nanotechnology deals with the production and stabilization of various types of nanoparticles ( 1 ) . plants or natural resources have been found to be a good alternative method for nanoparticles synthesis , since this method does not use any toxic chemicals and does not require the use of high pressure , energy , and temperature .
this green chemistry process has numerous benefits , including environmental friendliness , cost - effectiveness , and suitability for pharmaceutical and biomedical applications . at present , several groups of researchers concentrate on biomimetic approaches such as plant or plant leaf extracts , nuts , microorganisms and yeast to synthesize the metal nanoparticles called " green chemical or phytochemical " approach ( 2 - 5 ) .
antibacterial activity of the silver ion has been well established ( 6 ) and ag is currently used to control bacterial growth in a variety of applications , including dental work , catheters , and burn wounds ( 7,8 ) . in fact , ag ions and ag - based compounds are highly toxic to microorganisms , showing strong biocidal effects on as many as 12 species of bacteria including e. coli ( 9 ) . apart from these antimicrobial activities , ag - nps are also known to possess antifungal , anti - inflammatory , antiviral , anti - angiogenesis and antiplatelet properties ( 10 , 11 ) .
the  betel  ( piper betle ) is the leaf belonging to the family  piperaceae . the p. 
 betle leaves possess antifungal , antiseptic , antihelmintic , antihypertensive etc .
, 2011 claim that ethanolic extract of p. betle leaves show antibacterial activity against some food borne pathogens ( 13 ) .  
the aim of this study was to synthesize agnps using aqueous leaves extract of p. betle , characterized the agnps and evaluated the antibacterial activity of ag - extract nanoparticles against some gram positive and gram negative bacteria .

 collection of materials 
  fresh young leaves of p. betle were collected from the local market of noakhali , bangladesh .
silver nitrate ( agno3 ) , nabh4 and aniline were collected from the laboratory of department of pharmacy , noakhali science and technology university ( nstu ) .  

preparation of p. betle leaf extract 
  about 30 g of freshly , taxonomically authenticated healthy leaves of p. betle were collected in january 2014 from noakhali district of bangladesh .
the leaves were washed thoroughly with double distilled water , cut into fine pieces and boiled with 150 ml double distilled water for 8 - 10 min .
finally , extract of leaves was stored at refrigerator at 4 c till further use . 

biosynthesis of silver nanoparticles 
  ag nanoparticles were made according to the recipe described in the literature ( creighton , 1979 ; suh js et al . ;
briefly , a 100-ml aqueous solution of 1.0 x 10 m silver nitrate was mixed with a 300-ml aqueous solution of 2.0 x 10 m sodium borohydride .
triply distilled water was used for solutions , and both solutions were chilled to ice temperature before mixing . by mixing both solutions , ag ions were reduced and clustered together to form monodispersed nanoparticles as a transparent solution in aqueous medium .
the solution was stirred repeatedly whenever some dark color appeared for approximately an hour until it became stabilized . at this point
this solution of ag nanoparticles was so stable that it did not change color for as long as several months without any stabilizing agent .
because the particle concentration of the solution is only 3.3 nm , it was concentrated 10 times using a rotary vacuum evaporator . 

preparation of ag - extracts nanoparticles 
  these above two solutions were mixed continuously for 30 min by magnetic stirrer and then stood for 2 h. after that uncoated ag - extract nanoparticles were prepared .  

coating of ag - extract nanoparticles with polyaniline 
  coating on the extract - agnps were done according to the k. gopalakrishnan , 2012 ( 14 ) .
after that 68 ml ( 6 % ) of h2o2 was added slowly at room temperature .
the final product was filtered , washed with distilled water and then dried at room temperature . finally , samples were ready for further analysis ( antimicrobial activity ) .  

the reaction mixture turned into dark brown color from its initial brownish - yellow color within 20 min of mixing .
the reduction of pure silver ions was monitored by measuring uv - vis spectra of the reaction mixture .
the absorption spectra of the samples were taken 240 to 540 nm using a uv  vis spectrophotometer ( hitachi , model u-2800 spectrophotometer ) . water was used as the blank .
again , spectrum was recorded in ftir ( ir prestige-21 , shimadzu ) in the range of 4000500 cm at a resolution of 4 cm .
tem observations were carried out on an h-7100 electron microscope ( hitachi , tokyo , japan ) .  

test microorganisms 
  authentic pure cultures of staphylococcus aureus atcc 25923 , salmonella typhi atcc 14028 , escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were obtained from the department of microbiology , nstu , bangladesh . 

antimicrobial activity using kirby - bauer s disc diffusion method 
  antibacterial activities of the ag - extract nanoparticles were determined according to kirby - bauer s disc diffusion method ( 2006 ) with slight modifications .
the petri dishes were flooded with mueller hinton agar and after solidification of agar 0.1 ml of diluted inoculums were spread over mueller hinton agar in the dishes using sterile l spreader to achieve confluent growth of test organisms and allowed to dry for 10 min .
the sterile readymade discs loaded with p. betle extract , agnps solution , uncoated agnps , coated ag - extract nps .
the plates were then incubated at 37 c for 36 h. the plates were observed for the zone of inhibition .
after incubation at 35 c for 18 h , the different levels of zone of inhibition were measured .

 characterization of the synthesized ag nanoparticles 
 
 evaluation of ag - nps by visual assessment 
  the formations of silver nanoparticles were confirmed through visual assessment . within 20 min of the reaction , color of the mixture turned into dark brown from its initial brownish yellow color .
the appearance of dark brown color may be due to the reduction of agno3 ( 17 ) .  
( a)agnps solution ( b)aqueous extract of p. betle ( c ) ag - extract nps 
 evaluation of ag - nps by uv spectrum 
  uv  vis spectrum of reaction mixture at different wavelengths ranging from 300 to 700 nm showed strong absorption peak with centering at approx .
400 nm ( figure 2 ) indicated the formation of aqueous extract coated ag - nps .  
uv  visible spectrum of agnps 
 evaluation of ag - nps through ft - ir spectra 
  the ft - ir spectra were used to identify the possible biomolecules ( including functional group ) responsible for the reduction of the agions and capping of the p. betle formed ag - nps .
figure 3 , showed the ftir spectra of p. betle aqueous extract and bio - synthesized ag - nps .
ft - ir spectrum for curve ( a ) red color- p. betle aqueous extract ; and curve ( b ) ag - extract nanoparticles the possible functional groups of leaf extract involved in coating nanoparticle are identified by ftir analysis . the intense absorption peaks at 3400 cm ( curve - a , red color ) and 3291.67 cm ( curve - b , black color ) correspond to n - h stretching of primary amine .
the band observed at 3059.23 cm ( curve - b ) represents = c - h stretching .
the weak band observed at 2917.46 cm and 2848.98 cm ( both in curve - b ) indicates the h - c - h asymmetric and symmetric stretching of alkanes .
the band observed at 2340.72 cm and 2369.65 cm ( both in curve - a ) , which are nearly the same band in curve - b , denotes the presence of hydrogen bonded oh stretching of carboxylic acids in leaf extract which may be a reducing / coating agent for silver nanoparticles . the band at 1617.38 cm ( curve - a ) and 1600.02 cm ( curve - b ) represents n - h bending vibration of primary amine .
the peak occurs at 1496.83 cm ( curve - b ) for n - h bending vibration of sec .
amine and at 1447.64 cm ( curve - b ) c - h bending vibration of alkane ( ch3 ) .
the band observed at 1387.84 cm and 1379.16 cm ( both in curve - b ) represent n = o stretching of nitro groups of leaf extract coated on nanoparticles .
the arising of functional groups in ftir spectrum indicates proper coating of leaf extract on silver nanoparticles .
the bands at 1296.22 , 1249.93 , 1175.66 , 1155.41 cm ( curve - b ) , which are nearly the same in curve a , denotes c - h stretching vibration of ester . beside
, c - o stretch occurs at 1072.47 , 1026.17 ( curve - b ) where first is stronger and broader than the second .
the band at 843.89 , 829.43 , 602.78 , 668.36 cm shows c - h bending vibration of alkynes . the band at 751.31 cm and 691.51 cm represents the ortho substituted and mono substituted aromatic stretching respectively .
the ft - ir results imply that the ag - nps were successfully synthesized and capped with bio - compounds present in the p. betle extract by using a green method .
the tem image in figure 4 shows that ag - nps were well dispersed with a spherical structures and particle size ranging from 10 to 30 nm with some deviations . of the total particles , approximately 20 % particles were of 10 nm , 50 % were of 20 nm and remaining 30 % particles
tem image of bio functionalized agnps 
 assessment of antibacterial activities 
  antibacterial activity of p. betle formed ag - nps on s. aureus atcc 25923 , e. coli atcc 25922 , s. typhi atcc 14028 , p. aeruginosa atcc 27853   values are the mean of three replicates  sd ( standard error ) .  
antibacterial activity of p. betle formed ag - nps on ( a ) s. aureus atcc 25923 ( b ) e. coli atcc 25922 ( c ) s. typhi atcc 14028 ( d ) p.
aeruginosa atcc 27853 graphical representation of the antibacterial activity of p. betle formed ag - nps on s. aureus atcc 25923 , e. coli atcc 25922 , s. typhi atcc 14028 , p. aeruginosa atcc 27853 the antibacterial activity of the aqueous extracts of p. betle leaves were analyzed against both gram - positive and gram - negative bacteria , which are presented in ( figure 6 , table 1 ) .
after analyzing the above results , it is clear that gram positive bacteria s. aureus is more susceptible than other experimental species of gram negative bacteria - e. coli , s. typhi , p. aeruginosa .
maximum zone of inhibition 32.780.64 mm was found for s. aureus , whereas norfloxacin showed maximum 32.150.40 mm zone of inhibition for s. aureus .
again , maximum zone of inhibition 29.550.45 mm was found for s. typhi , 27.120.38 mm for e. coli and 21.950.45 mm for p. aeruginosa .
we have demonstrated a good method for developing a simple , safe , cost - effective , and ecofriendly preparation of agnps using aqueous extract of p. betle .
the biosynthesized silver nanoparticles have spherical shapes and the particle size ranges from 10 to 30 nm approximately .
the ftir spectra revealed the involvement of hydroxyl moieties and n - h bonding in the formation of ag - nps .
they showed potential antibacterial activity against both gram - positive and gram negative bacteria . in our study
we also found , gram positive bacteria are more susceptible on ag - extract nps rather than gram negative bacteria .
application of these synthesized agnps based on our findings may lead to valuable discoveries in various fields , including medical devices and in the pharmaceutical and biomedical industries .
the results obtained by this study can not be directly extrapolated to human ; further studies should be undertaken to elucidate the exact mechanism of action by which agnps - extracts exert their antimicrobial effect which can be used in drug development program for safe health care services .
 plants or natural resources have been found to be a good alternative method for nanoparticles synthesis . in this study , 
 polyaniline coated silver nanoparticles ( agnps ) synthesized from piper betle leaves extract were investigated for their antibacterial activity . 
 silver nanoparticles were prepared from the reduction of silver nitrate and nabh4 was used as reducing agent . 
 silver nanoparticles and extracts were mixed thoroughly and then coated by polyaniline . 
 prepared nanoparticles were characterized by visual inspection , ultraviolet - visible spectroscopy ( uv ) , fourier transform infrared spectroscopy ( ft - ir ) , transmission electron microscopy ( tem ) techniques . 
 antibacterial activities of the synthesized silver nanoparticles were tested against staphylococcus aureus atcc 25923 , salmonella typhi atcc 14028 , escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 . 
 uv  
 vis spectrum of reaction mixture showed strong absorption peak with centering at 400 nm . 
 the ft - ir results imply that ag - nps were successfully synthesized and capped with bio - compounds present in p. betle . 
 tem image showed that ag - nps formed were well dispersed with a spherical structures and particle size ranging from 10 to 30 nm . 
 the result revealed that ag - extract nps showed 32.780.64 mm zone of inhibition against s. aureus , whereas norfloxacin ( positive control ) showed maximum 32.150.40 mm zone of inhibition for s. aureus . 
 again , maximum zone of inhibition 29.550.45 mm was found for s. typhi , 27.120.38 mm for e. coli and 21.950.45 mm for p. aeruginosa . 
 the results obtained by this study ca nt be directly extrapolated to human ; so further studies should be undertaken to established the strong antimicrobial activity of ag - extract nps for drug development program . 