Accepted Manuscript Biocompatibility and antibacterial activity of the Adathoda vasica Linn extract mediated silver nanoparticles M. Latha, M. Priyanka, P. Rajasekar, R. Manikandan, Dr. N.M. Prabhu, Assistant Professor PII:
S0882-4010(15)30170-4
DOI:
10.1016/j.micpath.2016.01.013
Reference:
YMPAT 1758
To appear in:
Microbial Pathogenesis
Received Date: 26 October 2015 Revised Date:
12 January 2016
Accepted Date: 19 January 2016
Please cite this article as: Latha M, Priyanka M, Rajasekar P, Manikandan R, Prabhu NM, Biocompatibility and antibacterial activity of the Adathoda vasica Linn extract mediated silver nanoparticles, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.01.013. 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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Biocompatibility and antibacterial activity of the Adathoda vasica Linn extract mediated silver nanoparticles
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M. Latha1, M. Priyanka1, P. Rajasekar1, R. Manikandan2, N. M. Prabhu1*
Department of Animal Health and Management, Alagappa University, Karaikudi -630 004, India.
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Department of Zoology, University of Madras, Guindy campus, Chennai- 600 025
*Corresponding author
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Dr. N. M. Prabhu, Assistant Professor, Department of Animal Health and Management,
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Alagappa University, Karaikudi-630 003, Tamil Nadu, India.
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E-mail: [email protected], Mobile No: +91 9444154070.
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Abstract The aim of this study is to investigate the biocompatibility and anti-Vibrio efficacy of green synthesized silver nanoparticles (AgNPs) using an aqueous leaf extract of Adathoda vasica
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(A. vasica). The green synthesized silver nanoparticles were characterized by UV-vis, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy dispersive X-ray analysis
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(EDX). A. vasica AgNPs showed significant antibacterial activity against V. parahaemolyticus in agar bioassay and well diffusion method. Further, nanoparticles interactions with bacteria and
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its antibacterial activity were confirmed by CLSM analysis. In vivo evaluation results confirmed that synthesized A. vasica AgNPs had good antibacterial efficacy and also nontoxic to the
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Artemia nauplii.
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Key words: Adathoda vasica, Vibrio parahaemolyticus, antibacterial activity, Artemia.
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1. Introduction The shrimp aquaculture industry is rapidly expanding and accounts for 15% of the internationally traded seafood products (FAO, 2014). Vibriosis is the presumptive cause of
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strong economic losses in the shrimp industry. Recently, the identification of certain strains such as Vibrio parahaemolyticus, a causative agent of early mortality syndrome (EMS) caused large scale losses in shrimp production in China, Vietnam, Thailand and Malaysia FAO, 2013 [1].
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Vibrio parahaemolyticus is a prevalent seafood-borne entero pathogen with the appearance of pandemic O3:K6 strains in 1996. It’s also associated with mortalities in Siberian tooth carps,
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milk fish [2], abalone [3] and shrimp [4]. Fish result in by this classical Vibrio shows typical signs of a generalized septicemia with hemorrhage at the base of fins are frequently exophthalmia and corneal opacity in finfish [5]. Resistance to antimicrobial agents by Vibrios has emerged in recent years and is a major challenge for shrimp production [6]. Vibrio infection
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in the larval stages of cultured fish cause high mortality rate, sometimes it leads to the death of the entire population. Usage of vaccines, antibiotics, and chemotherapeutic for disease control resulted in bacterial resistance and cause potential hazards to the environment [7, 8]. Therefore,
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aquaculture requires supports to overcome this bacterial disease by continuous research with scientific and technical innovation.
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To keep sustainable production, health management approaches must go beyond antibiotics and chemotherapeutic and there has been urging for researchers to find a new drug against the pathogenic bacteria [9-11]. Recently, much attention has been paid towards the use of nanoparticles as another element set of antibiotics attributable to their distinct advantages over conventional antimicrobial agents [12]. A discipline of nanotoxicology should make an important contribution to the development of a sustainable and safe nanotechnology. Silver
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nanoparticles have gained much popularity because of their antimicrobial properties [13, 14]. In recent years, synthesis of noble metal nanoparticles using various species of plants has arisen as a new trend [15]. Plant based nanoparticles are also reported to have antibacterial effect [16].
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Biological route synthesis of nanoparticles has much attention focused by researchers because they are eco-friendly, economical and cost effective. Green synthesis of silver nanoparticles offers a simple and eco- friendly approach to form stable colloids of nontoxic AgNPs along with
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antibacterial activity.
In traditional medicine, different parts of A. vasica Linn (known as vasaka), a shrub widespread
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in India, has been used for treating many human diseases. Vasaka is a bitter quinazoline alkaloid [17, 18]. The leaves contain several alkaloids namely vasicinone, vasicinol, adhatodine, adhatonine, adhavasinone, anisotine and peganine. A. vasica leaf extract have been used for the anti-allergic, anti-asthmatic, anti-inflammatory, anti-microbial effect [19] and also used to
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control tuberculosis [20]. In this study, we synthesized biogenic-silver nanoparticles using A. vasica and evaluated for its antibacterial activities against V. parahaemolyticus. Furthermore, biocompatibility of the synthesized A. vasica AgNPs was confirmed using Artemia napulii as a
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model organism. 2. Materials and Methods
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2.1. Chemicals, medium and bacteria The silver nitrate (AgNO3), bacterial media and other components used in this study were purchased from Hi Media (Mumbai, India). Vibrio parahaemolyticus (ATCC -17802) was obtained from the Department of Biotechnology, Alagappa University, Karaikudi.
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2.2. Preparation of A. vasica leaf extract The plant (A. vasica) was collected in and around Karaikudi, Tamil Nadu India and identified and confirmed with the help of field botanist. The leaves were washed thoroughly in distilled
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water to remove impurities and cut into small pieces. For extraction, the leaf materials (10 g) were weighed and mixed with 500 ml of deionized water and boiled at 60 °C for 10 min in a
No. 1 filter paper and used for the further experiment. 2.3. Synthesis of silver Nanoparticles
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water bath. The prepared aqueous plant extract was allowed to cool and filtered with Whatman
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After standardization, 15 ml of clear leaf broth was added into the aqueous solution of 0.75 mM of AgNO3 and the mixture was incubated in dark at room temperature. The change of colour solution indicates the formation of silver nanoparticles. 2.4. Characterization of silver nanoparticles
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The reduction of pure Ag+ ions was monitored by measuring the UV-Vis spectrum of the reaction medium at different time intervals (5 min to 3 h) in Shimadzu UV-1800, measured between the wavelengths of 200-700 nm. In order to identify the AgNPs associated
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biomolecules, Fourier Transform Infrared (FTIR) spectral analysis was performed to the washed and purified AgNPs powder on the thermo scientific Nicolet 380 FTIR spectroscopy. Two
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milligrams of the sample were mixed with 200 mg KBr (FTIR grade) and pressed into a pellet and placed into the sample holder of FTIR spectra. To obtain good signal to noise ratio, 256 scans of AgNPs were taken in the range of 400-4000 cm-1 and the resolution was kept at 4 cm-1. To determine the nature of AgNPs, the air dried powder was analyzed in XRD (X’PERTPRO.PAN analytical Netherland) operating in transmission mode at 40 kV and 30 mA with Cu K radiation. Further to evaluate the size and shape of the synthesized nanoparticles TEM was
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performed on Technite 10 Philips instrument on carbon coated copper grids with an accelerating voltage of 80 Kv. SEM analysis was measured using Hitachi-S-3000H and characterization of intermediate compounds which formed during the silver ions reduction with the plant extract was
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carried out by EDX( Hitachi S3000H). 2.5. Antibacterial activity
The pathogenic bacterium was pre-grown in nutrient broth at 37 °C for 24 h. Sterilized
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Muller Hinton Agar (MHA) medium with different concentration of AgNPs (10, 20, 30, 40 and 50 µg/ml) was poured into petri dishes. In the agar bioassay method, 100 µl of V.
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parahaemolyticus at the density of 105 CFU/ml was spread on the lawn of MHA medium and incubated at 37 °C for 24 h and observed for the antibacterial effect. Positive and negative control plates were also maintained for comparison. In agar well diffusion assay, 100 µl nutrient broth culture of each bacterium (105 CFU/ml) was used to prepare bacterial lawns. Agar wells of
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6 mm diameter were prepared with the help of a sterilized stainless steel cork borer. Different concentrations of AgNPs (10, 20, 30, 40 and 50 µg/ml) were inoculated into the wells made on MHA. The plates were allowed for incubation at 37 °C for 24 h.
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2.6. Confocal Laser Scanning Microscopic Studies For CLSM analysis, different concentrations of AgNPs were added into bacterial suspension
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of 105 CFU/ml. After 24 h incubation, the bacterial cell suspensions were centrifuged at 5000 rpm for 5 min. The supernatant of these samples were discarded and the remaining bacterial pellets were suspended in 0.5 ml prepared fluorescent marker solution (Acridine orange 1 µl and 1 ml of PBS). The remaining pellet was suspended into 200 µl PBS and incubated for 15 min. Then the incubated bacterial suspension was centrifuged at 5000g for 5 min to remove the excess dyes. The antibacterial activity was observed under CLSM (Model: LSM710) (Carl Zeiss Jena,
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Germany). The 488-nm Ar laser and a 500- 640 nm band pass emission filters are used to excite and detect the stained cells. Control and treated bacterial cell images were recorded and processed using Zen 2009 image.
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2.7. In vivo analysis
In vivo experiments were executed with freshly hatched Artemia nauplii (San Francisco Bay Brand, San Francisco, CA, USA). To hatch out the Artemia nauplii, one hundred milligrams of
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decapsulated cysts were kept in 100 ml of sterile seawater with the salinity of 30 ppt and were well aerated for 24 h. The freshly hatched healthy Artemia nauplii were collected and used for
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challenging test by standard [21, 22]. Briefly, healthy Artemia nauplii were divided into 7 groups in triplicate and each group contains 30 numbers of nauplii. The experiment was performed with 24 well plates in 5 ml of 30 ppt sea water for 48 h to assess the antibacterial efficacy. V. parahaemolyticus 105 CFU/ml were used in this experiment with the concentration
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of 50 µg/ml AgNPs based on the minimum bactericidal concentration determined. The nauplii survival rate was observed at 6 h intervals over the period of 48 h and percentage was calculated by following this formula: Survival rate (%)=[number of live nauplii at the 6 h intervals/number
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of nauplii at the time of inoculation] X100. Experimental setup
Control ( without any application )
Group II
AgNPs (50 µg/ml) alone.
Group III
Plant extract (50µg/ml) alone.
Group IV
V. parahaemolyticus alone (50µg/ml).
Group V
V. parahaemolyticus + AgNPs (50 µg/ml)
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V. parahaemolyticus + plant extract (50µg/ml).
Group VII
AgNO3 alone (50µg/ml).
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Group I
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3. Results 3.1. Synthesis of plant based AgNPs A. vasica was used for the reduction of AgNO3 into nanoscale level. The synthesis of AgNPs
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was confirmed based on the color change from yellowish to reddish brown within 30 min due to reduction of silver ion, which might be an indication of green synthesis of AgNPs. The reduction was observed by a change in the color of the reaction mixture with an excitation of surface
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plasmon resonance (SPR) observed at 450 nm in UV-Visible spectrophotometer. It was observed
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that the plasmon intensity at the reaction time of 5 mins is near to that at 3 h (Fig.1). 3.2. Fourier Transform Infrared spectral analysis (FTIR)
FTIR spectra of a leaf extract (before reaction with AgNO3) and synthesized AgNPs (after reaction with AgNO3) are shown in Fig. 2a (A and B). The results showed a shift in peaks: 3424-
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3445 (bonds due to N-H stretching, amides), 2924, 2853, 2359, 2342 (bond due to C-H stretching, alkanes), 1617-1640 (characteristic of animo acids contains NH2 groups, amide I band), 1457, 1383(due to C-H deformation, ketones and esters) 1021- 1091 (bonds due to p-o
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Stretching). These data indicated the involvement of amides, carboxyl, amino groups and amino acid residues in A. vasica leaf extract.
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3.3. X-ray diffraction (XRD)
X- ray diffraction studies were performed to confirm the crystalline structure of A. vasica AgNPs. XRD spectrum of A. vasica AgNPs showed four distinct diffraction peaks at ray 38. 1, 46, 64. 56, 78 which corresponds to the (1 1 1), (2 2 0), (2 2 2) and (3 1 1) (Fig. 2b) planes which can be indexed to the FCC AgNPs based on the comparison with the standard as given by JCPDS (file no.04-0783).
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3.4. Transmission electron microscopy (TEM) Size and shape of the synthesized nanoparticles were determined by the TEM micrograph.
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TEM analysis showed that A. vasica AgNPs were spherical, mono highly dispersed and the particle size varied between 10-50 nm (Figure. 3).
3.5. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX)
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Fig. 3b image revealed the morphology of AgNPs, it was observed that they were approximately spherical in shape with smooth surface. The shape of the particles has correlated
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with the SPR band at 450 nm for AgNPs. The micrograph also revealed that the powder form particles are slightly agglomerated. Chemical composition of AgNPs synthesized by Adathoda leaf extract was analyzed using EDX. The EDX spectrum (Fig. 3c) indicates that the layer around silver nanoparticles is mainly comprised of O, Si, Cl and Ag.
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3.6. Antibacterial activity by agar bioassay and well diffusion assay The green synthesized silver nanoparticles using A. vasica showed excellent antibacterial
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activity against V. parahaemolyticus. The bacterial counts were decreased with increased concentration of the silver nanoparticles in agar bio assay analysis (Fig. 4a.). Interestingly,
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highest zone of inhibition was observed in well diffusion assay (50 µg/ml). This indicates that the biologically synthesized nanoparticles showed better antibacterial activity against gramnegative bacteria V. parahaemolyticus (Fig. 4b.). 3.7. Confocal Laser Scanning Microscopic Studies V. parahaemolyticus interaction with different concentrations (10 to 50 µg/ml) of silver nanoparticles was examined by CLSM. The live bacteria cells were emitted green due to acridine
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orange strain. The result of the CLSM analysis of V. parahaemolyticus with different concentration of silver nanoparticles showed bacterial interaction with AgNPs. Interestingly, the bacterial growth was reduced with increasing in the concentrations of silver nanoparticles (10,
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20, 30, 40 and 50 µg/ml). In control, the growth and number of cells were numerous when
bacterial structure was disrupted (fig. 5). 3.8. In vivo experiment challenging with Artemia nauplii
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compare with test experiments. Number of live bacterial cells reduces significantly and the
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Artemia napulii survival rate and morphological variations were observed after the administration of different source like leaf extract of A. vasica, AgNO3, AgNPs V. parahaemolyticus, AgNPs +V. parahaemolyticus, leaf extract + V. parahaemolyticus and control on 0th h up to 48th h (fig.6a). The treated Artemia were observed under the phase contrast
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microscope and resulted images confirmed the accumulation of the present inside the guts of Artemia. Fig. 6b shows the Artemia were unable to eliminate the ingested particles, which were thought to be due to the formation of massive particles in the guts. Although all the suspensions
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of AgNPs, leaf extract did not exhibit any significant acute toxicity within 48 h compared to V. parahaemolyticus. Group IV Artemia treated with V. parahaemolyticus showed complete
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mortality within 24 hrs. Whereas Artemia treated with silver nanoparticles alone Group II and Group III plant alone did not demonstrate any lethal effect. Interestingly, Group V Artemia treated with bacteria and green synthesized silver nanoparticles showed less mortality than group VI animal treated with plant extract and V. parahaemolyticus. Thus, the synthesized silver nanoparticles confirm the antibacterial effect against Vibrio parahaemolyticus (Fig .6a and 6b). Artemia nauplii treated with AgNPS and plant extract showed morphologically similar to the
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control groups. This proves the synthesized nanoparticles had no toxic effect against Artemia nauplii.
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4. Discussion Vibrio infection in the larval stages of cultured shrimps causes high mortality rate, sometimes it leads to the death of the entire population. High mortality and contagious bacterial diseases
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leads to the usage of large amounts of antibiotics and chemotherapeutic. In our present investigation, we synthesized the A. vasica extract mediated silver nanoparticles to control
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vibriosis in the culture system. The A. vasica (Acanthaceae) plant leaf contains alkaloids compounds such as vasicinone, vasicinol, adhatodine, adhatonine, adhavasinone, anisotine and peganine, which may be responsible for the green synthesis of A. vasica AgNPs. In the present study the colour of the solution changed into reddish brown within 30 minutes and a peak specific for the synthesis of silver nanoparticles were obtained at 450 nm in UV-vis
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spectrophotometer analysis. The plant extracts are widely being applied to synthesize AgNPs by reducing Ag+ ions into Ago that increase the optical density of the solution [23]. This was owing to the excitation of surface plasmon resonance (SPR) and the reduction of silver nitrate during
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the incubation period [24, 25].
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In this study, the size and morphology of nanoparticles were characterized by TEM and SEM. The size of A. vasica AgNPs synthesized was in the range of 10-50 nm. The small size and morphology surface planar accessibility of the AgNPs dictates its efficient antibacterial potential [26]. The nanoparticles was found to be highly dispersed and of spherical in morphology. SEM results indicated that the synthesized AgNPs were distinguishable, spherical in shape. Additionally, EDX results showed the presence of a strong signal from silver atoms (86.29 %).
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Synthesized A. vasica AgNPs show high inhibitory activity against pathogenic strain Gram negative V. parahaemolyticus. Similarly Kvitek [27] reported the bactericidal action AgNPs may attach with sulfur and thiol group compounds found in the respiratory enzymes of bacterial cells
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thus inhibiting the respiration process in bacteria [28-30]. It is well known that the biofilmforming potential of V. parahaemolyticus confers virulence [31] that is responsible for shrimp mortality [32].
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In the present investigation, we report the in-vivo anti-bacterial efficiency of synthesized A. vasica AgNPs using Artemia nauplii against the pathogenic V. parahaemolyticus. A. vasica
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AgNPs did not show any lethal effects on Artemia nauplii, whereas a higher rate of mortality was observed when exposing Artemia to V. parahaemolyticus. Biocompatibility is the ability of a material to do with an appropriate host response in specific application [33]. Thus, the real time study transport and biocompatibility of silver nanoparticles in early
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napulii (I in sector stage) development at resolution can offer new knowledge about their delivery and effect in vivo. Similar results present in vertebrate animal study [34]. When the silver nanoparticles were exposed to the Artemia nauplii showed the nontoxic effect followed by
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control. The highest percentage of viable Artemia was observed after exposure to the nanoparticles of (94%) while the lowest value (12%) was found when the Artemia were exposed
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to V. parahaemolyticus which was the most toxic to aquatic organisms. The present study concluded that the green synthesized silver nanoparticles using Adathoda leaf extract showed prominent results in controlling the V. parahaemolyticus. The obtained result of in vivo analysis revealed the antibacterial effect of AgNPs enhancing the survival rate of Artemia by decreasing the virulence of V. Parahaemolyticus. Conflict of interest
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We declare that there is no conflict of interest Acknowledgements The authors acknowledge the financial assistance provided by Department Science and
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Technology, New Delhi, for INSPIRE fellowship (DST/INSPIRE Fellowship/2011/[2], dated 26.09.2011), Department of Physics, Alagappa University, Karaikudi, for their help in X-ray diffraction spectrum analysis.
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Figure Caption Fig. 1. UV-vis spectra of aqueous silver nitrate with A. vasica leaf extract at different time intervals. The figure inset shows (a) synthesized silver nanoparticles (b) Aqueous AgNo3
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alone Fig. 2a. (A) FTIR spectrum of A. vasica leaf extracts (B) synthesized silver nanoparticles.
2b. XRD spectral patterns of green synthesized silver nanoparticles using A. vasica leaf. Fig. 3 The images showing the shape and size of AgNPs by TEM (a), SEM (b) and EDX
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spectrum (c).
Fig. 4a. Antibacterial activity of green synthesized silver nanoparticles using A. vasica at
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different concentration of silver nanoparticles (a) 0 µg/ml, (b) 10 µg/ml, (c) 20 µg/ml, (d) 30 µg/ml, (e) 40 µg/ml and (f) 50 µg/ml.
Fig. 4b. (a) Agar well diffusion assay of green synthesized silver nanoparticles using A. vasica leaf extract, bacterial culture, AgNO3, AgNPs.
(b) different concentration of silver nanoparticles (a) 0 µg/ml, (b) 10 µg/ml, (c) 20
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µg/ml, (d) 30 µg/ml, (e) 40 µg/ml and (f) 50 µg/ml.
Fig. 5. Confocal laser scanning microscopy antibacterial activity of green synthesized silver nanoparticles using A. vasica at different concentration (a) 0 µg/ml (b) 10 µg/ml, (c) 20 µg/ml, (d) 30 µg/ml, (e) 40 µg/ml and (f) 50 µg/ml.
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Fig. 6a. Biocompatibility and Morphological variations of Artemia naupilli treated with various sources (50 µg/ml) were observed using phase contrast microscope. (a) Control, (b)
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AgNO3 (50 µg/ml) alone, (c) V. parahaemolyticus (50 µg/ml) alone, (d) AgNPs+ V.
parahaemolyticus (50 µg/ml), (e) AgNPs alone (50 µg/ml), (f) Plant extract (50 µg/ml),
plant extract +V. parahaemolyticus (50 µg/ml).
Fig. 6b. Mortality and survival rate of various sources treated with Artemia nauplii (0 th h to 48 h ) (a) Control, (b) AgNO3 (50 µg/ml) alone,(c) V. parahaemolyticus alone, (50 µg/ml) (d) AgNPs+ V. parahaemolyticus (50 µg/ml), (e) AgNPs alone (50 µg/ml), (f) Plant extract(50 µg/ml), plant extract +V. parahaemolyticus (50 µg/ml).
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Highlights •
Silver nanoparticles preparation using Adathoda vasica. Synthesized nanoparticles acted as effective bactericidal agent.
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Vibrio parheamolyticus and silver nanoparticles inter action study in CLSM.
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An excellent Artemia naupilli biocompatibility was attained in vivo analysis.
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S0882-4010(15)30170-4
DOI:
10.1016/j.micpath.2016.01.013
Reference:
YMPAT 1758
To appear in:
Microbial Pathogenesis
Received Date: 26 October 2015 Revised Date:
12 January 2016
Accepted Date: 19 January 2016
Please cite this article as: Latha M, Priyanka M, Rajasekar P, Manikandan R, Prabhu NM, Biocompatibility and antibacterial activity of the Adathoda vasica Linn extract mediated silver nanoparticles, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.01.013. 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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Biocompatibility and antibacterial activity of the Adathoda vasica Linn extract mediated silver nanoparticles
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M. Latha1, M. Priyanka1, P. Rajasekar1, R. Manikandan2, N. M. Prabhu1*
Department of Animal Health and Management, Alagappa University, Karaikudi -630 004, India.
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Department of Zoology, University of Madras, Guindy campus, Chennai- 600 025
*Corresponding author
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Dr. N. M. Prabhu, Assistant Professor, Department of Animal Health and Management,
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Alagappa University, Karaikudi-630 003, Tamil Nadu, India.
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E-mail: [email protected], Mobile No: +91 9444154070.
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Abstract The aim of this study is to investigate the biocompatibility and anti-Vibrio efficacy of green synthesized silver nanoparticles (AgNPs) using an aqueous leaf extract of Adathoda vasica
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(A. vasica). The green synthesized silver nanoparticles were characterized by UV-vis, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy dispersive X-ray analysis
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(EDX). A. vasica AgNPs showed significant antibacterial activity against V. parahaemolyticus in agar bioassay and well diffusion method. Further, nanoparticles interactions with bacteria and
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its antibacterial activity were confirmed by CLSM analysis. In vivo evaluation results confirmed that synthesized A. vasica AgNPs had good antibacterial efficacy and also nontoxic to the
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Artemia nauplii.
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Key words: Adathoda vasica, Vibrio parahaemolyticus, antibacterial activity, Artemia.
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1. Introduction The shrimp aquaculture industry is rapidly expanding and accounts for 15% of the internationally traded seafood products (FAO, 2014). Vibriosis is the presumptive cause of
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strong economic losses in the shrimp industry. Recently, the identification of certain strains such as Vibrio parahaemolyticus, a causative agent of early mortality syndrome (EMS) caused large scale losses in shrimp production in China, Vietnam, Thailand and Malaysia FAO, 2013 [1].
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Vibrio parahaemolyticus is a prevalent seafood-borne entero pathogen with the appearance of pandemic O3:K6 strains in 1996. It’s also associated with mortalities in Siberian tooth carps,
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milk fish [2], abalone [3] and shrimp [4]. Fish result in by this classical Vibrio shows typical signs of a generalized septicemia with hemorrhage at the base of fins are frequently exophthalmia and corneal opacity in finfish [5]. Resistance to antimicrobial agents by Vibrios has emerged in recent years and is a major challenge for shrimp production [6]. Vibrio infection
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in the larval stages of cultured fish cause high mortality rate, sometimes it leads to the death of the entire population. Usage of vaccines, antibiotics, and chemotherapeutic for disease control resulted in bacterial resistance and cause potential hazards to the environment [7, 8]. Therefore,
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aquaculture requires supports to overcome this bacterial disease by continuous research with scientific and technical innovation.
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To keep sustainable production, health management approaches must go beyond antibiotics and chemotherapeutic and there has been urging for researchers to find a new drug against the pathogenic bacteria [9-11]. Recently, much attention has been paid towards the use of nanoparticles as another element set of antibiotics attributable to their distinct advantages over conventional antimicrobial agents [12]. A discipline of nanotoxicology should make an important contribution to the development of a sustainable and safe nanotechnology. Silver
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nanoparticles have gained much popularity because of their antimicrobial properties [13, 14]. In recent years, synthesis of noble metal nanoparticles using various species of plants has arisen as a new trend [15]. Plant based nanoparticles are also reported to have antibacterial effect [16].
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Biological route synthesis of nanoparticles has much attention focused by researchers because they are eco-friendly, economical and cost effective. Green synthesis of silver nanoparticles offers a simple and eco- friendly approach to form stable colloids of nontoxic AgNPs along with
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antibacterial activity.
In traditional medicine, different parts of A. vasica Linn (known as vasaka), a shrub widespread
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in India, has been used for treating many human diseases. Vasaka is a bitter quinazoline alkaloid [17, 18]. The leaves contain several alkaloids namely vasicinone, vasicinol, adhatodine, adhatonine, adhavasinone, anisotine and peganine. A. vasica leaf extract have been used for the anti-allergic, anti-asthmatic, anti-inflammatory, anti-microbial effect [19] and also used to
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control tuberculosis [20]. In this study, we synthesized biogenic-silver nanoparticles using A. vasica and evaluated for its antibacterial activities against V. parahaemolyticus. Furthermore, biocompatibility of the synthesized A. vasica AgNPs was confirmed using Artemia napulii as a
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model organism. 2. Materials and Methods
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2.1. Chemicals, medium and bacteria The silver nitrate (AgNO3), bacterial media and other components used in this study were purchased from Hi Media (Mumbai, India). Vibrio parahaemolyticus (ATCC -17802) was obtained from the Department of Biotechnology, Alagappa University, Karaikudi.
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2.2. Preparation of A. vasica leaf extract The plant (A. vasica) was collected in and around Karaikudi, Tamil Nadu India and identified and confirmed with the help of field botanist. The leaves were washed thoroughly in distilled
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water to remove impurities and cut into small pieces. For extraction, the leaf materials (10 g) were weighed and mixed with 500 ml of deionized water and boiled at 60 °C for 10 min in a
No. 1 filter paper and used for the further experiment. 2.3. Synthesis of silver Nanoparticles
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water bath. The prepared aqueous plant extract was allowed to cool and filtered with Whatman
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After standardization, 15 ml of clear leaf broth was added into the aqueous solution of 0.75 mM of AgNO3 and the mixture was incubated in dark at room temperature. The change of colour solution indicates the formation of silver nanoparticles. 2.4. Characterization of silver nanoparticles
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The reduction of pure Ag+ ions was monitored by measuring the UV-Vis spectrum of the reaction medium at different time intervals (5 min to 3 h) in Shimadzu UV-1800, measured between the wavelengths of 200-700 nm. In order to identify the AgNPs associated
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biomolecules, Fourier Transform Infrared (FTIR) spectral analysis was performed to the washed and purified AgNPs powder on the thermo scientific Nicolet 380 FTIR spectroscopy. Two
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milligrams of the sample were mixed with 200 mg KBr (FTIR grade) and pressed into a pellet and placed into the sample holder of FTIR spectra. To obtain good signal to noise ratio, 256 scans of AgNPs were taken in the range of 400-4000 cm-1 and the resolution was kept at 4 cm-1. To determine the nature of AgNPs, the air dried powder was analyzed in XRD (X’PERTPRO.PAN analytical Netherland) operating in transmission mode at 40 kV and 30 mA with Cu K radiation. Further to evaluate the size and shape of the synthesized nanoparticles TEM was
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performed on Technite 10 Philips instrument on carbon coated copper grids with an accelerating voltage of 80 Kv. SEM analysis was measured using Hitachi-S-3000H and characterization of intermediate compounds which formed during the silver ions reduction with the plant extract was
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carried out by EDX( Hitachi S3000H). 2.5. Antibacterial activity
The pathogenic bacterium was pre-grown in nutrient broth at 37 °C for 24 h. Sterilized
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Muller Hinton Agar (MHA) medium with different concentration of AgNPs (10, 20, 30, 40 and 50 µg/ml) was poured into petri dishes. In the agar bioassay method, 100 µl of V.
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parahaemolyticus at the density of 105 CFU/ml was spread on the lawn of MHA medium and incubated at 37 °C for 24 h and observed for the antibacterial effect. Positive and negative control plates were also maintained for comparison. In agar well diffusion assay, 100 µl nutrient broth culture of each bacterium (105 CFU/ml) was used to prepare bacterial lawns. Agar wells of
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6 mm diameter were prepared with the help of a sterilized stainless steel cork borer. Different concentrations of AgNPs (10, 20, 30, 40 and 50 µg/ml) were inoculated into the wells made on MHA. The plates were allowed for incubation at 37 °C for 24 h.
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2.6. Confocal Laser Scanning Microscopic Studies For CLSM analysis, different concentrations of AgNPs were added into bacterial suspension
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of 105 CFU/ml. After 24 h incubation, the bacterial cell suspensions were centrifuged at 5000 rpm for 5 min. The supernatant of these samples were discarded and the remaining bacterial pellets were suspended in 0.5 ml prepared fluorescent marker solution (Acridine orange 1 µl and 1 ml of PBS). The remaining pellet was suspended into 200 µl PBS and incubated for 15 min. Then the incubated bacterial suspension was centrifuged at 5000g for 5 min to remove the excess dyes. The antibacterial activity was observed under CLSM (Model: LSM710) (Carl Zeiss Jena,
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Germany). The 488-nm Ar laser and a 500- 640 nm band pass emission filters are used to excite and detect the stained cells. Control and treated bacterial cell images were recorded and processed using Zen 2009 image.
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2.7. In vivo analysis
In vivo experiments were executed with freshly hatched Artemia nauplii (San Francisco Bay Brand, San Francisco, CA, USA). To hatch out the Artemia nauplii, one hundred milligrams of
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decapsulated cysts were kept in 100 ml of sterile seawater with the salinity of 30 ppt and were well aerated for 24 h. The freshly hatched healthy Artemia nauplii were collected and used for
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challenging test by standard [21, 22]. Briefly, healthy Artemia nauplii were divided into 7 groups in triplicate and each group contains 30 numbers of nauplii. The experiment was performed with 24 well plates in 5 ml of 30 ppt sea water for 48 h to assess the antibacterial efficacy. V. parahaemolyticus 105 CFU/ml were used in this experiment with the concentration
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of 50 µg/ml AgNPs based on the minimum bactericidal concentration determined. The nauplii survival rate was observed at 6 h intervals over the period of 48 h and percentage was calculated by following this formula: Survival rate (%)=[number of live nauplii at the 6 h intervals/number
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of nauplii at the time of inoculation] X100. Experimental setup
Control ( without any application )
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AgNPs (50 µg/ml) alone.
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Plant extract (50µg/ml) alone.
Group IV
V. parahaemolyticus alone (50µg/ml).
Group V
V. parahaemolyticus + AgNPs (50 µg/ml)
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V. parahaemolyticus + plant extract (50µg/ml).
Group VII
AgNO3 alone (50µg/ml).
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Group I
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3. Results 3.1. Synthesis of plant based AgNPs A. vasica was used for the reduction of AgNO3 into nanoscale level. The synthesis of AgNPs
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was confirmed based on the color change from yellowish to reddish brown within 30 min due to reduction of silver ion, which might be an indication of green synthesis of AgNPs. The reduction was observed by a change in the color of the reaction mixture with an excitation of surface
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plasmon resonance (SPR) observed at 450 nm in UV-Visible spectrophotometer. It was observed
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that the plasmon intensity at the reaction time of 5 mins is near to that at 3 h (Fig.1). 3.2. Fourier Transform Infrared spectral analysis (FTIR)
FTIR spectra of a leaf extract (before reaction with AgNO3) and synthesized AgNPs (after reaction with AgNO3) are shown in Fig. 2a (A and B). The results showed a shift in peaks: 3424-
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3445 (bonds due to N-H stretching, amides), 2924, 2853, 2359, 2342 (bond due to C-H stretching, alkanes), 1617-1640 (characteristic of animo acids contains NH2 groups, amide I band), 1457, 1383(due to C-H deformation, ketones and esters) 1021- 1091 (bonds due to p-o
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Stretching). These data indicated the involvement of amides, carboxyl, amino groups and amino acid residues in A. vasica leaf extract.
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3.3. X-ray diffraction (XRD)
X- ray diffraction studies were performed to confirm the crystalline structure of A. vasica AgNPs. XRD spectrum of A. vasica AgNPs showed four distinct diffraction peaks at ray 38. 1, 46, 64. 56, 78 which corresponds to the (1 1 1), (2 2 0), (2 2 2) and (3 1 1) (Fig. 2b) planes which can be indexed to the FCC AgNPs based on the comparison with the standard as given by JCPDS (file no.04-0783).
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3.4. Transmission electron microscopy (TEM) Size and shape of the synthesized nanoparticles were determined by the TEM micrograph.
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TEM analysis showed that A. vasica AgNPs were spherical, mono highly dispersed and the particle size varied between 10-50 nm (Figure. 3).
3.5. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX)
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Fig. 3b image revealed the morphology of AgNPs, it was observed that they were approximately spherical in shape with smooth surface. The shape of the particles has correlated
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with the SPR band at 450 nm for AgNPs. The micrograph also revealed that the powder form particles are slightly agglomerated. Chemical composition of AgNPs synthesized by Adathoda leaf extract was analyzed using EDX. The EDX spectrum (Fig. 3c) indicates that the layer around silver nanoparticles is mainly comprised of O, Si, Cl and Ag.
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3.6. Antibacterial activity by agar bioassay and well diffusion assay The green synthesized silver nanoparticles using A. vasica showed excellent antibacterial
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activity against V. parahaemolyticus. The bacterial counts were decreased with increased concentration of the silver nanoparticles in agar bio assay analysis (Fig. 4a.). Interestingly,
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highest zone of inhibition was observed in well diffusion assay (50 µg/ml). This indicates that the biologically synthesized nanoparticles showed better antibacterial activity against gramnegative bacteria V. parahaemolyticus (Fig. 4b.). 3.7. Confocal Laser Scanning Microscopic Studies V. parahaemolyticus interaction with different concentrations (10 to 50 µg/ml) of silver nanoparticles was examined by CLSM. The live bacteria cells were emitted green due to acridine
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orange strain. The result of the CLSM analysis of V. parahaemolyticus with different concentration of silver nanoparticles showed bacterial interaction with AgNPs. Interestingly, the bacterial growth was reduced with increasing in the concentrations of silver nanoparticles (10,
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20, 30, 40 and 50 µg/ml). In control, the growth and number of cells were numerous when
bacterial structure was disrupted (fig. 5). 3.8. In vivo experiment challenging with Artemia nauplii
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compare with test experiments. Number of live bacterial cells reduces significantly and the
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Artemia napulii survival rate and morphological variations were observed after the administration of different source like leaf extract of A. vasica, AgNO3, AgNPs V. parahaemolyticus, AgNPs +V. parahaemolyticus, leaf extract + V. parahaemolyticus and control on 0th h up to 48th h (fig.6a). The treated Artemia were observed under the phase contrast
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microscope and resulted images confirmed the accumulation of the present inside the guts of Artemia. Fig. 6b shows the Artemia were unable to eliminate the ingested particles, which were thought to be due to the formation of massive particles in the guts. Although all the suspensions
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of AgNPs, leaf extract did not exhibit any significant acute toxicity within 48 h compared to V. parahaemolyticus. Group IV Artemia treated with V. parahaemolyticus showed complete
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mortality within 24 hrs. Whereas Artemia treated with silver nanoparticles alone Group II and Group III plant alone did not demonstrate any lethal effect. Interestingly, Group V Artemia treated with bacteria and green synthesized silver nanoparticles showed less mortality than group VI animal treated with plant extract and V. parahaemolyticus. Thus, the synthesized silver nanoparticles confirm the antibacterial effect against Vibrio parahaemolyticus (Fig .6a and 6b). Artemia nauplii treated with AgNPS and plant extract showed morphologically similar to the
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control groups. This proves the synthesized nanoparticles had no toxic effect against Artemia nauplii.
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4. Discussion Vibrio infection in the larval stages of cultured shrimps causes high mortality rate, sometimes it leads to the death of the entire population. High mortality and contagious bacterial diseases
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leads to the usage of large amounts of antibiotics and chemotherapeutic. In our present investigation, we synthesized the A. vasica extract mediated silver nanoparticles to control
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vibriosis in the culture system. The A. vasica (Acanthaceae) plant leaf contains alkaloids compounds such as vasicinone, vasicinol, adhatodine, adhatonine, adhavasinone, anisotine and peganine, which may be responsible for the green synthesis of A. vasica AgNPs. In the present study the colour of the solution changed into reddish brown within 30 minutes and a peak specific for the synthesis of silver nanoparticles were obtained at 450 nm in UV-vis
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spectrophotometer analysis. The plant extracts are widely being applied to synthesize AgNPs by reducing Ag+ ions into Ago that increase the optical density of the solution [23]. This was owing to the excitation of surface plasmon resonance (SPR) and the reduction of silver nitrate during
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the incubation period [24, 25].
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In this study, the size and morphology of nanoparticles were characterized by TEM and SEM. The size of A. vasica AgNPs synthesized was in the range of 10-50 nm. The small size and morphology surface planar accessibility of the AgNPs dictates its efficient antibacterial potential [26]. The nanoparticles was found to be highly dispersed and of spherical in morphology. SEM results indicated that the synthesized AgNPs were distinguishable, spherical in shape. Additionally, EDX results showed the presence of a strong signal from silver atoms (86.29 %).
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Synthesized A. vasica AgNPs show high inhibitory activity against pathogenic strain Gram negative V. parahaemolyticus. Similarly Kvitek [27] reported the bactericidal action AgNPs may attach with sulfur and thiol group compounds found in the respiratory enzymes of bacterial cells
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thus inhibiting the respiration process in bacteria [28-30]. It is well known that the biofilmforming potential of V. parahaemolyticus confers virulence [31] that is responsible for shrimp mortality [32].
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In the present investigation, we report the in-vivo anti-bacterial efficiency of synthesized A. vasica AgNPs using Artemia nauplii against the pathogenic V. parahaemolyticus. A. vasica
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AgNPs did not show any lethal effects on Artemia nauplii, whereas a higher rate of mortality was observed when exposing Artemia to V. parahaemolyticus. Biocompatibility is the ability of a material to do with an appropriate host response in specific application [33]. Thus, the real time study transport and biocompatibility of silver nanoparticles in early
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napulii (I in sector stage) development at resolution can offer new knowledge about their delivery and effect in vivo. Similar results present in vertebrate animal study [34]. When the silver nanoparticles were exposed to the Artemia nauplii showed the nontoxic effect followed by
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control. The highest percentage of viable Artemia was observed after exposure to the nanoparticles of (94%) while the lowest value (12%) was found when the Artemia were exposed
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to V. parahaemolyticus which was the most toxic to aquatic organisms. The present study concluded that the green synthesized silver nanoparticles using Adathoda leaf extract showed prominent results in controlling the V. parahaemolyticus. The obtained result of in vivo analysis revealed the antibacterial effect of AgNPs enhancing the survival rate of Artemia by decreasing the virulence of V. Parahaemolyticus. Conflict of interest
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We declare that there is no conflict of interest Acknowledgements The authors acknowledge the financial assistance provided by Department Science and
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Technology, New Delhi, for INSPIRE fellowship (DST/INSPIRE Fellowship/2011/[2], dated 26.09.2011), Department of Physics, Alagappa University, Karaikudi, for their help in X-ray diffraction spectrum analysis.
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Figure Caption Fig. 1. UV-vis spectra of aqueous silver nitrate with A. vasica leaf extract at different time intervals. The figure inset shows (a) synthesized silver nanoparticles (b) Aqueous AgNo3
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alone Fig. 2a. (A) FTIR spectrum of A. vasica leaf extracts (B) synthesized silver nanoparticles.
2b. XRD spectral patterns of green synthesized silver nanoparticles using A. vasica leaf. Fig. 3 The images showing the shape and size of AgNPs by TEM (a), SEM (b) and EDX
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spectrum (c).
Fig. 4a. Antibacterial activity of green synthesized silver nanoparticles using A. vasica at
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different concentration of silver nanoparticles (a) 0 µg/ml, (b) 10 µg/ml, (c) 20 µg/ml, (d) 30 µg/ml, (e) 40 µg/ml and (f) 50 µg/ml.
Fig. 4b. (a) Agar well diffusion assay of green synthesized silver nanoparticles using A. vasica leaf extract, bacterial culture, AgNO3, AgNPs.
(b) different concentration of silver nanoparticles (a) 0 µg/ml, (b) 10 µg/ml, (c) 20
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µg/ml, (d) 30 µg/ml, (e) 40 µg/ml and (f) 50 µg/ml.
Fig. 5. Confocal laser scanning microscopy antibacterial activity of green synthesized silver nanoparticles using A. vasica at different concentration (a) 0 µg/ml (b) 10 µg/ml, (c) 20 µg/ml, (d) 30 µg/ml, (e) 40 µg/ml and (f) 50 µg/ml.
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Fig. 6a. Biocompatibility and Morphological variations of Artemia naupilli treated with various sources (50 µg/ml) were observed using phase contrast microscope. (a) Control, (b)
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AgNO3 (50 µg/ml) alone, (c) V. parahaemolyticus (50 µg/ml) alone, (d) AgNPs+ V.
parahaemolyticus (50 µg/ml), (e) AgNPs alone (50 µg/ml), (f) Plant extract (50 µg/ml),
plant extract +V. parahaemolyticus (50 µg/ml).
Fig. 6b. Mortality and survival rate of various sources treated with Artemia nauplii (0 th h to 48 h ) (a) Control, (b) AgNO3 (50 µg/ml) alone,(c) V. parahaemolyticus alone, (50 µg/ml) (d) AgNPs+ V. parahaemolyticus (50 µg/ml), (e) AgNPs alone (50 µg/ml), (f) Plant extract(50 µg/ml), plant extract +V. parahaemolyticus (50 µg/ml).
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Highlights •
Silver nanoparticles preparation using Adathoda vasica. Synthesized nanoparticles acted as effective bactericidal agent.
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Vibrio parheamolyticus and silver nanoparticles inter action study in CLSM.
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An excellent Artemia naupilli biocompatibility was attained in vivo analysis.
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