Eco-Friendly Synthesis of Silver Nano Particles Using Carica papaya Leaf Extract

Silver nanoparticles were synthesized using eco-friendly method with extract of Carica papaya as reducing and stabilizing agent. The silver precursor used was silver nitrate solution. A visible colour change from colourless to reddish brown confirmed the formation of the nanoparticles and the UV-Vis spectroscopy showed surface plasmon resonance of 435 nm for the silver nanoparticle. The mean particle size was 250 nm while the polydispersity index was 0.22. The antimicrobial activity of the synthesized nanoparticles was studied against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The silver nanoparticles biosynthesized showed satisfactory antimicrobial activity against the test isolates. Antimicrobial property of the nanoparticles was similar (P > 0.05). Generally, MIC values of the samples against the microorganisms tested ranged from 25 100 mg/ml. Pseudomonas aeruginosa was most sensitive while Staphylococcus aureus and Bacillus subtilis were least sensitive to the silver nanoparticles.

But most of the chemical methods used for the synthesis of nanoparticles involve the use of toxic hazardous chemicals that create biological risk and sometimes these chemical processes are not eco-friendly. Therefore, there is a growing need to develop cost-effective, non-toxic and eco-friendly methods for the synthesis of silver nanoparticles using simple techniques and readily available equipment. The use of plants and microorganisms in the synthesis of nanoparticles has emerged as an eco-friendly and exciting approach [2] [3].
In recent times, plant extract has been used as reducing and capping agent for the synthesis of nanoparticles. The use of plant extract is more beneficial as it does not involve sophisticated processes such as intracellular synthesis and multiple purification steps or the maintenance of microbial cell culture [4]. Plants extracts from Ocimum tenuiflorum, Solanum tricobatum, Syzygium cumini, Centella asiatica and Citrus sinensis have been used as reducing agents in the synthesis of silver nanoparticles (Ag NPs) from silver nitrate solution [5]. Synthesis of silver nanoparticles has also been done using Citrullus lanatus [6], Murraya koenigii [7] and Eriobotrya japonica leaf extract [1].
Nanoparticles are now considered viable alternatives to antibiotics as they seem to possess a high potential to address the problem of the emergence of bacterial multidrug resistance [8]. Silver nanoparticles have attracted much attention in the science [9]. Silver has always been used against various infections and functions as both antiseptic and antimicrobial agent against gram-positive and gram-negative bacteria [10]. Silver nanoparticles were considered, in recent years, particularly attractive for the production of a new class of antimicrobials [8] [11] [12], opening up a completely new way to combat a wide range of pathogenic bacteria.
This study aims to synthesise silver nanoparticles using Carica papaya leaf extract and also to determine the antimicrobial of the nanoparticles synthesised.

Materials and Method
Silver nitrate (Sigma U.S.) and plant extract (Carica papaya) were the materials used. Other chemicals and reagent were of laboratory grade.

Microorganisms Used
Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Staphylococcus aureus collected from Microbiology Postgraduate laboratory, University of Uyo purified by sub-culturing several times to obtain pure cultures. The plant leaves were thoroughly washed with tap water to remove dust particles and other unwanted materials accumulated on the leaves. The dust free leaves were pulverized and kept to dry under shade in the Pharmaceutics laboratory for 24 h. The dried leaves were then powdered by using an electric blender.

Extraction Procedure
50 g of the powdered plant material was put in 500 mL conical flask and 250 mL of distilled water was added. The conical flask was covered with aluminum foil and kept in a reciprocating shaker for 24 h for continuous agitation at 150 rpm for thorough mixing. Then, the extract was filtered by using muslin cloth followed by Whatman no 1 filter paper. The resultant solution was kept for the nanoparticle synthesis.

Characterisation of Silver Nano-Composites: UV-VIS Spectroscopy to Determine Surface Plasmon Resonance for Silver Nanoparticles
UV-Vis spectral analysis was done using a double-beam spectrophotometer (Hitachi, U-3010) with the samples dispersed in distilled water and kept in a quartz cuvette with a path length of 10 mm. The photoluminescence emission spectra from the samples (dispersed in distilled water) were recorded by a spectrofluorometer (LS 55, Perkin Elmer) at room temperature using a four sided polished quartz cuvette with a path length of 10 mm.

Antimicrobial Studies of Carica Silver-Nanocomposites
The silver nanoparticles biosynthesised from the Carica papaya leaf extract was screened for antimicrobial activity using the agar well diffusion method described by Okeke et al., 2001 [13].
0.1 ml of each of the organisms was aseptically spread on the surface of the Muller-Hinton agar plate using sterile bench Hockey stick. These plates were left on the bench for thirty minutes to pre-diffuse into the medium. A sterile cock borer of 5 mm was used to bore holes on the agar plates. The silver nanoparticles concentrations were graded as 500 mg/ml, 400 mg/ml, 200 mg/ml, 100 mg/ml. About 0.5 ml volume of each diluted silver nanoparticle was used to fill the agar wells made in the Muller-Hinton agar plates. The plates were allowed to stand for one hour to allow the extract to diffuse into the medium.
1% Silver nitrate was used as control. All plates were incubated at 37˚C for 24 -48 hours.
Antimicrobial activities of the silver nanoparticles and the control against microbial isolates were determined by measuring the inhibition zone diameter in cm.
The Minimum inhibitory concentrations were determined by preparing different concentrations 200 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml and mixed with the medium and then the organisms were streaked on the plates and incubated for 24 hours at 37˚C. The minimum inhibitory concentration was determined by checking the plate for the line of streaking of the minimum concentration of the silver nanoparticle without growth.

Results and Discussion
The results for characterization of synthesized nanoparticles are shown in Table  1. The results for the determination of antimicrobial activities of synthesized silver nanoparticles are shown in Table 2 and Table 3.

Antimicrobial Studies
The antimicrobial activity of silver nanoparticles was carried out against both Gram positive and Gram negative bacteria. The synthesized silver nanoparticles exhibited good antibacterial activity against both Gram positive and Gram negative bacteria.
Silver has been known to impart antimicrobial activity to bacteria. Dilute solutions of silver nitrate were used as far back as the 19 th century for the treatments of infections [14]. Hence, silver nitrate solution was used as a control system in this research work.

Colour Change
Reduction of silver ions into silver nanoparticle by the plant extract was confirmed by a colour change from colourless to reddish brown. The colour change was due to the surface plasmon resonance (SPR) phenomenon. The metal nanoparticles have free electrons, giving the SPR absorption band due to the combined vibration of electrons of metal nanoparticles in resonance with light.

Conclusions
The rapid biosynthesis of silver nanoparticles using the leaf extract of Carica papaya provides an efficient, cost-effective and eco-friendly route for the synthesis. The colour change from colourless to reddish brown observed is the characteristics of silver nanoparticles due to SPR phenomenon. UV-Vis spectroscopy confirmed the formation of silver nanoparticles with absorption peak at 435 nm for the entire nanoparticle. The antimicrobial activity of synthesized nanoparticle was studied against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The nanoparticles synthesized have satisfactory inhibitions against the four mentioned microorganisms with Pseudomonas aeruginosa being the most sensitive.