Antibacterial Activity of Neem Extract and its Green Synthesized Silver Nanoparticles against Pseudomonas aeruginosa

Azadirachta indica, generally called as neem, margosa or Indian lilac is an Indian subcontinent native species, it ie well known for its various bioactivity In this study, neem leaves were collected and extracted. The extract was subjected for TLC and antibacterial activity against Pseudomonas aeruginosa. The extract was subjected for green synthesis of silver nanoparticles and it was able to reduce silver to silver nanoparticle of size around 65nm. The silver nanoparticle was also exhibiting antibacterial activity against P.aeruginosa.

Over two millennia, neem trees products have been utilized in India as traditional medicines due to their novel properties 1 . Basically, neem is called arishtha in Sanskrit 2,3 which means 'reliever of sickness' 3 and in India, Neem is also called as the 'village pharmacy' 4 for its properties to relieve from various infections, pains and fever. These most popularly known Neem tree is scientifically called as Azadirachta indica and is found in tropical regions indigenous to Indian subcontinents 5 . The other species closely related to A. indica is Melia azedarac also known as the Persian lilac 3 . The biological activities of neem include antiinflammatory, anti-pyretic, hypoglycaemic, antifungal, anti-gastric ulcer, diuretic, antibacterial, antitumour, antimalarial, anticancer, hypolipidemic etc 1,3,6,7 . Thus in Ayurveda, Neem (Nimba) has been long used for the control of malarial fever, blood disorders, leprosy, wounds, eye disorders and ulcer 8 and hence is referred to as 'Sarva-Rogha-Nirvarini' which means 'curing ailment' in Ayurveda 3,8,9 . In a few experimental reports, the different approaches for the production of polymeric resins using neem is documented 10 . Neem is best known for its pesticide activity 11,12 . One of the primary mode of action of neem against insects is the disruption of its metamorphosis and the bitter flavour aids in keeping them away from feeding the host plants making it an exemplary antifeedent and repellant 13 . Recently neem has acquired a great deal of global attention towards it 12 for its versatile ability in medicine and agriculture. As a sign of global acceptance, US national academy of sciences has titled neem as 'The tree for solving global problems' 9 and the ability of neem extracts to prevent from tooth decaying, inflammation of  gums are proved by various tests in Germany and it is currently exploited as an active ingredient in toothpastes in Germany and India. In 1985, neem based insecticide called Margosan-O was accepted by the Environmental protection agency for non-food uses and ever since various other neem based products were also approved by various administrations and agency for food and feed crops 14 . Neem can also be used to produce nanoparticles for its ability to reduce the metallic ions to yield nanoparticles 15 . Silver salts is known widely for its anti-bacterial effects having high toxicity towards micro organisms and is used for the same in many applications like dental work, catheters and burn wounds 16,17 . The novel properties of silver are also exploited in the field of nanotechnology. Nanoparticles are clusters of atoms that are very small ranging from the size 1-100nm and are known for their high surface area to volume ratio which aids its applications in many fields 18 . Silver nanoparticles (AgNPs) are used as anti-microbial agents in health, food, textile industries and their products are also approved by US FDA, US EPA, SIAA of Japan, FITI testing and research institute, etc 19 . Though there are various ways to synthesise nanoparticles, the most nontoxic and eco-friendly method is the bio-based method which utilises various biologically products such as microbes and plants 20,21 . Hence this study focuses on the evaluation of the bioactivity of the neem extract and its ability to produce silver nanoparticles. The antibacterial effect of both the extract and the AgNPs is also discussed. Senthilkumar

sample collection and extraction
Leaves of A. indica was collected from Chennai, Tamil Nadu, India and cleaned, then it was shade dried. The powdered leaves were added with ethanol and left for 3 days. The solvent was then filtered using Whatmann no.1 filter and then dried.

Thin layer chromatography
Thin Layer Chromatography was performed with TLC silica plate (Merk F245) for the sample and various solvents like chloroform, methane, ethanol, dichloromethane and hexane were used as mobile phase. The plates were exposed to iodine agar well diffusion assay Agar well diffusion method was done to identify and evaluate the anti-bacterial activity of the extract obtained against Pseudomonas aeruginosa 22 .

synthesis of agnPs
The extract was mixed with 3mM Silver nitrate solution in water in the ratio of 1:5 and was kept in the dark for 24h in room temperature in order to produce and settle silver nanoparticles.

Characterization of agnPs
The neem extract and the nanoparticles so produced were subjected to UV-Visible spectroscopy (Shimadzu 1800, Japan) and Fourier Transform Infra-red Spectroscopy (Shimadzu, Japan). Silver nanoparticles were subjected to EDX spectroscopy and Scanning electron microscopy (SEI and BSI).

agar well diffusion assay of agnPs
The nanoparticles were subjected to agar well diffusion assay to evaluate the antibacterial activity of it against Pseudomonas aeruginosa 22 with a concentration range from 2 to 16µg/ml. The positive control used was Ciproflaxacin.

Thin layer chromatography
The ethanol extracted samples were run using TLC with various mobile phases and the plates were exposed to iodine. Four bands were seen when chloroform was used as the mobile phase ( Figure 1c).

agar well diffusion assay
When the extract was subjected to antibacterial assay, it was found that the concentrations above 15µg/ml were acting against the bacterial cultures from the zone of inhibition (Table. 1).

Characterisation of agnPs
The absorbance spectra for the neem extract and the nanoparticles were obtained and it was found that the peaks near 360, 375, 380 and 440nm in the spectrum obtained for neem extract (Figure 2a) had a shift to 385, 440, 495 and 500nm in the spectrum obtained for the AgNPs (Figure 2b).
In another study 24 on the biologically synthesised nanoparticles, absorption maxima was seen at the range of 440-500nm which is in par with the current study.
The IR spectrum of both the neem extract and the neem based silver nanoparticles were evaluated (Figure 3a, b). They both had a few bands in common with amide groups (at around 1640 cm -1 ) 25 , hydroxyl group ( around 3330 and 1045 cm -1 ) 26 . The extract was found to have band at 1085 which corresponds to the -C-N bonds of alcohols, esters, ethers and carboxylic acid while the AgNP had bands at 1339 and 1396 which corresponds to the C-N stretching of aromatic amine groups 27 . From the IR specctrum it can be confirmed that the amino acid residue and the protein groups present might have the strongest ability to bind with the metal nanoparticle succesfully aiding in both formation and stabilisation of the particles 28 .
The spectrum obtained confirms the presence of silver in the analysed sample with the presence of the typical range for the absorption of metallic silver nanocrystals at 3KeV (Figure 4b).
The weak signals showing the presence of C, O suggests the capping of AgNPs with the organic compound from neem extract 29,30 .
The Scanning electron micrographs of the silver nanoparticle shows the size of the ranging from 46 to 94nm and the presence of highly aggregated nanoparticles. (Figure 4a). In a study, less than 20nm spherical nanoparticles were produced using neem gum 30 and 12nm sized nanoparticles were prepared using neem plant extracts 31 .

agar well diffusion assay of agnPs
From Figure 5, it is evident that the antibacterial activity of the AgNPs increased with the increase in concentration when checked against Pseudomonas aeruginosa. This shows that the nanoparticles are bactericidal.

ConClusion
From the above study it is clear that the neem extract and its silver nanoparticles have antibacterial activity against P.aeruginosa. The nanoparticles were found to be capped by the organic compounds present in the neem extract which could be a reason for the production of nanoparticles and also for its stability.