Green Synthesis of Silver Nano Particles as Novel Antifungal Agents

1. International Journal of Trend in International Open Access Journal International Open Access Journal | www.ijtsrd.com International Journal of Trend in Scientific Research and Development (IJTSRD) Research and Development (IJTSRD) www.ijtsrd.com ISSN No: 2456 ISSN No: 2456 - 6470 | Volume - 2 | Issue – 6 | Sep 6 | Sep – Oct 2018 Green Synthesis of Silver Nano Particles a Synthesis of Silver Nano Particles as Novel Antifungal Agents s Novel Antifungal Agents R. Nazreen, R. Ushasri Scholar, Department of Applied Microbiology, PG.Scholar, Department of Applied Microbiology, JBAS College for Women JBAS College for Women, Chennai, Tamil Nadu, India ABSTRACT Aspergillus species are causative agents of invasive fungal infections in immunocompromised patients and associated with pulmonary diseases, mycotic diseases, keratitis mycotic keratitis, otomycosis and nasal sinusitis. At least 30 Aspergillus species are associated with human diseases. Aspergillus niger is a member of the genus Aspergillus which includes a set of fungi that are generally considered asexual, although perfect forms. Aspergillus flavus is a fungus grows by producing thin thread like branched Aspergillus flavus is a filamentous mould.A. fumigatus is characterized by green echinulate conidia in chains basipetally from greenish phialides, 6 to 8 by 2 to 3µm. Rhizopus is filamentous fungus found in soil decaying fruit and vegetables, Rhiz are most common in habitants of bread hence called as bread molds. Candidiasis is a fungal infection caused by yeasts that belong to the genus Candida. Silver is the one of the important nano five hundred tons of silver nanoparticles production per year it has been associated with strong bactericidal effects and antifungal activities. The aim of this study was to synthesize nanoparticles using plant extracts and determine antifungal activity by standard methods. METHODS Sabourd Dextrose agar was prepared. Fungal cultures were sub cultured and observed for microscopic and macroscopic characters. The leaves of Neem, Lemon, Black berry, Tamarind and Almond were collected and powdered. The powdered plant material was extracted using sterile water. Silver nano particles were prepared using crude extracts of Neem, Lemon, Black berry, Tamarind and Almond. RESULTS Aspergillus niger was found to be resistant to Aqueous leaf extracts of Neem, Lemon and Tamarind Aqueous leaf extracts of Neem, Lemon and Tamarind where this fungus was found to be sensitive tu maximum(250µl )and minimum concentration of Black berry and Almond concentration of ( 100 µl) with values of 24 mm,21 mm and 23 mm , 21 mm. Aspergillus flavus was found to be resistant to aqueous leaf extracts of Neem, Lemon, and Tamarind in concentration ranging from 250µl to 100 µl formation.A.fumigatus was found to be resistant to aqueous leaf extracts of neem, lemon and tamarind in concentration ranging from 250 µl to 10 no zone formation.Candida albicans was found to be resistant to aqueous leaf extracts of Neem, Lemon and Tamarind in concentration ranging from 250 to 100 µl showing no zone formation.Rhizopus spp was found to be resistant to aqueous leaf extr Lemon and Tamarind in concentration ranging from 250 to 100µl showing no zone formation.Rhizopus spp was found to be resistant to aqueous leaf extracts of Neem, Lemon and Tamarind in concentration ranging from 250 to 100µl showing no zone formation. Aspergillus niger , A.flavus, A. fumigatus, Rhizopus and Candida albicans were found to be resistant to aqueous silver nanoparticles leaf extracts of Lemon and Tamarind in concentration ranging from 250µ to 100µl showing no zone formation and fou sensitive to leaf extracts of Black berry, Almond and Neem . Keyword: SDA, plant extracts, spectrophotometer, SEM, MHA INTRODUCTION Fungi are ubiquitous and found in air, soil, water, and plants. Fungi are beneficial and harm ful are of great importance in production of industrially important secondary metabolites. Fungi cause many important secondary metabolites. Fungi cause many Aspergillus species are causative agents of invasive fungal infections in immunocompromised patients and associated with pulmonary diseases, mycotic diseases, keratitis mycotic keratitis, otomycosis and nasal sinusitis. At least 30 Aspergillus species are associated with human diseases. Aspergillus niger is a member of the genus Aspergillus which includes a set of fungi that are generally considered asexual, although perfect forms. Aspergillus flavus is a fungus grows by producing thin thread like branched hyphae. Aspergillus flavus is a filamentous mould.A. fumigatus is characterized by green echinulate conidia in chains basipetally from greenish phialides, 6 to 8 by 2 to 3µm. Rhizopus is filamentous fungus found in soil decaying fruit and vegetables, Rhizopus species are most common in habitants of bread hence called as bread molds. Candidiasis is a fungal infection caused by yeasts that belong to the genus Candida. Silver is the one of the important nano-material with where this fungus was found to be sensitive tu maximum(250µl )and minimum concentration of Black berry and Almond concentration of ( 100 µl) with values of 24 mm,21 mm and 23 mm , 21 mm. Aspergillus flavus was aqueous leaf extracts of Neem, Lemon, and Tamarind in concentration ranging from 250µl to 100 µl formation.A.fumigatus was found to be resistant to aqueous leaf extracts of neem, lemon and tamarind in concentration ranging from 250 µl to 100 µl showing no zone formation.Candida albicans was found to be resistant to aqueous leaf extracts of Neem, Lemon and Tamarind in concentration ranging from 250 to 100 µl showing no zone formation.Rhizopus spp was found to be resistant to aqueous leaf extracts of Neem, Lemon and Tamarind in concentration ranging from 250 to 100µl showing no zone formation.Rhizopus spp was found to be resistant to aqueous leaf extracts of Neem, Lemon and Tamarind in concentration ranging from 250 to 100µl showing no zone leaf leaf extracts extracts in in showing showing no no zone zone cles production per year it has been associated with strong bactericidal effects and antifungal activities. The aim of this study was to synthesize nanoparticles using plant extracts and determine antifungal activity by standard Aspergillus niger , A.flavus, A. fumigatus, Rhizopus and Candida albicans were found to be resistant to aqueous silver nanoparticles leaf extracts of Lemon and Tamarind in concentration ranging from 250µ to 100µl showing no zone formation and found to be sensitive to leaf extracts of Black berry, Almond and Dextrose agar was prepared. Fungal cultures were sub cultured and observed for microscopic and macroscopic characters. The leaves of Neem, Lemon, Black berry, Tamarind and Almond were collected and powdered. The powdered plant material was sterile water. Silver nano particles were prepared using crude extracts of Neem, Lemon, SDA, plant extracts, spectrophotometer, Fungi are ubiquitous and found in air, soil, water, and plants. Fungi are beneficial and harm ful. Fungi are of great importance in production of industrially Aspergillus niger was found to be resistant to @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct 2018 Oct 2018 Page: 1338

2. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 infections to Humans such as Aspergillosis, Aspergilloma and candidiasis. Fungal infections are most common in immune compromised individuals. Aspergillus niger Aspergillus niger is a genus Aspergillus which includes a set of fungi that are generally considered asexual, although perfect forms.A.nger is thermo tolerant and found everywhere and found to be resistant to freezing tempe Aspergilli are ubiquitous in nature. Aspergillus niger can be classified as a member of Dueteromycetes They are geographically widely distributed and have been observed in a broad range of habitats because they can colonize a wide variety of substrates. niger is commonly found as a saprophyte growing on dead leaves, stored grain, compost piles, and othe decaying vegetation. Aspergillus flavus Aspergillus flavus is a fungus. It grows by producing thread like branching filaments known as hyphae. Filamentous fungi such as A. flavus are sometimes called molds. A network of hyphae known as the mycelium secretes enzymes that break down complex food sources.When young, the conidia of A. flavus appear yellow green in color. As the fungus ages the spores turn a darker green. Aspergillus fumigatus A. fumigatus is identified based on morphology of conidia and conidiophores. characterized by green echinulate conidia in chains basipetally from greenish Phalides, 6 to 8 by 2 to 3µm. A. fumigatus is pigment less and produce white conidia. Rhizopus SPP Rhizopus is filamentous fungus found in soil decaying fruit and vegetables, Rhizopus species are most common in habitants of bread hence called as bread molds. They cause most severe infections in human beings. Candida albicans Candidiasis is a fungal infection caused by yeasts that belong to the genus Candida. There are over 20 species of Candida yeasts that can cause infection in humans, the most common of which is albicans. Candida yeasts normally reside in the intestinal tract and can be found on mucous membranes and skin without causing infection. There membranes and skin without causing infection. There infections to Humans such as Aspergillosis, Aspergilloma and candidiasis. Fungal infections are most common in immune compromised individuals. are two types of candidiasis such as yeat infection or oral thrush and invasive candidiasis. are two types of candidiasis such as yeat infection or oral thrush and invasive candidiasis. The ‘green environment eco friendly chemistry and chemical technologies which are popular and much require as a result of world problems associated with environmental conditions. Silver is the one of the important nano five hundred tons of silver nanoparticles pro per year it has been associated with strong bactericidal effects and antifungal activities. Green techniques have been used as biological technique for the synthesis of silver Nano particles as alternate methods to conventional methods. Silver nanoparticles can be produced at low concentration of leaf extract without using any additional harmful chemical/physical methods. The method applied here is simple, cost effective, easy to perform and sustainable. Generally the synthesis of nanoparticles carried out using three different approaches, including physical, chemical, and biological methods. In physical methods nanoparticles, nanoparticles are prepared by evaporation condensation using a tube furnace at atmospheric pressure. Convention physical methods including spark discharging and pyrolysis were used for the synthesis of AgNPs The advantages of physical methods are speed, radiation used as reducing agents, and no hazardous chemicals involved, but the downsides are low yield and high energy consumption, solvent contamination and lack of uniform distribution METHODS SUBCULTURE OF FUNGAL SPECIES The fungal cultures were inoculated in to sabauraud dextrose agar and incubated at room temperature for 24 hrs to 48 hrs and observed funga colonies were observed microscopically to identify the morphology of fungi. PREPARATION OF EXTRACTS OF NEEM, LEMON, TAMARIND, BLACK BERRY AND ALMOND. BLACK BERRY AND ALMOND. The ‘green environment eco friendly processes in chemistry and chemical technologies which are popular and much require as a result of world problems associated with environmental conditions. Silver is the one of the important nano-material with five hundred tons of silver nanoparticles production per year it has been associated with strong bactericidal effects and antifungal activities. is a member member of of the the which includes a set of fungi that are generally considered asexual, although perfect forms.A.nger is thermo tolerant and found everywhere and found to be resistant to freezing temperature. Aspergillus niger can be classified as a member of Dueteromycetes They are geographically widely distributed and have been observed in a broad range of habitats because they can colonize a wide variety of substrates. A. is commonly found as a saprophyte growing on dead leaves, stored grain, compost piles, and other Green techniques have been used as biological technique for the synthesis of silver Nano particles as alternate methods to conventional methods. Silver anoparticles can be produced at low concentration of leaf extract without using any additional harmful chemical/physical methods. The method applied here is simple, cost effective, easy to perform and is a fungus. It grows by producing thread like branching filaments known as hyphae. Generally the synthesis of nanoparticles has been carried out using three different approaches, including physical, chemical, and biological methods. In physical methods nanoparticles, nanoparticles are prepared by evaporation condensation using a tube furnace at atmospheric pressure. Conventional physical methods including spark discharging and pyrolysis were used for the synthesis of AgNPs are sometimes called molds. A network of hyphae known as the retes enzymes that break down complex food sources.When young, the conidia of A. flavus appear yellow green in color. As the fungus ages the is identified based on morphology of conidiophores. characterized by green echinulate conidia in chains basipetally from greenish Phalides, 6 to 8 by 2 to 3µm. A. fumigatus is pigment less and produce white The advantages of physical methods are speed, radiation used as reducing agents, and no hazardous chemicals involved, but the downsides are low yield igh energy consumption, solvent contamination and lack of uniform distribution conidia and A. A. fumigatus fumigatus is is SUBCULTURE OF FUNGAL SPECIES The fungal cultures were inoculated in to sabauraud dextrose agar and incubated at room temperature for 24 hrs to 48 hrs and observed fungal growth. The colonies were observed microscopically to identify filamentous fungus found in soil decaying species are most common in habitants of bread hence called as bread molds. They cause most severe infections in human PREPARATION EXTRACTS OF NEEM, LEMON, TAMARIND, OF AQUEOUS AQUEOUS LEAF LEAF is a fungal infection caused by yeasts that . There are over 20 yeasts that can cause infection in humans, the most common of which is Candida yeasts normally reside in the intestinal tract and can be found on mucous @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct 2018 Oct 2018 Page: 1339

3. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 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 o 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 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 contamination. GREEN SYNTHESIS NANOPARTICLES (Ag NP) Aqueous solution (1 mM) of silver nitrate (AgNO3) 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 spec 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 in color. The colloidal 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 SPECTRA ANALYSIS Samples (1 mL) of the suspensions 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 nanometer in a spectrophotometer ANTIFUNGAL ACTIVITY Antifungal activities of 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 don for antifungal activity. The fungal test organisms such as Aspergillus niger, Aspergillus flavus, , Aspergillus flavus, Aspergillus , 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 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 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 ut contamination. NEEM GREEN NANOPARTICLES (Ag NP) SYNTHESIS OF OF SILVER SILVER mM) of silver nitrate (AgNO3) Erlenmeyer flasks and LEMON 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 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 idal solution containing silver nanoparticles of leaf extracts was estimated by UV visible spectrophotometric analysis. The colloidal mixture was sealed and stored in refrigerator for TAMARIND VIS SPECTRA ANALYSIS 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 nanometer in a spectrophotometer BLACKBERRY s of synthesized synthesized silver 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 before proceeding for antifungal activity. The fungal test organisms such ALMOND @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct 2018 Oct 2018 Page: 1340

4. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 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 with crude aqueous leaf extracts of respective plants into four wells using same 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 of 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. The MFC plates were incubated at room temperature for 24hrs. The plates were observed for minimum fungal inhibition concentration minimum fungal inhibition concentration Candida albicans were used to prepare lawn on Sabourd dextrose agar 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 h crude aqueous leaf extracts of respective plants into four wells using same 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 Green synthesis of Nano particles Green synthesis of Nano particles 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 f 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 ctivity. The MFC plates were incubated at room temperature for 24hrs. The plates were observed for UV – spectrophotometric analysis spectrophotometric analysis crude leaf extracts SEM image of Almond silver nano particle leaf 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. Antifungal activity of crude silver nano particles synthesized by leaves of Almond, Lemon, Neem, @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct 2018 Oct 2018 Page: 1341

5. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 looking conidium, while Aspergillus niger 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 in concentration of ( 100µl) with values of 24mm,21 mm and 23 mm , 21 mm. This study was compared with that ofprevious similar work using similar work .The aqueous extracts of Acacia albida was reported to be hihly potent with maximum antifungal activity (18mm) followed by (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 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 extrac 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 fumigatus was found to be sensitive 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 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) inhibitory concentration of 100 was found to be sensitive to aqueous leaf extract of blackberry at concentration of 250µl(19mm) and minimum inhibitory concentration of 100µl(17mm). minimum inhibitory concentration of 100µl(17mm). Aspergillus niger looks more like a toilet brush, with its globose base at the end of 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 xtracts in concentration of ( 100µl) with values mm , 21 mm. This study was previous similar work using The aqueous extracts of Acacia albida was reported to be hihly potent with maximum activity (18mm) followed byP. juliflora The aqueous extracts of Acacia albida was reported to be highly potent with maximum antifungal was found to be resistant to aqueous leaf extracts of 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 at the concentration of 100 µl (17mm). A. flavus was found to be sensitive to aqueous leaf. The present was compared with previous work. was found to be resistant to aqueous leaf extracts of neem, lemon and tamarind in concentration 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). A. 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. was found to be resistant to aqueous leaf 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) inhibitory concentration of 100µl(19mm). Rhizopus was found to be sensitive to aqueous leaf extract of blackberry at concentration of 250µl(19mm) and and and minimum minimum DISCUSSION A. niger but the black conidia and spores confirm the species to A. niger.Penicillium has a paint brush but the black conidia and spores confirm the has a paint brush @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct 2018 Oct 2018 Page: 1342

6. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 Candida albicans was found to be resistant to aqueous leaf extracts of Neem, Lemon and concentration ranging from 250 to 100 µl showing no zone formation. The fungus was found to be highly sensitive to 250µl(30mm) and minimum inhibitory concentration of 100µl(23mm). Candida albicans found to be sensitive to aqueous leaf extra blackberry at concentration of 250µl(19mm) and minimum inhibitory concentration of 100µl(12mm) . The current study was compared with previous work exhibiting the zone of inhibition recorded at 500 mg/ml concentration was higher than that of 250, 100, 10 mg/ml concentration for all the extracts. As the amount of the extract increased, the inhibitory effect had increased Aspergillus niger was found to be resistant to aqueous silver nanoparticles leaf extracts of Lemon and Tamarind in concentration ranging from 250µ to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles of almond leaf 250µl(28mm) and minimum inhibition concentration of 100µl(20mm). A. niger was to be sensitive to aqueous silver nanoparticles leaf extract concentration of 250µl(28mm)and inhibition at concentration of 100µl(17mm). was found to be sensitive to aqueous silver nanoparticles leaf extract of Neem at concentration of 250µl(16mm) and minimum inhibitory concentration of 100µl(14mm) This work was found to be similar to previous work which showed the antifungal activity of silver nanoparticle had been evaluated against Aspergillus niger. The zones of inhibition of Aspergillus niger against AgNPs, ethanol, plant extract and chloramphenicol (standard) was observed The silver nanoparticles showed strong inhibitory (+) action and no zone of inhibition was seen for ethanol. A.flavus was found to be resistant to aqueous silver nanoparticles leaf extracts of Lemon and tamarind in concentration ranging from 250µl to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles leaf extract at the concentration of 250µl(29mm) and minimum inhibition concentration of 100µl(27mm). A. flavus was found to be sensitive to aqueous silver nanoparticles leaf extract concentration of 250µl(20mm) inhibition concentration of 100µl(17mm). A. flavus was found to be sensitive to aqueous leaf extra nee at concentration 0f 250µl (17mm) and minimum nee at concentration 0f 250µl (17mm) and minimum was found to be resistant to aqueous inhibition concentration at 100µl (14mm). The current study focused on MIC values and well diffusion method in whichAgNPs exhibited higher antifungal activity even at low concentration (0.1. The antifungal potency of the plant extract and AgNPs increased with increasing concentrations. A.fumigatus was found to be resistant to aqueous silver nanoparticles leaf extracts of Lemon and tamarind in concentration ranging from 250µl to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles leaf extract at the concentration of 250µl (28mm) and minimum inhibitio of 100µl(23mm). A. fumigatus sensitive to aqueous silver nanoparticles leaf extract of blackberry at concentration of 250µl(24mm) and minimum inhibition concentration of 100µl(16mm). A. fumigatus was found to be sensitive to extract of neem at concentration 0f 250µl (17mm) and minimum inhibition concentration at 100µl (12mm). The current observations were found to be similar to that of previous work Rhizopus spp was found to be resistant to aqueous silver nanoparticles leaf extracts of Lemon and tamarind in concentration ranging from 250µl to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles leaf extract at the concentration of 250µl(3mm) and minimum inhi 100µl(27mm). Rhizopus spp was found to be sensitive to aqueous silver nanoparticles leaf extract of blackberry at concentration of 250µl(19mm) and minimum inhibition concentration of 100µl(17mm). Rhizopus spp was found to be sensitive to aqueous leaf extract of Neem at concentration 0f 250µl(18mm) and minimum inhibition 100µl(15mm). The previous work reported that AgNPs showed better against Aspergillus sp. evidenced by minimum inhibitory concentration (MIC) value 21.8 ng/mL The results showed that the AgNPs were fungicidal against both the tested fungus at very low concentrations and the fungicidal activity was dependent on the tested fungus species. These results were confirmed by plating the content of each well on dextrose agar medium, and there was no growth for any of the strains resultant from the MIC point. These enhanced effects of AgNPs might MIC point. These enhanced effects of AgNPs might inhibition concentration at 100µl (14mm). The current study focused on MIC values and well diffusion method in whichAgNPs exhibited higher antifungal activity even at low concentration (0.1. The ungal potency of the plant extract and AgNPs increased with increasing Tamarind in concentration ranging from 250 to 100 µl showing no zone formation. The fungus was found to be highly sensitive to 250µl(30mm) and minimum inhibitory Candida albicans was their their corresponding corresponding found to be sensitive to aqueous leaf extract of blackberry at concentration of 250µl(19mm) and minimum inhibitory concentration of 100µl(12mm) . The current study was compared with previous work the zone of inhibition recorded at 500 mg/ml concentration was higher than that of 250, 100, 10 mg/ml concentration for all the extracts. As the amount of the extract increased, the inhibitory effect was found to be resistant to aqueous leaf extracts of Lemon and tamarind in concentration ranging from 250µl to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles leaf extract at the concentration of 250µl (28mm) and minimum inhibition concentration A. fumigatus was found to be sensitive to aqueous silver nanoparticles leaf extract of blackberry at concentration of 250µl(24mm) and minimum inhibition concentration of 100µl(16mm). was found to be sensitive to aqueous leaf extract of neem at concentration 0f 250µl (17mm) and minimum inhibition concentration at 100µl (12mm). The current observations were found to be similar to was found to be resistant to aqueous silver nanoparticles leaf extracts of Lemon and ing from 250µ to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles of almond leaf 250µl(28mm) and minimum inhibition concentration of 100µl(20mm). was to be sensitive to aqueous silver oparticles leaf extract concentration of 250µl(28mm)and inhibition at concentration of 100µl(17mm).A.niger was found to be sensitive to aqueous silver nanoparticles leaf extract of Neem at concentration of of of blackberry blackberry at at minimum minimum was found to be resistant to aqueous rticles leaf extracts of Lemon and tamarind in concentration ranging from 250µl to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles leaf extract at the concentration of 250µl(3mm) and minimum inhibition concentration of nhibitory concentration of 100µl(14mm) This work was found to be similar to previous work which showed the antifungal activity of silver nanoparticle had been evaluated against . The zones of inhibition of was found to be sensitive to aqueous silver nanoparticles leaf extract of blackberry at concentration of 250µl(19mm) and minimum inhibition concentration of 100µl(17mm). was found to be sensitive to aqueous leaf extract of Neem at concentration 0f 250µl(18mm) and minimum inhibition 100µl(15mm). The previous work reported that AgNPs showed better ethanol, plant extract and chloramphenicol (standard) was observed The silver nanoparticles showed strong inhibitory (+) action and no zone of inhibition was seen for ethanol. concentration concentration at at was found to be resistant to aqueous silver ts of Lemon and tamarind in concentration ranging from 250µl to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles leaf extract at the concentration of 250µl(29mm) and minimum on of 100µl(27mm). A. flavus was found to be sensitive to aqueous silver nanoparticles leaf extract concentration of 250µl(20mm) inhibition concentration of 100µl(17mm). A. flavus was found to be sensitive to aqueous leaf extract of antifungal and and Rhizopus sp. antifungal properties properties as evidenced by minimum inhibitory concentration ng/mL The results showed that the AgNPs were fungicidal against both the tested fungus at very low concentrations and the fungicidal activity was dependent on the tested fungus species. hese results were confirmed by plating the content of each well on dextrose agar medium, and there was no growth for any of the strains resultant from the of of blackberry and and blackberry at at minimum minimum @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct 2018 Oct 2018 Page: 1343

7. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 be due to the antifungal properties of silver nanoparticles (Shreya Meddaet al., 2015) Candida albicans was found to be resistant to aqueous silver nanoparticles leaf extracts of Lemon and tamarind in concentration ranging from 250µl to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles leaf extract at the concentration of 250µl (30mm) and minimum inhibition concentration of 100µl (20mm). Candida albicans was found to be sensitive to aqueous silver nanoparticles leaf extract of blackberry at concentration of 250µl (19mm) and minimum inhibition concentration of 100µl (15mm Candida albicans was found to be s aqueous leaf extract of neem at concentration 0f 250µl (14mm) and minimum inhibition concentration at 100µl (12mm). The present study was based on previous work in which the aqueous extract exhibited strong antifungal activity against C. albica antifungal ability was again determined by disk diffusion protocol with the aid of measuring the zone of inhibition Maximum diameter of 15.60 mm was observed at concentration nanoparticles. SUMMARY AND CONCUSSION The fungal cultures were inoculated in to sabauraud dextrose agar and incubated at room temperature for 24 hrs to 48 hrs and observed fungal growth. The colonies were observed microscopically to identify the morphology of fungi.The fresh leaves of Neem, Lemon, Tamarind, Black berry and Almond were collected from S.I.E.T collage 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 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 supernants of each plant leaf was collected in sterile conical flask 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 contamination. Aqueous solution (1 mM) of silver nitrate (AgNO3) was prepared in 250 mL Erlenmeyer flasks and aqueous leaf extracts were added for silver nitrate aqueous leaf extracts were added for silver nitrate be due to the antifungal properties of silver reduction. The composite mixture was then kept in oven for complete redction of silver nitrate. The was observed from yellowish brown ro reddish brown followed by spectrophotometry for 30 minutes. The reaction was carried out in darkness at room temperature so as to prevent photoreactivation of silver nitrate along with controls. The confirmation er nanoparticles synthesis was based on change in colour. The colloidal solution containing silver nanoparticles of leaf extracts was estimated by UV visible spectrophotometric analysis. The colloidal mixture was sealed and stored in refrigerature for fungal activity. Samples (1 mL) of the suspensions were collected to analyse complete bioreduction of Ag+ in aqueous solution by diluting 2ml of deionized water followed by scanning in UV visible spectra between 200 to 700 nanometer in a spectrophotometer reduction. The composite mixture was then kept in oven for complete redction of silver nitrate. The colour change was observed from yellowish brown ro reddish brown followed by spectrophotometry for 30 minutes. The reaction was carried out in darkness at room temperature so as to prevent photoreactivation of silver nitrate along with controls. The confirmation of silver nanoparticles synthesis was based on change in colour. The colloidal solution containing silver nanoparticles of leaf extracts was estimated by UV visible spectrophotometric analysis. The colloidal mixture was sealed and stored in refrigerature for antifungal activity. Samples (1 were collected to analyse complete bioreduction of Ag+ in aqueous solution by diluting 2ml of deionized water followed by scanning in UV visible spectra between 200 to 700 nanometer in a spectrophotometer Antifungal activities of nanoparticles from different leaf extract were determined the current study was also done using crude aqueous leaf extract without nanoparticles. 20 ml of sabouraud dextrose agar was poured into petriplate and sterility check was done before proceding for antifungal activity. The fungal test organisms such as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Rhizopus Spp and Candida albicans were used to prepare lawn on sabourauds dextroseagar plates. 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 with crude aqueous leaf extracts of respective plants into four wells using same 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. 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 of potato dextrose broth. Serial dilution was done in order to obtainminimum concentration. 10ml of each fungal broth culture was loaded into respective wells containing respective leaf extracts with and without nanoparticles showing highest antifungal activity. The MFC plates were incubated at room temperature for plates were incubated at room temperature for ., 2015) was found to be resistant to aqueous leaf extracts of Lemon and tamarind in concentration ranging from 250µl to 100µl showing no zone formation. The fungus was found to be highly sensitive to aqueous silver nanoparticles leaf extract at the concentration of n concentration was found to be sensitive to aqueous silver nanoparticles leaf extract of blackberry at concentration of 250µl (19mm) and minimum inhibition concentration of 100µl (15mm). was found to be sensitive to aqueous leaf extract of neem at concentration 0f 250µl (14mm) and minimum inhibition concentration at 100µl (12mm). The present study was based on the aqueous extract exhibited Antifungal nanoparticles from different leaf extract were determined the current study was also done using crude aqueous leaf extract without nanoparticles. 20 ml of sabouraud dextrose agar was poured into terility check was done before proceding for antifungal activity. The fungal test organisms such as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Rhizopus Spp and Candida albicans were used to prepare lawn on sabourauds dextroseagar 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, activities of synthesized synthesized silver silver C. albicans. The antifungal ability was again determined by disk diffusion protocol with the aid of measuring the zone of inhibition Maximum diameter of 15.60 mm was observed at concentration of of 50 50 µg silver s were inoculated in to sabauraud dextrose agar and incubated at room temperature for 24 hrs to 48 hrs and observed fungal growth. The colonies were observed microscopically to identify the morphology of fungi.The fresh leaves of Neem, ck berry and Almond were collected from S.I.E.T collage 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 ere also loaded with crude aqueous leaf extracts of respective plants into four wells using same concentration. The plates were incubated at 37ºC and examined for the zones of inhibition in the form of clear area. The diameter of s measured using a scale in eaf powder in 100ml of sterile distilled water overnight. The flask containing leaf solution was filtered using gauze and centrifuged. The supernants of each plant leaf was collected in sterile conical flask and filtered. A 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 ose broth. Serial dilution was done in order to obtainminimum concentration. 10ml of each fungal broth culture was loaded into respective wells containing respective leaf extracts with and without nanoparticles showing highest antifungal activity. The with 10ml of 0.1N silver nitrate solution and kept in dark room for synthesis of silver nanoparticles. The filtrates were rs to obtain crude aqueous leaf extracts. The effectiveness and accuracy in results mM) of silver nitrate (AgNO3) mL Erlenmeyer flasks and @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct 2018 Oct 2018 Page: 1344

8. International Journal of Trend in Scientific International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456 Research and Development (IJTSRD) ISSN: 2456-6470 3.Daniel M. C., Astruc 104:293–346. 4.Wong T. S., Schwaneberg Biotechnol. 2003; 14:590– 5.Preparation, characterization and applications. John Wiley., Fendler J. nanostructured films. 1998:463. 6.Tsuji M., Hashimoto M., Nishizawa Y., Tsuji T., Chem. Lett. 2003; 32:1114 7.Kundu S., Maheshwari R. Nanotechnology. 2008; 8.Okitsu K., Mizukoshi Y., Yamamoto T. Maeda Y., Nagata Y., 61:3429–3431. 9.Narayanan K. B., Sakthivel Interface. Sci. 2010;22(156):1 10.Barie P S. Multidrug-resistant organisms and antibiotic management. Am. 2012; 92:345–391. 11.Bhaduri G A, Little R, Khomane R U, Kulkarni B D, Mendis B synthesis of silver nanoparticles using sunlight. Photochem Photobiol A: Chemistry. 9. 12.Clinical and Laboratory Institute. Method for antifungal disk diffusion susceptibility testing of yeasts: approved standard M44-A2. Wayne: Clinical Standards Institute; Standards Institute; 24hrs. The plates were observed for minimum fungal inhibition concentration Based on the results obtained the present could be concluded as silver Nano particles synthesized by leaf extracts of Tamarind, lemon, black berry, Neem and Almond. exhibited antifungal activity. Antifungal activity was done using crude aqueous extract with and without Nano particles. Almond leaf extract sintering Nano particles were found to be highly potential followed by Black berry and Neem against fungi. . The leaf extracts of Tamarind, and Lemon had no antifungal activity. The crude Aqueous extracts of Tamarind, and Lemon had no antifungal activity. Almond aqueous leaf extracts showed highest antifungal activity followed by Black B comparative study was done and concluded that Aqueous crude silver Nano leaf extracts of Almond, Black Berry and Neem were found to be highly potential when compared to crude aqueous leaf extracts of Almond, Black Berry and Neem plants. Acknowledgement We thank Ms Summera Rafiq Head & Associate Professor, P.G.Dept of Applied Microbiology JBAS College for Women for providing facilities to carry out this work References 1.Roco M. C. Curr. Opin. Biotechnol. 14:337–346. 2.Zhang L., Gu F. X., Chan J. M., Wang A. Langer R. S., Farokhzad O. C.. Clin. Pharmacol. Ther. 2008; 83:761–780. 24hrs. The plates were observed for minimum fungal D. Chem. Rev. 2004; S., Schwaneberg U. Curr. Opin. –596. Based on the results obtained the present could be concluded as silver Nano particles synthesized by leaf Preparation, characterization and applications. John Wiley., Fendler J. H., Nanoparticles and 1998:463. , black berry, Neem and Almond. exhibited antifungal activity. Antifungal activity was done using crude aqueous extract with and without Nano particles. Almond leaf extract sintering Nano particles were found to be highly Tsuji M., Hashimoto M., Nishizawa Y., Tsuji 32:1114–1115. and Neem against Kundu S., Maheshwari ; 19(6):065604. V., V., Saraf Saraf fungi. . The leaf extracts of Tamarind, and Lemon had no antifungal activity. The crude Aqueous extracts of Tamarind, and Lemon had no antifungal activity. Almond aqueous leaf extracts showed highest antifungal activity followed by Black Berry. A comparative study was done and concluded that Aqueous crude silver Nano leaf extracts of Almond, Black Berry and Neem were found to be highly potential when compared to crude aqueous leaf extracts of Almond, Black Berry and Neem plants. Okitsu K., Mizukoshi Y., Yamamoto T. A., Maeda Y., Nagata Y., Lett. Materials. 2007; B., Sakthivel N. Adv. Colloid. (156):1–13 resistant organisms and management. Surg Clin North A, Little R, Khomane R B, Lokhande S D, Mendis B G, et al. Green synthesis of silver nanoparticles using sunlight. J Photochem Photobiol A: Chemistry. 2013; 258:1– We thank Ms Summera Rafiq Head & Associate Professor, P.G.Dept of Applied Microbiology JBAS College for Women for providing facilities to carry Clinical and Laboratory Standards Standards Method for antifungal disk diffusion susceptibility testing of yeasts: approved standard Wayne: Clinical Curr. Opin. Biotechnol. 2003; and and Laboratory Laboratory 2008. 2008. M., Wang A. Z., Clin. Pharmacol. @ IJTSRD | Available Online @ www.ijtsrd.com www.ijtsrd.com | Volume – 2 | Issue – 6 | Sep-Oct 2018 Oct 2018 Page: 1345