Accepted Manuscript Biological activities of green silver nanoparticles synthesized with Acorous calamus rhizome extract Jayachandra Reddy N, Rani M, Arvind Kumar Gupta, S. Sudha Rani PII:

S0223-5234(14)00751-X

DOI:

10.1016/j.ejmech.2014.08.024

Reference:

EJMECH 7257

To appear in:

European Journal of Medicinal Chemistry

Received Date: 13 March 2014 Revised Date:

4 August 2014

Accepted Date: 6 August 2014

Please cite this article as: J.R. N, R. M, A.K. Gupta, S Sudha Rani, Biological activities of green silver nanoparticles synthesized with Acorous calamus rhizome extract, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.08.024. 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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Biological activities of green silver nanoparticles synthesized with Acorous calamus rhizome extract

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N. Jayachandra Reddy, M. Rani, Gupta Arvind Kumar, S. Sudha Rani* Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry

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University, Pondicherry, India.

Corresponding author:




Dr. S. Sudha Rani Assistant Professor

Pondicherry University

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Department of Biochemistry and Molecular Biology

Pondicherry 605 014, India.

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Phone: +91 9443768726

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E-mail: [email protected], [email protected]

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ABSTRACT Nanomedicine utilize biocompatible nanomaterials for diagnostic and therapeutic purposes. This study reports the synthesis of silver nanoparticles using aqueous rhizome extract of Acorus

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calamus (ACRE) and evaluation of antioxidant, antibacterial as well as anticancer effects of synthesized A. calamus silver nanoparticles (ACAgNPs). The formation of ACAgNPs was confirmed by UV-visible spectroscopy and their average size was found to be 31.83 nm by DLS particle size analyzer. Scanning electron micrograph (SEM) revealed spherical shape of ACAgNPs and energy dispersive spectroscopy (EDX) data showed the presence of metallic

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silver. Fourier transform infrared spectroscopy (FTIR) analysis indicated the presence of phenol/alcohol, aromatic amine and carbonyl groups in ACRE that were involved in reduction

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and capping of nanoparticles. ACRE and ACAgNPs exhibited substantial free radical quenching ability in various in vitro antioxidant assays performed in this study. ACAgNPs also displayed appreciable antibacterial activity against three different pathogenic bacteria and the growth kinetic study with E. coli designated the inhibition of bacterial growth at the log phase. The cytotoxic effect of ACAgNPs was assessed by MTT assay in HeLa and A549 cells. The IC50 value of ACAgNPs respectively after 24 and 48 h was found to be 92.48 and 69.44µg/ml in

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HeLa cells and in A549 cells it was 53.2 and 32.1µg/ml. Apoptotic cell death in ACAgNPs treated cells was indicated by acridine orange/ethidium bromide (AO/EB) and annexinV-Cy3 staining techniques. Staining with propidium iodide (PI) and 4', 6-diamidino-2-phenylindole, dilactate (DAPI) also confirmed nuclear changes such as condensation and fragmentation.

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Further, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed distribution of ACAgNPs treated cells in the late apoptotic stage. These findings emphasize that

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such biocompatible green nanoparticles with multifaceted biological activities may find their applications in the field of nanomedicine. KEYWORDS: Acorus calamus rhizome; Silver nanoparticles; Antibacterial; Cytotoxic effect; DNA fragmentation; Apoptosis.

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1. Introduction Nanomedicine is a rapidly developing and promising field that makes best use of inert

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metals like silver, gold, and platinum to synthesize metallic nanoparticles with high therapeutic potential for various biomedical applications. Silver with its potent antimicrobial activity has been used in the synthesis of silver nanoparticles which finds extensive use in the preparation of

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creams, topical ointments and medical implants [1-2]. Though the synthesis of silver nanoparticles has been carried out by various methods such those based on reduction in solution

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[3], chemical and photochemical reactions [4], decomposition of silver compounds by thermal method [5] and microwave assisted process [6], they involve the use of noxious chemicals. The green synthesis methods [7-10] using plant extracts have been shown to be more advantageous owing to their simple methodology and eco-friendlily nature [11]. Green synthesis of silver

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nanoparticles using various medicinal plants including, Besella alba, Helianthus annus, Saccharum officinarum, Oryza sativa, Sorghum bicolour, Zea Mays [12] tea leaf extract [13], Crataegus douglasii fruit [14] have been reported. Such green synthesized silver nanoparticles

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from Dillenia indica [15], Origanum vulgare [16], Morinda pubescens [17], Podophyllum hexandrum [18] have also been shown to exhibit in vitro antioxidant and anticancer activities.

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With these evidences, this study was designed to synthesize silver nanoparticles by using

aqueous Acorus calamus L (sweet flag) rhizome extract (ACRE) and assess their antioxidant, antibacterial and anticancer potential in vitro. A. calamus is considered as a highly valid herb in Ayurveda, the Indian traditional system of medicine. Its rhizome has been used to treat a number of ailments including chronic diarrhea, dysentery, liver troubles, rheumatism, sinusitis, eczema, epilepsy, mental ailments, glandular and abdominal tumors, bronchial catarrh and intermittent

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fever. The rhizome acts as a rejuvenator for the nervous system [19] and is also widely employed in the modern herbal medicinal preparations owing to its sedative, laxative, diuretic and carminative properties [20]. The rhizome has a rich profile of bioactive compounds including

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alkaloids, flavonoids, triterpenes and phenolic compounds that act as natural antioxidants [2122].

Here we present data on the green synthesis of silver nanoparticles (ACAgNPs) from

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ACRE, their physical characterization and their antioxidant, antibacterial and cytotoxic effects in vitro. The physicochemical properties of ACAgNPs were investigated by UV – visible SEM-EDX

(scanning

electron

microscope -

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spectroscopy,

energy

dispersive

X-

ray spectroscopy), FTIR (fourier transform infrared spectroscopy), TGA-DSC (thermal gravimetric analysis- differential scanning calorimetry) and DLS (dynamic light scattering) particle size analyzer. The ACAgNPs were assessed for their antioxidant activities by various in

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vitro tests including ferric ion reducing, DPPH (2, 2-diphenyl-1-picrylhydrazyl), superoxide and hydrogen peroxide radical scavenging assays. The antibacterial activity of ACAgNPs was tested against three different bacterial strains. The in vitro cytotoxic effect of ACAgNPs was evaluated

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in human cervical cancer (HeLa) and human lung adenocarcinoma (A549) cell lines. Apoptotic cell death in ACAgNPs treated cells was examined by AO/EB (acridine orange/ethidium

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bromide), annexin V, propidium iodide (PI), DAPI (4, 6-diamidino-2-phenylindole, dihydrochloride) staining techniques and by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay.

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2. Materials and Methods Silver nitrate (AgNO3) and iron III chloride (FeCl3) were purchased from Merck, India. MTT (3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), DMEM (dulbecco's modified

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eagle's medium), potassium bromide (KBr) and annexinV-Cy3 were purchased from SigmaAldrich, Bangalore, India. Folin-ciocalteau, rutin, querectin, DPPH, nitro blue tetrazolium (NBT), NADH (nicotinamide adenine dinucleotide), acridine orange (AO), ethidium bromide

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(EB), propidium iodide (PI) and DAPI (4, 6-diamidino-2-phenylindole, dihydrochloride) were purchased from Hi-Media. The TUNEL kit (APO BrdU TUNEL and Propidium Iodide

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(PI/RNase)) assay kit) was supplied by Invitrogen detection technologies, UK. A. calamus rhizome was purchased from Pondicherry local market, India. Double distilled water was used for all the experiments. All other chemicals used in this study were of analytical grade. 2.1. Preparation of the Extract

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ACRE was prepared by mixing 1g of finely powdered A. calamus rhizome with 100 ml of double distilled water and incubating at room temperature for 20 h. The extract was then heated at 60 oC for 10 min and filtered with Whatman No. 1 filter paper. The ACRE thus obtained was

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stored at 4 °C in refrigerator until further use. 2.2. Phytochemical screening

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Qualitative and quantitative phytochemical analysis of ACRE was performed by standard procedures [23].

2.3. Synthesis of ACAgNPs ACAgNPs were synthesized by mixing aqueous AgNO3 solution (1 mM) and ACRE in the ratio of 5:1 and incubating the mixture at room temperature for 24 h. Following incubation, the ACAgNPs formed were collected by centrifugation at 18,000 rpm. The collected pellet was

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washed three times with double distilled water, transferred to a petriplate and dried at room temperature.

2.4.1. UV-visible spectral analysis

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2.4. Characterization

The synthesized ACAgNPs were analyzed by UV-visible spectrophotometer (UV-1700 Shimadzu) in 300-700 nm range at regular 4 h intervals up to 24 h.

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2.4.2. SEM/EDX analysis

In this experiment a thin film of dry ACAgNPs powder was prepared on a copper coated carbon

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grid, dried under mercury lamp for 5 min and analyzed by SEM/EDX analyzer (Hitachi S-4500) for morphology and elemental composition. 2.4.3. FTIR spectral analysis

Dried powders of ACRE and ACAgNPs were subjected to FTIR analysis in a FTIR spectrometer

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(Thermo Nicolet Nexus 670 equipped with KBr optics and a DTGS detector) to identify the biomolecules in ACRE and those associated with ACAgNPs. The equipment was operated with a resolution of 4 cm-1 and scanning range of 500 – 4000 cm-1.

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2.4.4. Particle size analysis

The synthesized ACAgNPs in solution were analyzed by DLS particle size analyzer [ZETA

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Seizers Nanoseries (Malvern Instruments Nano ZS)] to determine the size distribution of nanoparticles.

2.4.5. TGA-DSC analysis

Dry powder of ACAgNPs was subjected to increasing temperatures in the range of 0 to 800 oC in a SDT Q600 and Q20 DSC thermal gravimetric analyzer to assess the thermal stability and associated weight loss of nanoparticles.

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2.5. In vitro antioxidant assays 2.5.1. DPPH free radical scavenging assay DPPH radical scavenging assay for ACRE and ACAgNPs was performed as described by Chang

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et.al [24]. In this assay, different concentrations of ACRE or ACAgNPs (100 µg to 3 mg/ml) were separately mixed with 3 ml of 0.1 mM DPPH and incubated in dark for 15 min. The absorbance of samples was then measured in a UV-visible spectrophotometer at 517 nm using

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methanol as blank and DPPH as control. Rutin was used as a positive control in this test. The percentage of inhibition was calculated by the following formula:

% of inhibition =

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Absorbance control-Absorbance test

* 100

Absorbance control 2.5.2. Reducing power assay

The total ferric ion reducing power of ACRE and ACAgNPs was determined according to the

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method described by Makari et al [25]. In this method, 0.5 ml of ACRE or ACAgNPs with different concentrations (100-500 µg/ml) in different tubes were mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1 % potassium ferricyanide. The mixture was incubated

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for 20 min at 50 oC followed by the addition of 2.5 ml of 10 % trichloroacetic acid (TCA) and

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then centrifuged at 3000 rpm for 10 min. To 2.5 ml of supernatant, 2.5 ml of double distilled water and 0.5 ml of FeCl3 (0.01%) solution were added and the absorbance was measured at 700 nm against phosphate buffer blank. Quercetin was used as positive control in this test. Increase in absorbance is directly proportional to the reducing power of the samples. 2.5.3. Superoxide radical scavenging assay Superoxide anion radical scavenging activity of ACRE and ACAgNPs was determined by following the method described by Nishimiki et al [26]. In this method the superoxide radicals 7

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generated from phenazine methosulfate-nicotinamide adeninedinucleotide (PMS/NADH) nonenzymatic system reacts with NBT and produce purple color which is measured in a spectrophotometer. In this assay, 1 ml of the reaction mixture containing different concentrations

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(100 µg to 3 mg/ml) of ACAgNPs or ACRE along with phosphate buffer (100 mM, pH 7.4), NADH (468 µM), NBT (156 µM), PMS (60 µM) was incubated at room temperature for 5 min. The absorbance of the reaction mixture was measured at 560 nm against appropriate blank and

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rutin was used as a positive control. 2.5.4. Hydrogen peroxide scavenging assay

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Hydrogen peroxide radical scavenging activity of ACAgNPs and ACRE was examined by the method of Avani Patel et al [27]. In this method, 300 µl of ACAgNPs or ACRE at different concentrations (10-80 µg/ml) were added to 600 µl of 100 mM H2O2 in phosphate buffer and the absorbance was measured at 230 nm with a separate reagent blank for each concentration. Rutin

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was used as a standard and the activity was expressed as percentage inhibition with the following formula:

% of inhibition = [A0-A1/A0]*100

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Where A0= absorbance of control and A1= absorbance of sample or standard 2.6. Antibacterial activity

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2.6.1. Disc diffusion method

The antibacterial activity of ACAgNPs and ACRE was assessed by the Kirby-Bauer disc diffusion technique against three different bacterial strains namely Bacillus subtilis, Bacillus cereus and Staphylococcus aureus. Sterile Whatman filter paper discs of 5 mm diameter were loaded with 20 µg of the sample and placed on nutrient agar plates inoculated with bacterial cultures. The plates were then incubated at 37 ºC for 24 h and the zone of inhibition was

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measured by using antibiotic zone scale (Hi media) [28]. Standard streptomycin antibiotic discs were used as positive control in this test. 2.6.2. Growth kinetic studies

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The effect of ACAgNPs on different phases of bacterial growth was evaluated by adding ACAgNPs (40µg/ml) to overnight culture of E.coli in a 500 ml culture flask and kept in a incubator shaker at 27 oC. The absorbance of the bacterial culture was measured at 600 nm at 1 h

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intervals for 24 h. The growth curve of bacteria indicating the four phases of growth namely lag, log, stationary and decline phase was constructed by plotting absorbance versus time.

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2.7. Cell culture

HeLa and A549 cells were cultured in DMEM containing L-glutamine and 4.5 g/L glucose along with 10 % fetal bovine serum (FBS), penicillin G (100 units/ml) and streptomycin sulfate (0.1 mg/ml) in a humidified atmosphere of a 5 % CO2 at 37 oC.

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2.7.1. MTT assay

The cytotoxic effect of ACAgNPs in HeLa and A549 cells was evaluated by MTT assay by following the standard protocol [29]. The cultured cells were trypsinized and the cell count was

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adjusted to 1 lakh/ml with DMEM containing 10 % FBS. Cells were seeded at 104cells/well in 96 well plates and incubated for 24 h. The cells were then treated with ACAgNPs at different

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concentrations (25, 50, 75, 100, 125, 150, 175 and 200 µg/ml) and incubated for 24 and 48 h at 37 oC in 5% CO2. MTT (5 mg/ml in Phosphate buffered saline (PBS)) was then added and the cells were incubated again for 4 h at 37 oC in 5% CO2. The formazan crystals formed by mitochondrial reduction of MTT were solubilized in DMSO (dimethyl sulphoxide) and the absorbance was read at 570 nm with reference wavelength at 650 nm. The percentage of

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inhibition caused by ACAgNPs after 24 and 48 h of incubation was calculated by using the following formula and expressed as the IC50 value. Absorption control – absorption test * 100

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% of inhibition = Absorption control 2.7.2. AO/EB staining

The potential of ACAgNPs to induce apoptosis in HeLa and A549 cells was determined by the

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AO/EB double staining technique. HeLa and A549 cells were seeded in six well plates

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(105cells/well) and were treated with ACAgNPs at appropriate IC50 concentrations. The plates were incubated for 24 and 48 h at 37 OC with 5% CO2. After incubation AO/EB staining solution was added and the cells were examined under Nikon Eclipse Ti Japan, fluorescence microscope for identifying apoptotic cell death. 2.7.3. AnnexinV-Cy3 staining

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AnnexinV-Cy3 staining technique was used to confirm apoptotic cell death induced by ACAgNPs in HeLa and A549 cells. The annexinV-Cy3 staining was performed according to the instructions given by the suppliers without any modifications. HeLa and A549 cells

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(105cells/well) were separately seeded in six well plates and treated with appropriate IC50

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concentrations of ACAgNPs and incubated for 24 h at 37 OC with 5% CO2. After incubation, the cells were treated with annexineV-Cy3 stain and examined under Nikon Eclipse Ti Japan, fluorescence microscope. 2.7.4. PI staining PI

staining

technique

was

used

to

assess

the

morphological

changes

such

as

condensed/fragmented nuclei in cells undergoing apoptosis. HeLa and A549 cells were separately seeded in six well plates at 105cells/well and treated with ACAgNPs at the appropriate

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IC50 concentrations and incubated for 24 and 48 h at 37 OC with 5% CO2. The cells were then washed with PBS, fixed with 4% paraformaldehyde followed by 70% ethanol. Finally the cells were stained with 50 µg/ml of PI, incubated for 20 min and were examined under Nikon Eclipse

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Ti Japan, fluorescence microscope. 2.7.5. DAPI staining

DAPI staining is used to evaluate apoptosis associated morphological changes like nuclear

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fragmentation or condensation. In this assay, HeLa and A549 cells at 5x104cells/well were seeded separately in the six well plates and treated with appropriate IC50 value of ACAgNPs and

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incubated for 24 and 48 h at 37 OC with 5% CO2. After incubation cells were washed with PBS and fixed with 4% paraformaldehyde and again with 70% ethanol. Cells were stained with DAPI at the concentration of 1 mg/ml and incubated for 20 min in dark. Stained images were recorded with Nikon Eclipse Ti Japan, fluorescence microscope.

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2.7.6. TUNEL assay

DNA damage in cells undergoing apoptotic cell death was analyzed by TUNEL assay. The kit had all the necessary materials to label DNA strand breaks and for the detection of apoptotic cell

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death in cells and the assay was performed in a flow cytometer by following the protocol provided by the suppliers without any modifications. In this assay, about 1×106 HeLa and A549

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cells were treated separately with ACAgNPs at appropriate IC50 concentrations and incubated for 24 h at 37 OC with 5% CO2. After incubation, the cells were washed with PBS and fixed with ice cold 1% (w/v) paraformaldehyde in PBS followed by 70% ethanol. The cells were then centrifuged, washed with PBS and treated with DNA labeling solution provided in the kit and incubated at 4 oC for 1h. The cells were then treated with antibody solution, incubated for 30 min

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and then PI/RNase solution was added. After 30 min incubation at room temperature in dark the cells were analyzed in guava easy Cyt 8HT Flow Cytometer.

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3. Results and Discussion

The qualitative phytochemical analysis of ACRE showed the presence of vital secondary metabolites such as alkaloids, flavonoids, phenols, reducing sugars, terpenoids, anthraquinones,

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cardiac glycosides, amino acids, xantho proteins, oils, phlobatannins. Quantitatively ACRE

equivalent (RE)/g of alkaloids.

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contained 25 mg gallic acid equivalent (GAE)/g of phenolic compounds and 9 mg rutin

The formation of ACAgNPs was indicated by the change in color of the reaction mixture from colorless to yellowish brown and this color change was due to surface plasma resonance and reduction of silver ions by ACRE. Preliminary information on the shape and size distribution

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of nanoparticles may be obtained from UV- visible spectral data [30]. In this study the UVvisible spectrum of ACAgNPs was recorded at 4 h intervals up to 24 h which showed a single broad peak with the λ max at 421 nm. The broadening of the spectrum between 350 nm to 480 nm

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suggested the poly dispersed nature of ACAgNPs (Fig.1). The increase in the intensity of absorbance at 421 nm observed at different time intervals indicated the potency of reduction as a

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function of time and the best reaction time was found to be between 4 to 8 h (Fig.1). Similar UVvisible spectral data has been observed with silver nanoparticles synthesized from Terminalia chebula fruit extract exhibiting a broad peak with λ max at 452 nm [31].

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Fig. 1

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SEM image of ACAgNPs (Fig. 2A) indicated that the nanoparticles were spherical in shape with mild agglomeration. This finding can be correlated with the single SPR spectrum obtained for ACAgNPs in the UV- visible spectrophotometric analysis. Fig. 2B represents the

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EDX spectra of ACAgNPs which gave a strong signal in the region of silver. Our finding can be correlated with the previous reports on the formation of spherical shaped silver nanoparticles

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from the leaf extracts of Ceratonia siliqua [32] and Euphorbia hirta [33] that were confirmed by SEM analysis. Fig. 2

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FTIR spectrum of ACRE and ACAgNPs was recorded to determine the presence of various functional groups. The spectrum obtained for ACRE showed bands at 3378, 2919, 1660,

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1400, 1202, 905 cm-1 and for ACAgNPs the bands were observed at 3324, 3234, 2919, 1624, 1373, 1148, 1022 cm-1 (Fig. 3). The band vibrations observed at 3378, 2919 in ACRE spectrum indicated the O-H stretching of the phenol group and alkyl C-H group respectively. The bands at 1660, 1400, 1202 cm-1 represented the carbonyl stretching of -C=O, aromatic stretching of -C-N

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and –C-O or –C-O-C respectively which corroborate earlier report [34]. The change in the stretching vibrations observed with ACAgNPs at 3324 and 3234 cm-1 indicated the phenolic group with a shift from 3378 cm-1 of ACRE. Similarly, bending of carbonyl group that was

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observed at 1624 cm-1 with ACAgNPs was shifted from 1660 cm-1 observed with ACRE. From the data obtained in the FTIR analysis, it can be predicted that the functional groups including

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phenol/alcohol, aromatic amine and carbonyl groups from ACRE mediated the reduction, capping and stabilization of ACAgNPs. ACRE has been reported to contain various bioactive compounds including beta asarone, calamenol, alphapinene, eugenol, methyl eugenol, calamone, azuline, sugars and flavones [20-22] and this supports our FTIR results. Fig. 3

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ACAgNPs were found to be poly dispersed in nature when analyzed by DLS particle size

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analyzer and the average size of the particles was 31.83 nm (Fig.4A)

The thermal stability of ACRE and ACAgNPs was examined by TGA and DSC analysis (Fig.4B and 4C). The TGA curves indicated the degradation of ACRE and ACAgNPs with increasing temperature. The TGA curve for ACRE exhibited sharp degradation with a total

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weight loss of 75% in the temperature range of 50-800 oC (Fig.4B). On the other hand, ACAgNPs showed gradual degradation with a total weight loss of 45% in the temperature range of 50-800 oC (Fig.4C). An initial weight loss of 6% was observed with ACAgNPs at around 70 o

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C and this may be due to loss of moisture. Further degradation of ACAgNPs that was observed

between 150 oC to 370 oC with an associated weight loss of 14.74 % may be attributed to the

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surface desorption of associated bio-organic compounds derived from ACRE. ACAgNPs did not show any further steep degradation between 400-800oC indicating the stability of silver. In the DSC curve, the ACAgNPs showed an endothermic peak at 314.63 oC. The denaturation enthalpy of ACAgNPs is closer to the single stage decomposition temperature of bio-organic compounds which occurred at 250-315 oC as shown by the TGA data. The co-ordination between DSC and TGA curves indicated that the phytocompounds from ACRE that were responsible for reduction of Ag+ to Ago in ACAgNPs could be thermally less stable. Similar findings have been reported 15

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for silver nanoparticles synthesized from Coccinia grandis leaf extract which showed similar decomposition and denaturation temperature in TGA and DSC analysis respectively [35].

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Fig.4

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The free radical quenching ability of ACAgNPs as compared to ACRE was assessed by using different in vitro techniques. As silver can exist in two oxidation states namely Ag+ and

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Ag2+, silver nanoparticles may possess the ability to quench free radicals by donating or accepting electrons based on the reaction conditions. The free radical quenching effect of ACAgNPs and ACRE was assessed by DPPH assay in which the purple colored DPPH radical gets bleached to form a corresponding yellow colored hydrazine molecule on accepting electrons

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or hydrogen ions. Fig. 5A depicts an increasing trend in the DPPH radical scavenging activity of both ACRE and ACAgNPs with increasing concentrations. ACAgNPs showed 10% higher activity than ACRE with 77% activity as compared to that of standard rutin. Our finding is

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supported by an earlier observation where silver nanoparticles prepared from Cleistanthus collinus showed enhanced DPPH radical scavenging activity as compared to plant extract [36].

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The ferric ion reducing activity of ACRE and ACAgNPs are represented as dose response

bar chart in Fig 5B. Results indicated that both ACAgNPs and ACRE exhibited similar reducing powers and the activity was found to increase with increasing concentrations. The superoxide radical scavenging effect of ACAgNPs and ACRE was assessed by the PMS- NBT reduction system. Superoxide anions impose potent damage to cells as they can readily react with DNA and proteins and this necessitate their clearance from living system. As shown in Fig 5C, the

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superoxide radical scavenging activity of ACRE and ACAgNPs displayed an increasing trend with increasing concentrations. Interestingly ACRE showed better activity than ACAgNPs which was comparable to the activity of the reference compound, rutin. Similar results were observed in

activity than the green synthesized silver nanoparticles [37].

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our earlier work where Piper longum fruit extract showed potent superoxide radical scavenging

The antioxidant potential of ACRE and ACAgNPs was further analyzed by H2O2 radical

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scavenging assay. In biological systems, formation of H2O2 can lead to the production of hydroxyl radicals that can inflict severe damage to cell membranes. In this study, ACLE was

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found to be more effective in quenching H2O2 and the percentage of inhibition was found to be comparable to that of standard antioxidant rutin while ACAgNPs exhibited moderate activity ( Fig. 5D). In an earlier work acetone and methanol extracts of Shorea roxburghii stem bark have been shown to exhibit moderate H2O2 quenching ability but lesser as compared with that of

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Fig. 5

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vitamin C [38].

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The antibacterial activity of ACRE and ACAgNPs was analyzed against Bacillus subtilis, Bacillus cereus and Staphylococcus aureus by disc diffusion method. The culture plates that

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were treated with ACAgNPs exhibited moderate antibacterial activity. The zone of inhibition caused by ACAgNPs was found to be 1.5, 1.7 and 1.6 cm respectively for Bacillus cereus, Bacillus subtilis and Staphylococcus aureus as compared to the standard antibiotic streptomycin which produced respective clearance zones of 3.4, 3.1 and 2.6 cm at the same concentration of 20µg used in this study (Table 1).

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Table-1

Bacillus subtilis

ACRE

20

Bacillus Staphylococcus cereus aureus Diameter of zone (cm) 1.2 0.8 1.0

ACAgNPs

20

1.7

1.5

Streptomycin

20

2.6

3.1

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Concentration (µg/ml)

1.6

3.2

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Test sample

ACAgNPs exhibited better antibacterial activity than ACRE (Fig.6A). In a previous

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study, similar antibacterial activity was exhibited by silver nanoparticles synthesized from Vitex negundo extract against gram negative Escherichia coli and gram positive Staphylococcus aureus [39]. In another report, papaya fruit derived silver nanoparticles have been shown to display potent antibacterial activity against Escherichia coli and Pseudomonas aeruginosa and

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the activity was comparable to that of standard antibiotics tetracycline and rifamycine [40]. Even though silver has been recognized for its antimicrobial activity for several decades, the advances in the synthesis of silver nanoparticles as powerful bactericides has been made recently [41]. The

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silver nanoparticles exhibit antibacterial activity by attaching to the bacterial cell membrane [42]. Since the bacterial plasma membrane is the site of respiratory chain components, energy

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transducing systems and for active transport of molecules and ions [43], any modification in the structure of the membrane would eventually result in inhibition of bacterial growth. The antibacterial activity of ACAgNPs with respect to its effect on various phases of

bacterial growth was analyzed with E.coli culture and the growth curve is shown in Fig.6B. ACAgNPs inhibited the bacterial growth at the log phase which is otherwise the active phase when the bacterial cells show exponential growth. Thus the growth kinetics analysis with E.coli

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which showed significant reduction in the number of viable cells confirmed the antibacterial effect of ACAgNPs.

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Fig. 6

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Anti proliferative effect of ACAgNPs was assessed in HeLa and A549 cells by MTT

assay. The cells were treated with various concentrations of ACAgNPs ranging from 25 mg L-1 to 200 mg L-1 for 24 and 48 h and the % inhibition of cell proliferation are shown in Fig.7. The IC50 values of ACAgNPs were 92.48 and 69.44 µg/ml in HeLa cells and in A549 cells the values were 53.2 and 32.1 µg/ml respectively at 24 and 48 h. These values were found well within the

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clinically acceptable concentration of 100 mg L-1 suggesting a potential anticancer effect of ACAgNPs.

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Fig. 7

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The cytotoxic potential of ACAgNPs was further analyzed by AO/EB staining for apoptotic cell death in HeLa and A549 cells. The cells were treated with appropriate IC50

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concentrations of ACAgNPs for 24 and 48 h and evaluated for apoptotic changes under fluorescent microscope. AO is a vital dye that stains both live and dead cells as it can penetrate normal cell membrane. On the other hand EB will stain only cells that have lost membrane integrity. In this study, live cells exhibited green fluorescence and those cells undergoing cell death showed orange colored apoptotic bodies formed as a result of nuclear shrinkage and blebbing (Fig. 8a). Our findings can be correlated with similar observations made with Annona squamosa green silver nanoparticles that induced apoptosis in MCF-7 cells [44]. 22

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To substantiate the ability of ACAgNPs to induce apoptosis, further analysis was carried out by using annexinV-Cy3 double staining technique in HeLa, and A549 cells. The cells were treated with appropriate IC50 concentration of ACAgNPs and after 24 h of incubation the cells

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were treated with the staining solution containing annexinV-Cy3 conjugate and 6-CFDA. At the onset of apoptosis phosphatidyl serine gets transported from inner leaflet of plasma membrane to outer leaflet and are bound by annexins in the presence of Ca2+ ions. Such cells undergoing

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apoptosis appear red due to the bound fluorescent conjugate Cy3. On the other hand, live cells appear green with 6-CFDA that penetrate live cells and get hydrolyzed by esterases into 6-CF

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that emits green fluorescence. In this study the merged images of green and red fluorescence of HeLa and A549 cells are shown in Fig. 8b.

While the control cells showed only green

fluorescence, ACAgNPs treated cells exhibited red and green fluorescence as these cells were bound by both Cy3(red) and 6-CF(green) suggesting that ACAgNPs induced apoptotic cell

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To confirm that the apoptotic cell death induced by ACAgNPs was associated with nuclear condensation and fragmentation, the cells were analyzed separately by PI and DAPI staining techniques. In this assay, HeLa and A549 cells after exposure to ACAgNPs for 24 and

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48 h at appropriate IC50 concentrations were stained with PI or DAPI and examined under fluorescence microscope. PI is an intercalating agent that binds to DNA between bases. The increased number of PI positive cells (Fig. 9A) observed in this study indicated that ACAgNPs

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induced nuclear changes in cells undergoing apoptosis. In a recent study, similar findings have been reported in HCT15 colon cancer cells treated with green synthesized Vitex negundo silver

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nanoparticles which showed apoptotic changes involving nuclear condensation by PI staining [45].

In DAPI staining technique, dsDNA is preferentially stained by DAPI which associates with A-T clusters in the minor groove of DNA. Cells treated with ACAgNPs showed changes in

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nuclear morphology with apoptotic changes including chromatin condensation, fragmentation of the nucleus while untreated cells appeared normal (Fig. 9B). Previously, silver nanoparticles synthesized from Thuja occidentalis leaf extract have been shown to induce nuclear

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condensation and fragmentation in HeLa cells [46].

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TUNEL assay was performed to confirm the cytotoxic potential of ACAgNPs in HeLa

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and A549 cells and to assess the percentage of cells in the early and late stages of apoptosis. In this flow cytometry analysis, cells undergoing apoptosis with DNA fragmentation or strand breaks are quantified by incorporation of Br-dUTP into exposed 3’-OH DNA ends and detected by fluorochrome-conjugated anti BrdU antibody. The distribution of HeLa and A549 cells in different quadrants are shown in Fig.10A and Fig. 10B respectively. The accumulation of ACAgNPs treated HeLa cells was found to be 12% in the first quadrant Q1 (early apoptosis) and 57% in the second quadrant Q2 (late apoptosis). On the other hand, the distribution of ACAgNPs

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treated A549 cells in Q1 and Q2 was 0.1 and 29% respectively and about 17% cells were found to be accumulated in Q3 suggesting possible necrosis. In an earlier study encapsulated Gelsemium sempervirens PLGA nanoparticles inducing DNA damage in A375 cells has been

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observed by TUNEL assay [47]. Our findings from the TUNEL assay along with our observations made in AO/EB, annexineV-Cy3, PI and DAPI staining techniques confirm the cytotoxic effects of ACAgNPs in cancer cells through induction of apoptosis.

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4. Conclusion Nanomedicine employs innovative approaches to develop nanoparticles with varied biological activities.

Data from this study revealed the advantages of greener method for

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synthesis of stable silver nanoparticles with biomedical applications. The synthesized ACAgNPs capped with secondary metabolites from ACRE, exhibited tremendous antioxidant, antibacterial and anticancer activities. The cytotoxic effects shown by ACAgNPs in HeLA and A549 cells

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involved apoptotic changes as confirmed by various staining techniques and flow cytometry. This simple and ecofriendly method could be extended to the preparation of other kinds of

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nanoparticles with broad range of applications in the field of material sciences as well as in medicine. Acknowledgements

The authors acknowledge the DST, Government of India, New Delhi, for financial support in the

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form of DST-FIST. The authors thank Pondicherry University DBT-IPLS (BUILDER) program for Fluorescence microscope facility, Department of Biotechnology for FACS facility and the Central Instrumentation Facility (CIF).

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1. UV-visible spectra of ACAgNPs at different time intervals. 2. (A) SEM micrograph of ACAgNPs (B) EDX analysis of ACAgNPs exhibited the strong signal

3. FTIR spectra of ACAgNPs and ACRE.

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of Ag.

4. (A) Distribution of ACAgNPs with average size of 31.86 nm (B) Thermal analysis (TGA-

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DSC) of ACRE and (C) ACAgNPs.

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5. (A) DPPH assay of ACAgNPs, ACRE and rutin (B) Reducing power of ACAgNPs, ACRE and quercetin (C) Superoxide radicals scavenging activity of ACAgNPs, ACRE and rutin (D) Hydrogen peroxide scavenging activity of ACAgNPs, ACRE and rutin. 6. (A) Images of antibacterial activity of ACAgNPs, ACRE and Streptomycin- Bacillus subtilis

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(i,iv), Bacillus cereus (ii, v), Staphylococcus aureus (iii, vi). (P= ACRE, N= ACAgNPs, iv-vi= Streptomycin) (B) Effect of ACAgNPs on different phases of E. coli growth.

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7. Cytotoxic effect of ACAgNPs against HeLa and A549 cells at 24 and 48 hrs. 8. Fluorescent microscopy image of IC50 concentration of ACAgNPs treated on HeLa and A549

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cells (20x Magnification). (A) Fluorescent microscopy imaging of AO/EB stained (i) untreated HeLa cells (ii) ACAgNPs treated HeLa cells (iii) untreated HeLa cells (iv) ACAgNPs treated HeLa cells (v) untreated A549 cells (vi) ACAgNPs treated A549 cells (vii) untreated A549 cells (viii) ACAgNPs treated A549 cells. (B) AnnexinV-cy3 staining for apoptosis at 24 hrs (i) untreated HeLa cells (ii) ACAgNPs treated HeLa cells (iii) untreated A549 cells (iv) ACAgNPs treated A549 cells.

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9. Fluorescent microscopy image of IC50 concentration of ACAgNPs treated on HeLa and A549 cells (20x Magnification). (A) Propidium iodide staining of HeLa and A549 cells in both control and treated with ACAgNPs (i) untreated HeLa cells (ii) ACAgNPs treated HeLa cells (iii)

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untreated HeLa cells (iv) ACAgNPs treated HeLa cells (v) untreated A549 cells (vi) ACAgNPs treated A549 cells (vii) untreated A549 cells (viii) ACAgNPs treated A549 cells. (B) DAPI staining of HeLa and A549 cells in both control and treated with ACAgNPs (i) untreated HeLa

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ACAgNPs treated A549 cells.

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cells (v) untreated A549 cells (vi) ACAgNPs treated A549 cells (vii) untreated A549 cells (viii)

10. DNA fragmentation mediated apoptosis in HeLa and A549 cells by TUNEL assay at 24 hrs (A) HeLa cells (B) A549 cells.

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Table 1

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1. Zone of inhibition of different bacterial strains with ACAgNPs and ACRE.

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Highlights Acorus calamus rhizome extract was used to synthesize silver nanoparticles (ACAgNPs).

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Spherical ACAgNPs with average size 31.86 nm.

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ACAgNPs exhibited good antibacterial and antioxidant activity.

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Induction of apoptosis by ACAgNPs in HeLa and A549 cells was confirmed by different.

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staining techniques and TUNEL assay.

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