Accepted Manuscript Cytotoxicity and antimicrobial activities of green synthesized silver nanoparticles S. Lokina, A. Stephen, V. Kaviyarasan, C. Arulvasu, V. Narayanan PII:

S0223-5234(14)00130-5

DOI:

10.1016/j.ejmech.2014.02.010

Reference:

EJMECH 6727

To appear in:

European Journal of Medicinal Chemistry

Received Date: 20 August 2013 Revised Date:

6 February 2014

Accepted Date: 7 February 2014

Please cite this article as: S. Lokina, A. Stephen, V. Kaviyarasan, C. Arulvasu, V. Narayanan, Cytotoxicity and antimicrobial activities of green synthesized silver nanoparticles, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.02.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights  Ag Np was synthesized using fruit extract - reducing and stabilizing agent.

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 Stability of Ag Np confirmed by UV-Visible and HRTEM analyses after one month.  HRTEM images showed the spherical crystalline Ag Np as small as 0.9 nm.

 Ag Np showed excellent antimicrobial activities against various microorganisms.

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 Ag Np showed good cytotoxicity results towards MCF – 7 breast cancer cell lines.

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Cytotoxicity and antimicrobial activities of green synthesized silver nanoparticles

a

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S. Lokinaa , A. Stephenb , V. Kaviyarasanc, C. Arulvasud and V. Narayanana, *

Department of Inorganic Chemistry, University of Madras, Guindy Maraimalai Campus, Chennai 600025, India.

Department of Nuclear Physics, University of Madras, Guindy Maraimalai Campus, Chennai 600025, India.

c

CAS in Botany, University of Madras, Guindy Maraimalai Campus, Chennai 600025, India.

Department of Zoology, University of Madras, Guindy Maraimalai Campus, Chennai 600025,

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b

India.

Dr. V. Narayanan, Assistant Professor,

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Address for Correspondence*

Department of Inorganic Chemistry,

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School of Chemical Sciences,

University of Madras, Guindy Maraimalai Campus, Chennai – 25, Tamil Nadu, India.

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Phone: 91 44 22202793; Fax: 91 44 22300488. *Corresponding author E-mail: [email protected] (V. Narayanan),

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ABSTRACT Bio–inspired silver nanoparticles are synthesized using Malus domestica (apple) extract. Polyphenols present in the apple extract act as a reducing and capping agent to produce the silver

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nanoparticles. UV-Visible analysis shows the surface plasmon resonance (SPR) absorption at 420 nm. The FTIR analysis was used to identify the functional groups responsible for the bioreduction of silver ion. The XRD and HRTEM images confirm the formation of silver

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nanoparticles. The minimal inhibitory concentration (MIC) of silver nanoparticles was recorded against most of the bacteria and fungus. Further, MCF–7 human breast adenocarcinoma cancer

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cell line was employed to observe the efficacy of cancer cell killing.

Keywords: Silver nanoparticles, apple, antimicrobial activity, cytotoxicity, MCF-7 cells. 1. Introduction

Nanotechnology is one of the promising fields for generating new applications in

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medicine. The metallic nanoparticles have been synthesized by various methods. The novel biosynthetic route using fruit and plant extracts has been proved superior to other methods. The silver nanoparticles have some distinctive properties like conductivity, catalytic activity,

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chemical stability and antimicrobial activity. The biosynthesis of silver and gold nanoparticles using geranium and neem leaf extracts was reported by Shankar et al. [1,2].

The green

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chemistry utilizes nontoxic chemicals and environmental friendly silver nanoparticles from abundantly available apple fruit extract. The proverb “an apple a day keeps the doctor away,” addresses the health effects of the apple [3]. There is the evidence that apples possess phenolic compounds which may be cancer-protective and demonstrate antioxidant activity [4]. The important phenolic phytochemicals present in apple are quercetin, kaempferol, myricetin, epicatechin, and procyanidin B2 [5]. The primary phenolic acid, chlorogenic acid is found in

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pulp and skin of an apple. When an apple is red in colour, it is because of more anthocyanins. Epicatechin is the primary polyphenol present in apples. Recent research studies shows that polyphenols in the skin of an apple absorb the UV-B radiation and prevent it from damaging the

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photosynthetic cells in the apple skin like natural sunscreen [6, 7]. In the apple seeds, 98% of the flavonoid phloridzin is found. Research suggests that apples may reduce the risk of prostate cancer, colon cancer and lung cancer. Compared to many other fruits and vegetables, apples

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contain relatively low amounts of vitamin C but are a rich source of other antioxidant compounds [8]. The polyphenols in apples can prevent spikes in blood sugar by various

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mechanisms. Flavonoids like quercetin can inhibit enzymes (alpha-amylase and alphaglucosidase) which are involved in the breakdown of carbohydrates into sugar. The fiber content in apples reduces the cholesterol by preventing reabsorption, and they are bulky for their calorific content.

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The modern nanotechnology has facilitated the production of silver nanoparticles with low toxicity to human and greater efficacy against bacteria. Nanoparticles are attractive alternative to antibiotics by showing improved activity against multidrug resistant bacteria. In

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general, silver ions can bind with a variety of negatively charged molecules like RNA, DNA and proteins. These biologically synthesized silver nanoparticles were found to be highly toxic

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against different multi drug resistant human pathogens. The various silver-based compounds and materials containing ionic silver (Ag+) [9,10] or metallic silver (Ag0) [11] have been synthesized and shown to exhibit antimicrobial activity against various bacteria. The inactivation of bacteria are associated with nanoparticles concentration [12], nanoparticle shape, bacterial type [13,14], the presence of Ag+ and nanoparticle size [15-17]. The bacterial growth at a given concentration

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of silver has been found to be dependent on the initial number of cells. The antibacterial efficacy of silver nanoparticles increases because of their larger total surface area per unit volume [18]. The effect of antimicrobial activity of the silver nanoparticles was observed in

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Staphylococcus aureus, Citrobacter koseri, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. The minimum inhibitory concentration was determined by Resazurin microtitre assay (REMA). DNA and bacterial membrane proteins possess sulfur

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and phosphorus compounds. Silver has higher affinity to react with these compounds [19]. Cancer is a major health problem and it arises from one single cell. The transformation of a

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normal cell into a tumour cell occurs in a multistage process and the changes are due to the interaction between genetic factors of a person and external agents of three categories like physical carcinogens, chemical carcinogens and biological carcinogens. In worldwide, each year 7.6 million people die from cancer. According to WHO, if it continue rising without any

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immediate action, 13.1 million people may die in 2030. Tobacco use, alcohol use, lack of physical activity, low intake of fruit and vegetable are some of the important risk factors, the reason for 30% of worldwide cancer deaths. Diagnosis of tumors in the human body was very

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difficult [20,21] at their earlier stage and there was a search of new treatment for treating this deadly disease. Radiotherapy, chemotherapy and surgery are some of the cancer treatments

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which are used to improve the patient’s life. Recently, nanoparticles are also used to overcome this problem. The nanoscale devices can easily enter the cells and they made an interaction with DNA, proteins, enzymes and cell receptors. The nanoparticles can detect the cancer disease in a very small volume of cells or tissue [22,23]. In this study we focused on the cytotoxicity of silver nanoparticles on cultured MCF-7 cell line using different concentrations. It is the acronym of Michigan Cancer Foundation – 7 (Cancer Institute in Detroit). MCF-7 cell line was first

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obtained from the breast tissue of a 69-year old (Caucasian woman) Frances Mallon. Her cells only gave a current knowledge about breast cancer [24]. Prior to MCF-7, it was found very difficult for cancer researchers to obtain a mammary cell line. They are useful for in vitro breast

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cancer studies because the cell line has retained about the mammary epithelium. They enhance the ability of the MCF-7 cells to process estrogen via estrogen receptors in the cytoplasm of the cell. MTT (3-(4, 5- dimethylthiazol-2yl)-2, 5- diphenyltetrazolium bromide) method was used to

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assess the antiproliferative effect [25].

In the present study, we wish to report for the first time green synthesis of silver

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nanoparticles using the extract of apple (Malus domestica). The optical absorption spectrum of green synthesized silver nanoparticles is recorded by using UV-visible spectrophotometer. Morphological characterizations are performed using XRD, SEM and TEM. The spherical shaped silver nanoparticles showed its toxicity against MCF–7 human breast adenocarcinoma

bacteria and fungus. 2. Results and discussions

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2.1. UV-Visible spectroscopy

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cancer cell lines. They showed excellent antimicrobial effect to inhibit different pathogenic

The apple extract was mixed with the aqueous solution of silver nitrate. The colour

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change from yellow to black indicates the formation of stable silver nanoparticles. Figure 1a shows the optical photograph of pure apple extract (a) and after addition of silver ions to apple extract (b). The free electrons of silver nanoparticles give rise to a surface plasmon resonance absorption band [26-28]. The synthesis of silver nanoparticles was evaluated at different contact time by taking absorbance using UV-Vis spectroscopy. It was noted that the sharpness of the absorption peak increases with increase of time. The absorbance of pure apple extract was taken

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at the beginning and after the addition of 0.01 M AgNO3 to apple extract, the spectra was recorded every 60 min of time interval. At the initial stage, there was no characteristic plasmon resonance peak. The surface plasmon resonance band starts to occur at 420 nm [29,30] at the

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infinite time which indicates the formation of spherical silver nanoparticles (Fig. 1b). Jain et al. reported the similar results for the synthesis of silver nanoparticles [31]. 2.2. FTIR spectral analysis

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FTIR measurements were used to identify the potential functional groups of the biomolecules present in the apple fruit extract. FTIR spectrum shows absorption bands at 3498,

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2925, 1624, 1384, 1047, 816, 774 and 582 cm-1 (Fig. 2). The bands at 3498 cm-1 and 2925 cm-1 correspond to O-H stretching vibration and aldehydic C-H stretching vibration respectively. The band at 1624 cm-1 could be attributed to C=C aromatic vibrations [32]. The band at 1047 cm-1 was assigned for C-N stretching vibration of aliphatic amine [33]. The band at 816 and 582 cm-1

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assigned for the presence of alkyl halides. The spectral analysis indicate that –OH group present in the apple extract is involved in the reduction of Ag+ to Ag0 through the oxidation of alcohol to aldehyde group. The reaction between AgNO3 and alcoholic groups present in the apple

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extract can be represented as,

2Ag+NO3- + ROH → 2Ag0 + RO + 2HNO3

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FTIR analysis confirmed that the carbonyl group of amino acids and proteins has the stronger ability to bind with silver nanoparticles and could form a layer on the surface of silver nanoparticles. Hence, the surface capped biomolecules prevent agglomeration and thereby stabilize the silver nanoparticles. This suggests that the biological molecules could act as both reducing and stabilizing agent for silver nanoparticles [34, 35].

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2.3. X-ray diffraction analysis The XRD pattern of silver nanoparticles is given in Fig. 3. The diffracted intensities were recorded from 10° to 70°. Three distinct diffraction peaks at 38.1°, 44.3° and 64.5°

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corresponding to the (111), (200) and (220) Bragg reflections of face centered cubic (fcc) crystal structure of Ag are observed (JCPDS No-65-2871). The other peaks are due to the presence of carbon, which comes from the biomaterial. The peak at (111) plane is highly intense and the

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peaks at (200), (220) planes are less intense. The interplanar spacing (dcalculated) values are 2.359, 2.043 and 1.445 Å for (111), (200) and (220) planes respectively. The dcalculated values from XRD

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pattern of silver nanoparticles are matched with standard silver values [34]. The crystallite size of silver nanoparticles is calculated as ~15 nm from the full–width at half maximum (FWHM) of the high intense diffraction peak using Scherrer’s formula, D = 0.9λ/ β cosθ

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in which, D is average crystallite domain size perpendicular to the reflecting planes, λ is X-ray wavelength, β is FWHM of diffraction peak and θ is diffraction angle. 2.4. Morphological analysis

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Figure 4a clearly shows the FE-SEM image of silver nanoparticles synthesized from apple fruit extract. It revealed that the synthesized nanoparticles are spherical in shape and also

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they are well dispersed without aggregation. The average diameter of silver nanoparticles synthesized is found to be in the range of 10-40 nm. Figure 4b clearly shows the EDAX spectrum of the silver nanoparticles reduced with apple extract. The highly intense peak shown the presence of silver nanoparticles and the other small peaks indicated the presence of organic materials [36]. The morphology of the silver nanoparticles was observed from high resolution TEM image at 20 nm. All the nanoparticles are uniform and well dispersed without any larger

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aggregations. Most of the nanoparticles are nearly spherical in shape (Fig. 5a). The uniform and clear lattice fringes in selected area electron diffraction (SAED) pattern (Fig. 5b) confirmed the

2.5. Antimicrobial and antifungal activities of silver nanoparticles 2.5.1. Microorganisms and culture medium

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crystallinity and face centered cubic structure of the silver nanoparticles.

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All the microorganisms were obtained from gene bank Chandigarh and used to test the antimicrobial activity of the silver nanoparticles synthesized from apple fruit extract. They were

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incubated at 37 ˚C for 24 h in Nutrient broth and C. albicans in Sabouraud dextrose broth at 37 ˚C for 48 h. The culture suspensions were prepared and adjusted by comparing against 0.4–0.5 Mc Farland turbidity standard tubes. The silver nanoparticles were dissolved in dimethyl sulfoxide (DMSO) and sterilized by filtration through a 0.22 m membrane filter. Each sample

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was filled into the wells of agar plates directly. The plates were swabbed with the bacteria culture and they were incubated for 24 h at 37 ºC. The fugues were incubated for 48 h at 37 ºC. The inhibition zones were evaluated at the end of the incubation period. To test the inhibitory

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activity of DMSO, the inhibited zones were compared with the reference discs. 2.5.2. Antibacterial and antifungal activity

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Antibacterial activities of silver nanoparticles were tested by the well diffusion method using nutrient agar against bacteria such as Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 25175), Citrobacter koseri (ATCC BAA-895), Pseudomonas aeruginos (ATCC 10145) and the antifungal activities of the silver nanoparticles were tested by using sabouraud dextrose agar against human pathogenic fungus Candida albicans (ATCC 90028). The radial growth of the colony was recorded on completion of the incubation and the mean diameter at a

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concentration of 10 mg/ml was recorded. The highest antimicrobial activity was observed against Pseudomonas aeroginosa (27 mm), followed by Staphylococcus aureus (22 mm), Bacillus cereus (20 mm), and the least was noticed against Citrobacter kosseri (15 mm). Silver has been

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used for its well known antimicrobial properties since Roman time, however the advances in generating Ag nanoparticles have made possible a revival of the use of silver as a powerful bactericide.

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2.5.3. Determination of minimum inhibitory concentration (MIC) using Resazurin

2.5.3.1. Preparation of Resazurin solution

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Microtitre Assay (REMA)

To determine the MIC of the silver nanoparticles, Isosensitest medium was used to carry out the test and it showed similar results for most of the tested bacterial strains. A final concentration of 5 × 105 cfu/mL was adopted to ensure a uniform number of bacteria. The

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resazurin solution was prepared by dissolving 270 mg in 40 mL of sterile distilled water. A vortex mixer was used to ensure that it was a well-dissolved and homogenous solution. The test was carried out in a sterile 96 well plates. The test material (100 µL) in 10% DMSO or sterile

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water was pipetted out into the first row of the plate. 50 µL of nutrient broth or normal saline was

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added to other wells. The multichannel pipette was used to perform the continuous dilutions and tips were discarded after use. 10 µL of Resazurin indicator solution and 30 µL of 3. 3 × strength isosensitised broths were added to each well. To achieve a concentration of 5 × 105 cfu/mL, the bacterial suspension (5 × 106 cfu/mL) was added to each well at the end. A set of control was followed in each plate. One column with all solutions and the culture (except the test sample as the positive control), second column with all solutions with the DMSO (except the test compound) and third column with all solutions (except the bacterial solution). The triplicate

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plates were placed in an incubator for 18–24 h at 37 °C. Any colour change from purple to pink or colourless was recorded as positive. The MIC value was taken from the lowest concentration.

2.5.3.2. Minimum inhibitory concentration of silver nanoparticles

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The average of three values has shown the MIC for the test material and bacterial strain.

The MIC study was conducted to investigate the antimicrobial activity of silver

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nanoparticles against bacterial strains such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli Citrobacter koseri and Bacillus cereus, and against the fungus Candida

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albicans. The colour change from purple to pink or colorless was recorded as positive (Fig. 6a and 6b).The MIC value was taken from the lowest concentration and the average of the three values was calculated. Figure 7 compared the MIC of silver nanoparticles against various microorganisms [37, 38]. The silver nanoparticle synthesized using apple extract showed higher activity against Staphylococcus aureus (ATCC 25923) (MIC: 0.0781 mg/ml), followed by

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Pseudomonas aeruginosa (ATCC 10145) (MIC: 0.3125 mg/ml), Bacillus cereus (ATCC 11778) and Candida albicans (ATCC 10231) (MIC: 1.25 mg/ml). Very low activity was found against

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Citrobacter koseri (ATCC BAA-895) and Escherichia coli (ATCC 25922) (MIC: 2.5 mg/ml). 2.5.3.3. Mechanism of antibacterial activity of silver nanoparticle on bacterial cells

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The size of silver nanoparticles is about 1-20 nm. They easily entered into bacterial cell (size is 100-1000 nm) and then combined with thiol, hydroxyl, and carboxyl group in the cell and deactivates the function by releasing the silver ion. Silver nanoparticles combined with respiratory enzyme, protease enzyme and DNAs of bacteria to cause suffocation, indigestion and inhibition of cell replication respectively. Bacterial cell functions were disturbed by these silver nanoparticles which damaged the cell and lead to the death of bacterial cell (Scheme 1).

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2.6. Cytotoxicity of silver nanoparticles against MCF-7 breast cancer cells

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The antiproliferative effect was assessed by MTT (3-(4, 5- dimethylthiazol-2yl)-2, 5diphenyltetrazolium bromide) method.

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2.6.1. Principle

Coloured formazan dye by the cleavage of tetrazolium salts corresponding to

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mitochondrial dehydrogenase activity of cells and hence, the conversion occurs only in viable cells. The formazan crystals are not water soluble in nature, with the addition of solubilising solution they form a colored complex which can be read at 570 nm in an ELISA reader. a) MTT (0.5 mg/ml), Five mg of MTT was dissolved in 10 ml of serum free DMEM medium.

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b) Solubilizing solution (20% sodium dodecyl sulfate (SDS) in 50% dimethylformamide (DMF)).

c) Five ml of DMF was made up to 10 ml with distilled water and 2 g of SDS were added

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2.6.2. Procedure

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and mixed well.

The direct-dose response relationship of the sample was shown by cytotoxicity

analysis. Silver nanoparticles were able to inhibit the cell line’s growth [39]. The Dulbecco’s modified eagle medium (DMEM) contains 10% Phosphate – buffered saline (PBS). The cancer cells (5000 cells/ml) were plated in 96 well plates with DMEM and incubated at 37 ºC for 24 h under 5% CO2, 95% O2. After that the DMEM medium was removed from the cell culture plates and the cells were washed with PBS. Then serum free medium was added and kept for 1 h in the

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incubator. It was removed from both control and sample plates. 100 µg/100 µl of silver nanoparticles containing medium was added to the sample plates and the control cultures were treated with DMSO. The maximum concentration of DMSO added to the medium in this study

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was 0.01%. The MTT solution (100 µl of 0.5 mg/ml) was added to each well and the cultures were incubated for 4 h. After incubation, 100 µl of 20% SDS in 50% DMF was added and the formed crystals were dissolved gently be pipetting 2 to 3 times. The absorbance at 570 nm for

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each well was measured by a micro plate reader. Figure 8(a)-(c) shows the cytotoxicity of silver nanoparticles at various time intervals. The cell viability of the silver nanoparticles at different

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incubation time was depicted by Figure 9. The calculated IC50 value of this experiment is 10 µg/ml concentrations. The calculation for the growth inhibition rate was given below. A

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of treated cells

Percentage of growth inhibition = ------------------------------------ × 100 A of control cells 570

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3. Conclusion

In this study, we have reported successful synthesis of silver nanoparticles using the extract of apple which played a crucial role in protecting our environment as green. UV-Vis

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spectroscopy reveals the surface plasmon resonance property, while FE-SEM, HRTEM and

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EDAX images reveal the nanoscale size of the prepared samples. Most of the studies have demonstrated and proved the efficacy of silver nanoparticles as antimicrobial agent. The polyphenol and flavonoid content of apple extract play a vital role in various biological activities. The present study showed significant cytotoxic effects exerted by biologically synthesized silver nanoparticles against MCF – 7 breast cancer cells. 4. Experimental 4.1. Synthesis of silver nanoparticles

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An Apple (Malus domestica) was washed in distilled water, cut into fine pieces, crushed and filtered through Whatman No.1 filter paper. The filtrate was centrifuged and immediately used for further experiment. 50 ml of 0.01 M silver nitrate was added into 25 ml of aqueous

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extract of apple for the synthesis of silver nanoparticles. The reaction mixture was shaken well and allowed to settle at room temperature. The color change indicates the formation of silver nanoparticles. Due to the higher reactivity, the silver nanoparticles rapidly undergo

nanoparticles.

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4.2. Characterization of silver nanoparticles

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agglomeration. Hence, the lyophilisation process was used to obtain the crystalline silver

The bioreduction of silver ions was monitored by recording the UV-Vis spectra at different time intervals from 200 to 800 nm to confirm the formation of silver nanoparticles. It has been done by using a Perkin-Elmer UV-Visible spectrophotometer of model 1800. The X-ray

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generator of model 3000 from Rich Siefert, Germany was used with Cu-Kα1 radiation (λ = 1.54056 Å) to determine the phase and crystal structure of silver nanoparticles. Bruker FTIR spectrophotometer was used for analyzing the functional groups present in silver nanoparticles.

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The morphological analyses of the prepared samples were analyzed by FE-SEM, HITACHIU6600 model. HRTEM, SAED and EDAX measurements were performed on a FEI-TECNAI-30

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model instrument operated at an accelerating voltage at 300 kV. For TEM analysis, samples were prepared by coating a drop of silver nanoparticles dispersion on to carbon-coated copper TEM grids.

Acknowledgment

We gratefully acknowledge National Centre for Nanoscience and Nanotechnology (NCNSNT), University of Madras for FESEM, HRTEM and EDAX analyses.

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Figure captions Fig. 1a: (a) Pure apple extract (b) After the addition of AgNO3 to apple extract

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Fig. 1b: UV- Vis absorption spectrum of silver nanoparticles synthesized from apple extract, I – Pure apple extract, Immediately after the addition of AgNO3, II - 1 h, III – 2 h IV – 3 h, V– Infinite time Fig. 2: FTIR spectrum recorded by making a KBr pellet with synthesized silver nanoparticles Fig. 3: XRD pattern of silver nanoparticles

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Fig. 4b: EDAX spectrum of silver nanoparticles

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Fig. 4a: FE-SEM micrograph of silver nanoparticles

Fig. 5a: HRTEM micrograph of silver nanoparticles at 20nm. Fig. 5b: SAED pattern of silver nanoparticles

Fig. 6a: MIC results of silver nanoparticles synthesized using apple extract against Staphylococcus aureus (SA) (ATCC 25923), Escherichia coli (EC) (ATCC25922), Bacilus cereus (BC) (ATCC 25922).

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Fig. 6b: MIC results of silver nanoparticles synthesized using apple extract against Candida albicans (CA) (ATCC 10231), Citrobacter koseri (CB) (ATCC BAA-895), Pseudomonas aeruginosa (PS) (ATCC10145). Fig. 7: MIC of silver nanoparticles synthesized using apple extract

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Fig. 8: Cytotoxicity of silver nanoparticles at (a) 24 h (b) 48 h (c) maximum

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Fig. 9: Cell viability of the silver nanoparticles at various incubation time.

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Scheme-1: Mechanism of antibacterial activity of silver nanoparticle on bacterial cells. (A) Bacterial cells take up Ag NPs. (B) Ag NPs interacts with respiratory and protease enzyme.

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(C) Destruction of bacterial cells.

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Cytotoxicity and antimicrobial activities of green synthesized silver nanoparticles.

Bio-inspired silver nanoparticles are synthesized using Malus domestica (apple) extract. Polyphenols present in the apple extract act as a reducing an...
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