,
lower MIC values were recorded followed by A. niger, Bipolaris
sp., Fusarium sp., Curvularia sp (Table 3).
© The Institution of Engineering and Technology 2017
Fig. 1  UV-vis absorption spectra of AgNPs synthesised from Helminthosporium sp. (I) and Chaetomium sp. (II)

Fig. 2  NTA of AgNPs synthesised from Helminthosporium sp. (I) and Chaetomium sp. (II)

Fig. 3  Zeta potential analysis of silver nanoparticles synthesised from Helminthosporium sp. (I) and Chaetomium sp. (II)

4 Discussion identify the possible interaction between silver and bioactive

The fungal filtrates of Helminthosporium sp. and Chaetomium sp.
were subjected to silver nitrate treatment, which exhibited the
change in colour from colourless to brown. The appearance of a
dark-brown colour in the solution was a clear indication of the
formation of AgNPs in the reaction mixture [31, 34, 35]. The
change in colour is due to reduction of silver ions into silver
nanoparticles [32]. AgNPs showed the UV-vis spectrum peak
between 400 and 460 nm, which is characteristic for presence of
AgNPs in solution and surface plasmon resonance is exhibited by
AgNPs [36]. Nanoparticles with Zeta potential values greater than  stretch vibration of C = O, = C-H, -C-Cl, respectively [42] which
+ 30 mV or less than −30 mV typically have high degrees of
stability [37–40]. Irrespective of low zeta potential values, both the
AgNPs suspensions were found to be highly stable over a
considerable period of time. This can be attributed to the capping
molecules present on the surface of nanoparticles. FTIR
measurements of the dried and powdered sample were made to
molecules, which may be responsible for synthesis and stabilisation
of AgNPs. In comparison with fungal filtrate, silver nanoparticles
synthesised from Helminthosporium sp. showed peaks at ∼3340 
cm−1, ∼ 2104 cm−1, ∼1636 cm−1, ∼608 cm−1, ∼544 cm−1 and
∼563 cm−1. Among these, the peak at around ∼3340 cm−1 can be
the attributed to the O-H stretch [41] while the peak at ∼2104 cm−1
was associated with stretch vibration of –C = C– stretch and
confirms the presence of alkyne group. Similarly, the peaks at
∼1636 cm−1, ∼608 cm−1, ∼544 cm−1 and ∼563 cm−1 is due to
indicates the presence of carbonyls, alkenes and alkyl halides as
capping agents on the silver nanoparticles [43]. Whereas, in case of
FTIR analysis for AgNPs obtained from Chaetomium sp. showed
peaks at ∼3297 cm−1, ∼2236 cm−1, ∼2112 cm−1, ∼1636 cm−1,
∼1237 cm−1, ∼613 cm−1, ∼574 cm−1. These peaks are associated
with the stretch vibration of N-H, C = N (1°, 2° amines and

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Fig. 4  FTIR analysis of silver nanoparticles synthesised from Helminthosporium sp. control (I) and experimental (II)

Fig. 5  FTIR analysis of silver nanoparticles synthesised from Chaetomium sp. control (I) and experimental (II)

Table 1 Antifungal activity of silver nanoparticles synthesised from Helminthosporium sp. alone and in combination with
standard antibiotics
Test organism Control AgNO3 AgNPs Zone of inhibition (mm) Ab2 (Ampho B) Ab2 + AgNPs
Ab1 (Keto) Ab1 + AgNPs
A. fumigatus Nil 11.33 ± 0.57 12 ± 0.66 13.66 ± 1.5 19 ± 1 14 ± 1 16.66 ± 0.57
A. flavus Nil 11.66 ± 0.57 12.33 ± 0.57 18.66 ± 0.5 22 ± 1 14.66 ± 0.57 17 ± 1
A. niger Nil 11 ± 1 11.66 ± 0.57 14.33 ± 0.5 22.33 ± 1.5 13.66 ± 0.57 16.33 ± 0.57
Culvularia sp. Nil 11.66 ± 0.57 11.33 ± 1.52 20 ± 0.5 20.33 ± 2 15.33 ± 0.5 18.66 ± 0.57
Bipolaris sp. Nil 11.33 ± 0.57 10.66 ± 0.57 14.33 ± 0.5 16 ± 1 15 ± 1 17.33 ± 0.57
Fusarium sp. Nil 12 ± 1 12.66 ± 0.57 14.66 ± 0.57 17.66 ± 0.57 16.33 ± 0.57 19.33 ± 0.57
(Ab – standard antibiotics, Keto – Ketoconazole, Ampho B – Amphotericin B. All values have been represented as mean ± standard deviation).

amides), -C = C-(nitriles), N-H (alkynes), C-N (1o amines), C-Cl, of the silver nanoparticles as a capping protein and also stabilises
C-Br (alkyl halides) [44, 45]. From overall observations, the the silver nanoparticles.
presence of protein is confirmed, which is responsible for coating Synthesised AgNPs showed notable antifungal activity against
all fungal pathogens. The activity of AgNPs was increased

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Table 2 Antifungal activity of silver nanoparticles synthesised from Chaetomium sp alone and in combination with standard
antibiotics
Fungal pathogen Control AgNO3 AgNPs Zone of Inhibition (mm) Ab2 (Ampho B) Ab2 + AgNPs
Ab1 (Keto) Ab1 + AgNPs
A. fumigatus Nil 12 ± 1 14 ± 1.5 15 ± 1 18.33 ± 0.5 12 ± 1 14 ± 1
A. flavus Nil 11.66 ± 0.57 12 ± 1 17.66 ± 0.57 19.33 ± 0.57 10.33 ± 0.5 11.33 ± 0.57
A. niger Nil 11.33 ± 0.57 12.66 ± 0.57 13 ± 1 16.66 ± 0.57 14 ± 1 17.33 ± 0.57
Culvularia sp. Nil 11.66 ± 0.57 12.52 ± 1.57 18.33 ± 0.5 19.33 ± 0.5 14.33 ± 0.57 19 ± 1
Bipolaris sp. Nil 11.33 ± 0.57 13 ± 1 14.33 ± 0.57 17 ± 1 15 ± 1 17.66 ± 0.57
Fusarium sp. Nil 11.66 ± 0.57 13.33 ± 0.57 14.66 ± 0.57 17.66 ± 0.57 14.66 ± 0.57 18.33 ± 0.5
(Ab – standard antibiotics, Keto – Ketoconazole, Ampho B – Amphotericin B. All values have been represented as mean ± standard deviation).

Table 3 Minimum inhibitory concentration of synthesised AgNPs using Helminthosporium sp. and Chaetomium sp against
fungal pathogens
Fungal pathogens Minimum inhibitory concentration in µg/ml
AgNPs by Helminthosporium sp. AgNPs by Chaetomium sp.
A. fumigatus 30 30
A. flavus 30 40
A. niger 40 30
Culvularia sp. 70 60
Bipolaris sp. 40 40
Fusarium sp. 50 40

Fig. 6  Possible mechanism of inhibitory action of silver nanoparticles on fungal cell

significantly in combination with standard antimycotic drugs.
Among this, combination of AgNPs synthesised from
Helminthosporium sp. with ketoconazole demonstrated remarkable
inhibitory zone against all tested Aspergillus sp. followed by
Fusarium sp., Bipolaris sp. and Culvularia sp. Similar results were
observed for AgNPs synthesised using Helminthosporium sp. in
combination with amphotericin B., while Chaetomium sp. fungal
extract- mediated synthesis of AgNPs showed significant inhibitory
zone against Culvularia sp. in combination with amphotericin B
followed by Fusarium sp., Bipolaris sp., A. niger, A. fumigates and
A. flavus. In addition to this, AgNPs clearly showed significant
antimicrobial activity in combination with antibiotics [46].
However, synergistic combinations between silver nanoparticles
and antibiotics make it possible to lower dosages, thus reducing
toxic effect. The prolonged use of such antibiotics develop fewer
chances of resistance to drugs with single component use. The
combination of antibiotics with nanoparticles enhance or may
restore activity of such antibiotics [47].
Many researchers have proposed hypothetical mechanism
involved in antifungal activity of silver nanoparticles. Literature
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survey showed that, there must be an interaction between
positively charged silver ions released from silver nanoparticles
with negatively charged fungal cell wall that leads to accumulation
of nanoparticles on fungal cell membrane [48]. Kim et al. [49]
reported that when fungal cell was treated with silver
nanoparticles, the latter were bound to DNA and caused damage.
Moreover, AgNPs also damaged cellular components leading to
cell death. The possible mechanism of inhibitory action of silver
nanoparticles on fungal cell, has been illustrated in Fig. 6.
5 Conclusion
The biosynthesis of silver nanoparticles from endophytic fungi has
been proved to be efficient, eco-friendly and simple. In the present
study, the endophytic fungus Helminthosporium sp. and
Chaetomium sp. showed the ability to synthesise silver
nanoparticles extracellularly, which is quite stable in nature.
Extracellular synthesis of nanoparticles using the cell free filtrate is
advantageous as it doesn't require another step of separating the
nanoparticles, and hence makes the downstream processing easier.
The synthesised silver nanoparticles demonstrated significant
5
antifungal activity against pathogens causing mycotic keratitis in
humans. The antifungal activity of ketoconazole and amphotericin-
B was remarkably increased in combination with silver
nanoparticles. Hence, antimycotic drug- loaded silver nanoparticles
can be used as potential antimycotic agent for treatment of mycotic
keratitis in humans.
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