Green Synthesis of Silver Nano Particles As Novel Antifungal Agents

International Journal of Trend in Scientific Research and Development (IJTSRD)
International Open Access Journal | www.ijtsrd.com
ISSN No: 2456 - 6470 | Volume - 2 | Issue – 6 | Sep – Oct 2018
Green Synthesis of Silver Nano Particles aass Novel Antifungal Agents

R. Nazreen, R. Ushasri
PG.Scholar,
Scholar, Department of Applied Microbiology,
JBAS College for Women
Women, Chennai, Tamil Nadu, India
ABSTRACT
Aspergillus species are causative agents of invasive where this fungus was found to be sensitive tu
fungal infections in immunocompromised patients maximum(250µl )and minimum concentration of
and associated with pulmonary diseases, mycotic Black berry and Almond leaf extracts in
diseases, keratitis mycotic keratitis, otomycosis and concentration of ( 100 µl) with values of 24 mm,21
nasal sinusitis. At least 30 Aspergillus species are mm and 23 mm , 21 mm. Aspergillus flavus was
associated with human diseases. Aspergillus niger is a found to be resistant to aqueous leaf extracts of Neem,
member of the genus Aspergillus which includes a set Lemon, and Tamarind in concentration ranging from
of fungi that are generally considered asexual, 250µl to 100 µl showing no zone
although perfect forms. Aspergillus flavus is a fungus formation.A.fumigatus was found to be resistant to
grows by producing thin thread like branched hyphae. aqueous leaf extracts of neem, lemon and tamarind in
Aspergillus flavus is a filamentous mould.A. concentration ranging from 250 µl to 10010 µl showing
fumigatus is characterized by green echinulate conidia no zone formation.Candida albicans was found to be
in chains basipetally from greenish phialides, 6 to 8 resistant to aqueous leaf extracts of Neem, Lemon and
by 2 to 3µm. Rhizopus is filamentous fungus found in Tamarind in concentration ranging from 250 to 100 µl
soil decaying fruit and vegetables, Rhiz
Rhizopus species showing no zone formation.Rhizopus spp was found
are most common in habitants of bread hence called to be resistant to aqueous leaf extracts
extr of Neem,
as bread molds. Candidiasis is a fungal infection Lemon and Tamarind in concentration ranging from
caused by yeasts that belong to the genus Candida. 250 to 100µl showing no zone formation.Rhizopus
Silver is the one of the important nano
nano-material with spp was found to be resistant to aqueous leaf extracts
five hundred tons of silver nanoparticles
cles production of Neem, Lemon and Tamarind in concentration
per year it has been associated with strong bactericidal ranging from 250 to 100µl showing no zone
effects and antifungal activities. The aim of this study formation.
was to synthesize nanoparticles using plant extracts
and determine antifungal activity by standard Aspergillus niger , A.flavus, A. fumigatus, Rhizopus
methods. and Candida albicans were found to be resistant to
aqueous silver nanoparticles leaf extracts of Lemon
METHODS and Tamarind in concentration ranging from 250µ to
Sabourd Dextrose agar was prepared. Fungal cultures 100µl showing no zone formation and found fou to be
were sub cultured and observed for microscopic and sensitive to leaf extracts of Black berry, Almond and
macroscopic characters. The leaves of Neem, Lemon, Neem .
Black berry, Tamarind and Almond were collected
and powdered. The powdered plant material was Keyword: SDA, plant extracts, spectrophotometer,
extracted using sterile water. Silver nano particles SEM, MHA
were prepared using crude extracts of Neem, Lemon,
Black berry, Tamarind and Almond. INTRODUCTION
Fungi are ubiquitous and found in air, soil, water,
RESULTS and plants. Fungi are beneficial and harm ful.
ful Fungi
Aspergillus niger was found to be resistant to are of great importance in production of industrially
Aqueous leaf extracts of Neem, Lemon and Tamarind important secondary metabolites. Fungi cause many
@ IJTSRD | Available Online @ www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct

Oct 2018 Page: 1338
International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470
2456
infections to Humans such as Aspergillosis, are two types of candidiasis such as yeat infection or
Aspergilloma and candidiasis. Fungal infections are oral thrush and invasive candidiasis.
most common in immune compromised individuals.
The ‘green environment eco friendly processes in
Aspergillus niger chemistry and chemical technologies which are
Aspergillus niger is a member of the popular and much require as a result of world
genus Aspergillus which includes a set of fungi that problems associated with environmental conditions.
are generally considered asexual, although perfect Silver is the one of the important nano-material
nano with
forms.A.nger is thermo tolerant and found everywhere five hundred tons of silver nanoparticles production
pro
and found to be resistant to freezing tempe
temperature. per year it has been associated with strong bactericidal
Aspergilli are ubiquitous in nature. Aspergillus niger effects and antifungal activities.
can be classified as a member of Dueteromycetes
They are geographically widely distributed and have Green techniques have been used as biological
been observed in a broad range of habitats because technique for the synthesis of silver Nano particles as
they can colonize a wide variety of substrates. A. alternate methods to conventional methods. Silver
niger is commonly found as a saprophyte growing on nanoparticles
anoparticles can be produced at low concentration of
dead leaves, stored grain, compost piles, and othe
other leaf extract without using any additional harmful
decaying vegetation. chemical/physical methods. The method applied here
is simple, cost effective, easy to perform and
Aspergillus flavus sustainable.
Aspergillus flavus is a fungus. It grows by producing
thread like branching filaments known as hyphae. Generally the synthesis of nanoparticles has been
Filamentous fungi such as A. flavus are sometimes carried out using three different approaches, including
called molds. A network of hyphae known as the physical, chemical, and biological methods. In
mycelium secretes
retes enzymes that break down complex physical methods nanoparticles, nanoparticles are
food sources.When young, the conidia of A. flavus prepared by evaporation condensation using a tube
appear yellow green in color. As the fungus ages the furnace at atmospheric pressure. Conventional
Convention
spores turn a darker green. physical methods including spark discharging and
pyrolysis were used for the synthesis of AgNPs
Aspergillus fumigatus
A. fumigatus is identified based on morphology of The advantages of physical methods are speed,
conidia and conidiophores. A. fumigatus is radiation used as reducing agents, and no hazardous
characterized by green echinulate conidia in chains chemicals involved, but the downsides are low yield
basipetally from greenish Phalides, 6 to 8 by 2 to and high
igh energy consumption, solvent contamination
3µm. A. fumigatus is pigment less and produce white and lack of uniform distribution
conidia.
METHODS
Rhizopus SPP SUBCULTURE OF FUNGAL SPECIES
Rhizopus is filamentous fungus found in soil decaying The fungal cultures were inoculated in to sabauraud
fruit and vegetables, Rhizopus species are most dextrose agar and incubated at room temperature for
common in habitants of bread hence called as bread 24 hrs to 48 hrs and observed fungal
funga growth. The
molds. They cause most severe infections in human colonies were observed microscopically to identify
beings. the morphology of fungi.
Candida albicans PREPARATION OF AQUEOUS LEAF

Candidiasis is a fungal infection caused by yeasts that EXTRACTS OF NEEM, LEMON, TAMARIND,
belong to the genus Candida.. There are over 20 BLACK BERRY AND ALMOND.
species of Candida yeasts that can cause infection in
humans, the most common of which is Candida
albicans. Candida yeasts normally reside in the
intestinal tract and can be found on mucous
membranes and skin without causing infection. There

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The fresh leaves of Neem,, Lemon, Tamarind, Black
berry and Almond were collected from S.I.E.T college
campus Chennai. The leaves were washed thoroughly
with distilled water and kept for shade drying. The
leaves of respective plants were ground to fine
powder followed by soaking of o 20gms of each fine
leaf powder in 100ml of sterile distilled water
overnight. The flask containing leaf solution was
filtered using gauze and centrifuged. The supernatants
of each plant leaf was collected in sterile conical flask
NEEM and filtered. A portion of filtrates were mixed with
10ml of 0.1N silver nitrate solution and kept in dark
room for synthesis of silver nanoparticles. The
filtrates were kept in oven for 48hrs to obtain crude
aqueous leaf extracts. The effectiveness and accuracy
in results without
ut contamination.
GREEN SYNTHESIS OF SILVER

NANOPARTICLES (Ag NP)
Aqueous solution (1 mM) of silver nitrate (AgNO3)
LEMON was prepared in 250 mL Erlenmeyer flasks and
aqueous leaf extracts were added for silver nitrate
reduction. The composite mixture was then kept in
oven for complete reduction of silver nitrate. The
color change was observed from yellowish brown ro
reddish brown followed by spectrophotometry
spec for 30
minutes. The reaction was carried out in darkness at
room temperature so as to prevent photo reactivation
of silver nitrate along with controls. The confirmation
of silver nanoparticles synthesis was based on change
TAMARIND in color. The colloidalidal solution containing silver
nanoparticles of leaf extracts was estimated by UV
visible spectrophotometric analysis. The colloidal
mixture was sealed and stored in refrigerator for
antifungal activity.
UV-VIS
VIS SPECTRA ANALYSIS
Samples (1 mL) of the suspensions
ensions were collected to
analyze complete bio reduction of Ag+ in aqueous
solution by diluting 2ml of deionized water followed
by scanning in UV visible spectra between 200 to 700
BLACKBERRY nanometer in a spectrophotometer
ANTIFUNGAL ACTIVITY
Antifungal activitiess of synthesized silver
nanoparticles from different leaf extract were
determined the current study was also done using
crude aqueous leaf extract without nanoparticles. 20
ml of sabourd dextrose agar was poured into Petri
plate and sterility check was done
don before proceeding
for antifungal activity. The fungal test organisms such
ALMOND as Aspergillus niger,, Aspergillus flavus, Aspergillus

Oct 2018 Page: 1340
2456
fumigatus, and Rhizopus Spp and Candida albicans
were used to prepare lawn on Sabourd dextrose agar
plates. Agar wells of 5mm size were prepared by
using sterilized stainless steel cork borer. Four wells
were loaded with different concentrations of silver
nanoparticles synthesized from different leaf extracts
in range of 250µl, 200µl, 150µl, 250µl. The plates
were also loaded withh crude aqueous leaf extracts of
respective plants into four wells using same Green synthesis of Nano particles
concentration. The plates were incubated at 37ºC and
examined for the zones of inhibition in the form of
clear area. The diameter of each zone of inhibition
was measured using a scale in mm.
MINIMUM FUNGICIDAL CONCENTRATION

The stock solutions of silver nanoparticles of
synthesized by leaf extract and without nanoparticles
of plant were diluted in 1ml of potato dextrose broth
followed by loading of wells in five rows with 100µl
off potato dextrose broth. Serial dilution was done in
order to obtained minimum concentration. 10ml of
each fungal broth culture was loaded into respective
wells containing respective leaf extracts with and
without nanoparticles showing highest antifungal
activity.
ctivity. The MFC plates were incubated at room
temperature for 24hrs. The plates were observed for
minimum fungal inhibition concentration
UV – spectrophotometric analysis
crude leaf extracts
SEM image of Almond silver nano particle leaf

extract
Antifungal activity of crude silver nano particles

synthesized by leaves of Almond, Lemon, Neem,
Black berry and Tamarind.

Oct 2018 Page: 1341
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looking conidium, while Aspergillus niger looks more
like a toilet brush, with its globose base at the end of
the stalk. Stachybotrys is much similar in colony
characters but can be differentiated. Aspergillus niger
was found to be resistant to Aqueous leaf extracts of
Neem, Lemon and Tamarind where this fungus was
found to be sensitive to maximum(250µl )and
minimum concentration of Black berry and Almond
leaf extracts
xtracts in concentration of ( 100µl) with values
of 24mm,21 mm and 23 mm , 21 mm. This study was
compared with that of previous similar work using
similar work .The
The aqueous extracts of Acacia albida
was reported to be hihly potent with maximum
antifungal activity (18mm) followed byP. by juliflora
(12.3 mm). The aqueous extracts of Acacia albida was
reported to be highly potent with maximum antifungal
activity (18mm)
Aspergillus flavus was found to be resistant to

aqueous leaf extracts of Neem,
Neem Lemon, and Tamarind
in concentration ranging from 250µl to 100 µl
showing no zone formation. The fungus was found to
be highly sensitive to aqueous almond leaf extract at
the concentration of 250 µl (22mm) and minimum
inhibition at blackberry leaf extract
extrac at the
concentration of 100 µl (17mm). A. flavus was found
to be sensitive to aqueous leaf. The present was
compared with previous work.
A.fumigatus was found to be resistant to aqueous leaf

extracts of neem, lemon and tamarind in concentration
ranging from 250µl to 100µl showing no zone
formation. The fungus was found to be highly
sensitive to aqueous almond leaf extract at the
concentration of 250 ml (26mm) and minimum
inhibition at concentration of 100µl (21mm).
(21mm A.
fumigatus was found to be sensitive to aqueous leaf
extract of blackberry at concentration of 250 µl
(20mm) and minimum inhibitory concentration of
100µl(16mm). This study was compared to previous
work and found to be efficient.
Rhizopus spp was found to be resistant to aqueous leaf

extracts
cts of Neem, Lemon and Tamarind in
concentration ranging from 250 to 100µl showing no
zone formation. The fungus was found to be highly
sensitive to aqueous almond leaf extract at the
concentration of 250µl(22mm) and minimum
inhibitory concentration of 100µl(19mm).
100 Rhizopus
DISCUSSION was found to be sensitive to aqueous leaf extract of
A. niger but the black conidia and spores confirm the blackberry at concentration of 250µl(19mm) and
species to A. niger.Penicillium has a paint brush minimum inhibitory concentration of 100µl(17mm).

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Candida albicans was found to be resistant to aqueous inhibition concentration at 100µl (14mm). The
leaf extracts of Neem, Lemon and Tamarind in current study focused on MIC values and well
concentration ranging from 250 to 100 µl showing no diffusion method in whichAgNPs exhibited higher
zone formation. The fungus was found to be highly antifungal activity even at low concentration (0.1. The
sensitive to 250µl(30mm) and minimum inhibitory antifungal
ungal potency of the plant extract and AgNPs
concentration of 100µl(23mm). Candida albicans was increased with increasing their corresponding
found to be sensitive to aqueous leaf extraextract of concentrations.
blackberry at concentration of 250µl(19mm) and
minimum inhibitory concentration of 100µl(12mm) . A.fumigatus was found to be resistant to aqueous
The current study was compared with previous work silver nanoparticles leaf extracts of Lemon and
exhibiting the zone of inhibition recorded at 500 tamarind in concentration ranging from 250µl to
mg/ml concentration was higher than that of 250, 100, 100µl showing no zone formation. The fungus was
10 mg/ml concentration for all the extracts. As the found to be highly sensitive to aqueous silver
amount of the extract increased, the inhibitory effect nanoparticles leaf extract at the concentration of
had increased 250µl (28mm) and minimum inhibition
inhibitio concentration
of 100µl(23mm). A. fumigatus was found to be
Aspergillus niger was found to be resistant to aqueous sensitive to aqueous silver nanoparticles leaf extract
silver nanoparticles leaf extracts of Lemon and of blackberry at concentration of 250µl(24mm) and
Tamarind in concentration ranging ing from 250µ to minimum inhibition concentration of 100µl(16mm).
100µl showing no zone formation. The fungus was A. fumigatus was found to be sensitive to aqueous leaf
found to be highly sensitive to aqueous silver extract of neem at concentration 0f 250µl (17mm) and
nanoparticles of almond leaf 250µl(28mm) and minimum inhibition concentration at 100µl (12mm).
minimum inhibition concentration of 100µl(20mm). The current observations were found to be similar to
A. niger was to be sensitive to aqueous silver that of previous work
nanoparticles
oparticles leaf extract of blackberry at
concentration of 250µl(28mm)and minimum Rhizopus spp was found to be resistant to aqueous
inhibition at concentration of 100µl(17mm).
100µl(17mm).A.niger silver nanoparticles
rticles leaf extracts of Lemon and
was found to be sensitive to aqueous silver tamarind in concentration ranging from 250µl to
nanoparticles leaf extract of Neem at concentration of 100µl showing no zone formation. The fungus was
250µl(16mm) and minimum inhibitory
nhibitory concentration found to be highly sensitive to aqueous silver
of 100µl(14mm) This work was found to be similar to nanoparticles leaf extract at the concentration of
previous work which showed the antifungal activity 250µl(3mm) and minimum inhibition
inhi concentration of
of silver nanoparticle had been evaluated against 100µl(27mm). Rhizopus spp was found to be sensitive
Aspergillus niger.. The zones of inhibition of to aqueous silver nanoparticles leaf extract of
Aspergillus niger against AgNPs, ethanol, plant blackberry at concentration of 250µl(19mm) and
extract and chloramphenicol (standard) was observed minimum inhibition concentration of 100µl(17mm).
The silver nanoparticles showed strong inhibitory (+) Rhizopus spp was found to be sensitive to aqueous
action and no zone of inhibition was seen for ethanol. leaf extract of Neem at concentration 0f 250µl(18mm)
and minimum inhibition concentration at
A.flavus was found to be resistant to aqueous silver 100µl(15mm). The previous work reported that
nanoparticles leaf extracts
ts of Lemon and tamarind in AgNPs showed better antifungal properties
concentration ranging from 250µl to 100µl showing against Aspergillus sp. and Rhizopus sp. as
no zone formation. The fungus was found to be highly evidenced by minimum inhibitory concentration
sensitive to aqueous silver nanoparticles leaf extract at (MIC) value 21.8 ng/mL The results showed that the
the concentration of 250µl(29mm) and minimum AgNPs were fungicidal against both the tested
inhibition concentration
on of 100µl(27mm). A. flavus fungus at very low concentrations and the fungicidal
was found to be sensitive to aqueous silver activity was dependent on the tested fungus species.
nanoparticles leaf extract of blackberry at These
hese results were confirmed by plating the content
concentration of 250µl(20mm) and minimum of each well on dextrose agar medium, and there was
inhibition concentration of 100µl(17mm). A. flavus no growth for any of the strains resultant from the
was found to be sensitive to aqueous leaf extra
extract of MIC point. These enhanced effects of AgNPs might
nee at concentration 0f 250µl (17mm) and minimum

Oct 2018 Page: 1343
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be due to the antifungal properties of silver reduction. The composite mixture was then kept in
nanoparticles (Shreya Meddaet al.,., 2015) oven for complete redction of silver nitrate. The
colour change was observed from yellowish brown ro
Candida albicans was found to be resistant to aqueous reddish brown followed by spectrophotometry for 30
silver nanoparticles leaf extracts of Lemon and minutes. The reaction was carried out in darkness at
tamarind in concentration ranging from 250µl to room temperature so as to prevent photoreactivation
100µl showing no zone formation. The fungus was of silver nitrate along with controls. The confirmation
found to be highly sensitive to aqueous silver of silver
er nanoparticles synthesis was based on change
nanoparticles leaf extract at the concentration of in colour. The colloidal solution containing silver
250µl (30mm) and minimum inhibitionn concentration nanoparticles of leaf extracts was estimated by UV
of 100µl (20mm). Candida albicans was found to be visible spectrophotometric analysis. The colloidal
sensitive to aqueous silver nanoparticles leaf extract mixture was sealed and stored in refrigerature for
of blackberry at concentration of 250µl (19mm) and antifungal
fungal activity. Samples (1 mL) of the suspensions
minimum inhibition concentration of 100µl (15mm
(15mm). were collected to analyse complete bioreduction of
Candida albicans was found to be ssensitive to Ag+ in aqueous solution by diluting 2ml of deionized
aqueous leaf extract of neem at concentration 0f water followed by scanning in UV visible spectra
250µl (14mm) and minimum inhibition concentration between 200 to 700 nanometer in a spectrophotometer
at 100µl (12mm). The present study was based on
previous work in which the aqueous extract exhibited Antifungal activities of synthesized silver
strong antifungal activity against C. albica
albicans. The nanoparticles from different leaf extract were
antifungal ability was again determined by disk determined the current study was also done using
diffusion protocol with the aid of measuring the zone crude aqueous leaf extract without nanoparticles. 20
of inhibition Maximum diameter of 15.60 mm was ml of sabouraud dextrose agar was poured into
observed at concentration of 50 µg silver petriplate and sterility
terility check was done before
nanoparticles. proceding for antifungal activity. The fungal test
organisms such as Aspergillus niger, Aspergillus
SUMMARY AND CONCUSSION flavus, Aspergillus fumigatus, Rhizopus Spp and
The fungal culturess were inoculated in to sabauraud Candida albicans were used to prepare lawn on
dextrose agar and incubated at room temperature for sabourauds dextroseagar plates. Agar wells of 5mm
24 hrs to 48 hrs and observed fungal growth. The size were prepared by using sterilized stainless steel
colonies were observed microscopically to identify cork borer. Four wells were loaded with different
the morphology of fungi.The fresh leaves of Neem, concentrations of silver nanoparticles synthesized
Lemon, Tamarind, Black ck berry and Almond were from different leaf extracts in range of 250µl, 200µl,
collected from S.I.E.T collage campus Chennai. The 150µl, 250µl. The plates were
ere also loaded with crude
leaves were washed thoroughly with distilled water aqueous leaf extracts of respective plants into four
and kept for shade drying. The leaves of respective wells using same concentration. The plates were
plants were ground to fine powder followed by incubated at 37ºC and examined for the zones of
soaking of 20gms of each fine leaf
eaf powder in 100ml inhibition in the form of clear area. The diameter of
of sterile distilled water overnight. The flask each zone of inhibition wass measured using a scale in
containing leaf solution was filtered using gauze and mm.
centrifuged. The supernants of each plant leaf was
collected in sterile conical flask and filtered. A The stock solutions of silver nanoparticles of
portion of filtrates were mixed with 10ml of 0.1N synthesized by leaf extract and without nanoparticles
silver nitrate solution and kept in dark room for of plant were diluted in 1ml of potato dextrose broth
synthesis of silver nanoparticles. The filtrates were followed by loading of wells in five rows with 100µl
kept in oven for 48hrsrs to obtain crude aqueous leaf of potato dextrose
ose broth. Serial dilution was done in
extracts. The effectiveness and accuracy in results order to obtainminimum concentration. 10ml of each
without contamination. fungal broth culture was loaded into respective wells
containing respective leaf extracts with and without
Aqueous solution (1 mM) of silver nitrate (AgNO3) nanoparticles showing highest antifungal activity. The
was prepared in 250 mL Erlenmeyer flasks and MFC plates were incubated at room temperature for
aqueous leaf extracts were added for silver nitrate

Oct 2018 Page: 1344
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24hrs. The plates were observed for minimum fungal 3. Daniel M. C., Astruc D. Chem. Rev. 2004;
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Biotechnol. 2003; 14:590–
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no antifungal activity. The crude Aqueous extracts of R. Nanotechnology. 2008;; 19(6):065604.
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Acknowledgement
11. Bhaduri G A, Little R, Khomane R B, Lokhande S
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Professor, P.G.Dept of Applied Microbiology JBAS
synthesis of silver nanoparticles using sunlight. J
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9.
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International Journal of Trend in Scientific Research and Development (IJTSRD)

International Open Access Journal | www.ijtsrd.com

ISSN No: 2456 - 6470 | Volume - 2 | Issue – 6 | Sep – Oct 2018

Green Synthesis of Silver Nano Particles aass Novel Antifungal Agents

@ IJTSRD | Available Online @ www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct

Candida albicans PREPARATION OF AQUEOUS LEAF

@ IJTSRD | Available Online @ www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct