Antimicrobial activity of Silver Nanoparticles  synthesized from Ficus benghalensis against Human Pathogens

 

Thukkaram Sudhakar¹*, Balashanmugam P², Jayapal Premkumar¹, Anisha A¹, Karthika D¹, Roshan Sapkota¹, Sakar Rijal¹

¹Department of Biomedical Engineering, School of Bio and Chemical Engineering, Sathyabama University, Chennai – 119

²National Post doc fellow, CHORD Division, CSIR, CLRI, Chennai

 

ABSTRACT:

In the context of present world Nanotechnology has found it's way in every important aspects of our daily lives from diagnostic techniques, drug delivery, sunscreens, antimicrobial bandages to disinfectants. In this study the synthesis of nanoparticles was done by reducing silver nitrate using the bacteria Bacillus species. Formation of nanoparticles was confirmed by UV-visible spectroscopy with maximum absorbance at 434nm, characteristic of silver nanoparticles by using various analytical studies. FTIR is taken to confirm the chemical bonding present in AgNPs. XRD and FE-SEM was used to determine the metallic characteristics of AgNPs as well as size and morphology of AgNPs. The size of AgNPs is 41.13nm. Disc diffusion method was done for antimicrobial activity, further comparative analysis and synergistic activity was determined. Comparative analysis of synthesized AgNPs was done with water, AgNO₃, extracted samples and AgNPs. Further synergetic activity was done to confirm the antimicrobial activity of AgNPs. AgNPs shows good antimicrobial activity against human pathogens so it can be used in drug delivery system.

 

 

 

 

INTRODUCTION:

Nanotechnology is scientific technology that deals with nanometer sized items. The exercise of nanomaterials in biotechnology unites the fields of biology and material science. Nanoparticles put forward an essentially useful platform, demonstrating unique properties with potentially wide-ranging application. such as chemicals, textile industry, medical diagnostic(future nanobots), drug and gene delivery, electronics diagnosis, artificial implants, tissue engineering[1], computing, biochemicals sensors[2], medical imaging and so on.

 

Biosynthesis of nanoparticles as an emerging highlight of the intersection of nanotechnology and biotechnology has received increased attention due to growing need to develop environmentally benign technologies in material synthesis. A great deal of effort has been put into the biosynthesis of inorganic material, especially metal nanoparticle using microorganisms and plants. It provides a platform to modify and improve the important properties of metals and has application in various fields. The use of silver nanoparticles gives product a silver finish. The antimicrobial property possed by the silver nanoparticles is one of the reason for development of nanosilver containing materials. Silver nanoparticles also exhibit strong optical features, catalytic Agent in chemical reaction, conducting inks because of their high conductivity beside their antimicrobial property. The antibacterial effects of Ag salts have been noticed since antiquity and Ag is currently used to control bacterial growth in a variety of applications, including dental work, catheters, and burn wounds. In fact, it is well known that Ag ions and Ag-based compounds are highly toxic to microbes, showing strong biocidal effects. The rate of reduction of metal ions using plants has been found to be much faster as compared to micro-organisms and stable formation of metal nanoparticles has been reported. The shape and size of the nanoparticles synthesized using plants can be controlled and modulated. many plants and their extracts have been used for synthesis of nanoparticles Azadirachta indica, Hibiscus rosasinensis, Ipomoea aquatic etc. In the present work, we investigated the synthesis of stable silver nanoparticles with the bioreduction method using Ficus benghalensis and evaluate their antibacterial properties Against Bacillus subtillis, Straphylococcus aureus, Pseudomonas aeruginosa, Klebsiella sp.

 

MATERIALS AND METHODS:

Preparation of extract of Ficus benghalensis:

Plant leaves from Ficus benghalensis were collected. The collected plant leaves were washed in tap water and rinsed in distilled water to remove dust and dirt particles. The freshly cleaned leaves were left to dry for 3-4 days. Dried plant leave were powdered using electrical mixture and the powdered were protected from sunlight for further use.

 

2gm of plant powder was taken and mixed with beaker containing 50ml of distilled water and it was boiled at 50-60°C for 15 minutes. Then the extract was filtered through Whattman no1 filter paper then the extract was stored.

 

Synthesis of silver nanoparticles:

Silver nitrate (AgNO₃) of analytical grade (AR) and purchased from E.MARCK (India). 1ml of extract is added to 9ml of milli ‘Q’ water into test and control test tubes. Weigh 0.5gms of silver nitrate crystals and add it in 5ml of milli ‘Q’ water, the control sample is kept undisturbed in dark room. To the test sample 1 milli molar of silver nitrate solution is added to test sample and kept in dark room for 12 hours. After 12 hours we can observe the colour change in the test sample.

 

Characterization of silver nanoparticles:

UV spectrophotometry:

Ag nanoparticles were characterized by UV spectroscopy, which is widely used technique for structural characterization of silver nano particle [3]. UV visible spectrophotometer with a resolution of 300 and 700nm was used. The reduction of pure Ag+ ions was monitored by measuring the UV- Vis spectral analysis has been done by using Hitachi-U-2900 spectrophotometer.

X-ray diffraction Measurement (XRD):

It is done for checking quality and formation of compound. It was done at a voltage of 40 kv and current of 30mA with Cu k alpha.

 

Field Emission Scanning Electron Microscope Analysis (FE-SEM):

Thin film of sample prepared on carbon coated copper grid by dropping small amount of sample on grid. The grids were allowed to dry by putting it under mercury lamp for five minutes [4].

 

EDAX Observation of Silver Nano particles For FE-SEM:

A semi conductor material is used to detect the X-rays together with processing electronics to analyze the spectrum.

 

Antibacterial screening:

Well-diffusion method was performed to determined antimicrobial activity against Bacillus subtillis and Staphylococcus aureus (gram positive bacteria) Pseudomonas aeruginosa, Klebsiella pneumoniae (gram negative bacteria). The culture of bacteria where swab bad uniformly on the individual plates using sterile cotton swabs on the Mueller Hinton Agar 4 wells were made on 6 mm in diameter in the Agar plates with help of gel puncture 40 microlitre of AgNPs 'A' , plant extract 'D', and antibiotic 'C' (streptomycin) and sterile silver nitrite 'B' was added.

 

RESULTS:

Synthesis of AgNPs:

Detailed study of extracellular biosynthesis of AgNPs of plant Ficus benghalensis showed good stability. Ag nitrate is used as reducing agent because of conducting stability. Two test tubes containing the filtrate of plant extract filtrated with Ag+ ions is incubated in the dark room temperature for 24 hrs. The changes in color from colorless to brown color confirm formation of AgNPs in reaction mixture.

 

UV visible spectroscopy[3,5]:

From the UV spectroscopy analysis and absorbance peak it can summarized that absorption peak is seen in the visible region. The absorption peak was found near 440nm after 12 hrs of incubation. UV absorption spectrum is basically used for determination of size and shape of nanoparticle when increase of silver nanoparticles reactivity would be less.

 

Figure 1. UV absorption of synthesised AgNPs from Ficus benghalensis

 

Field Emission Scanning Electron Microscopy (FESEM):

The FE-SEM image of AgNPs synthesized by green synthesis process by using Ficus benghalensis extract and 1mm AgNO₃ concentration was shown, silver nanoparticles were subjected to FE-SEM in the room temperature synthesized samples, of diameter of nanoparticles lies between 50-60nm. This technique visualize the size and morphology of particles and result obtained was particles are in spherical shape and averagely distributed in 60mm range.

 

Figure 2. FE-SEM analysis of silver nanoparticles (50-60nm)

 

Energy Dispersive X-Ray Analysis (EDAX):

EDAX reveals strong signal in the silver region and confirms the formation of silver nanoparticle. A typical absorption peak at 3kev confirms the metallic nanoparticles due to surface Plasmon resonance [6]. Other signal recorded due to enzymes or protein in Ficus benghalensis. This method also provides chemical analysis of the field view and confirms presence of specific elements, it is attached to SEM. The EDAX analysis displays signature spectra for silver and thus convincely evidence presence of noble metal.

 

X-Ray Diffraction Analysis (XRD):

Structural characterization have been performed using XRD analysis. To determine peak intensity position and full width at half maximum (FWHM) data was used with the Scherer; formula to determine main particle size.

 

 

 

Figure 3. EDAX analysis AgNPs

 

Figure 4. XRD analysis of AgNPs

 

 

Anti microbial activity of biologically synthesized AgNPs

 

Table 1. Zone of inhibition of synthesised AgNPs from Ficus benghalensis

 

Table 2. Comparative activity analysis of antimicrobial of AgNPs, plant extract antibiotic and AgNO₃

 

                              

K. pneumoniae                                                                                           S. aureus

                              

P. aeruginosa                                                                                                B. subtillis

 

Figure 5. Antibacterial activity of AgNPs from Ficus benghalensis against test pathogens

                           

         B. subtilus                                                                                            K. pneumoniae

 

   S. aureus                                                                                                P. aeruginosa

Figure 6. Comparative analysis of antimicrobial activity of AgNPs, plant extract antibiotic and AgNO₃

 

 

 

Synthesized silver nano particle showed excellent anti microbial activity against the Staphylococcus aureus, followed by Klebsiella pneumoniae, Bacillus subtills and Pseudomonas aeruginosa. Antibacterial effect was size and dose dependent and was more effective against gram negative bacteria than gram positive bacteria. It was observed that negative charge on cell surface of gram negative bacteria was higher than gram positive bacteria. Therefore, interaction between gram positive and silver nanoparticle was stronger than gram negative [7,8].

 

DISCUSSION:

Green nanoparticles were synthesized from Ficus benghalensis, when leaf extract was subjected to aqueous solution in 1 mM silver nitrate. A gradual colour change was observed after 12 hours which change to reddish brown. This change of colour could be due to the formation of AgNPs of various shape and size. The formation of the reduced AgNPs was further characterized by UV spectrometer analysis. It was generally recognized that UV visible spectroscopy a maximum absorption of AgNPs is around 440 nm which was co-relating [3,5].

Further various analytical studies was performed such as FE-SEM with the determined the size of nanoparticles which was around 50 – 60 nm and it was accordance with the work of Anuradha et al. (2010) [9]. EDAX study revealed the presence of specific and noble elements in AgNPs which were co-relating [6]. XRD study is very important to characterized the structure of crystalline material and used for the lattice parameters analysis of single crystals or the phase, texture are even analysis of sample. These results were in accordance with the result [8].

 

Antimicrobial study revealed that the synthesis nanoparticles from Ficus benghalensis was found to be very efficient against test pathogens such as Bacillus subtilus, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia, Shigella sp, a maximum zone of inhibition was seen in the concentration around 40 µl when compared to other concentration 10 - 30 µl against both gram positive and gram negative pathogens which was co-relating [3,10].

 

 

In comparative studies 40 µl of AgNPs AgNO₃ plant extract and streptomycin was added against various test pathogens by well diffused method. A maximum zone of inhibition was seen in standard antibiotic streptomycin and biologically synthesised nanoparticles. In future study synergistic activity of streptomycin and AgNPs can be performed at a pre-determined concentration to inhibit the growth of MDR strain. The novelty of work is to produce efficient AgNPs biologically and to inhibit the growth of resistance strain in a minimum concentration.

 

SUMMARY AND CONCLUSION:

The Ficus benghalensis aqueous extract has shown potential for extracellular synthesis of fairly monodispersed, silver nanoparticles in the range of 50–60 nm. The kinetics of silver nanoparticles synthesis using the cell filtrate indicates that the synthesis of nanoparticles would be suitable for developing a biological process. Furthermore, the extracellular synthesis would make the process simpler and easier for downstream processing. In future, it would be important to understand the biochemical and molecular mechanism of the synthesis of the nanoparticles by the cell filtrate in order to achieve better control over size and polydispersity of the nanoparticles.

 

The AgNPs shows highest antibacterial activity was observed in (gram-positive and gram-negative bacteria) Staphylococcus aureus, followed by Klebsiella pneumoniae Bacillus subtills and Pseudomonas aerginosa.. We believe that the silver nanoparticle has great potential for applications in catalysis, biomedical, and pharmaceutical industries.

 

All together the preliminary studied was carried out by above parameters and further studies will be need for prove better antibacterial and characterization studies in biologically synthesized silver nanoparticles.

 

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Accepted on 16.03.2017        © RJPT All right reserved