Synthesis of Plant Mediated gold Nanoparticles using Azima Tetracantha Lam . Leaves extract and Evaluation of their Antimicrobial Activities

Aim: The demand for nanoparticles is increasing day by day due to their wide range of applications in various areas including pharmaceutical industry. Nanoparticles are formally synthesized by chemical methods in which the toxic and flammable chemicals are used. Methods: This article reports about an effective, rapid and eco-friendly technique for the fabrication of gold nanoparticles from gold chloride solution using Azima tetracantha Lam. leaves extract. The effects of the leaves extract of Azima tetracantha, the concentration of Gold chloride solution, the time of the reaction and the effect of tem¬perature on the rate of the reaction were investigated. The synthesized gold nanoparticles (AuNPs) were characterized by using various techniques such as Dynamic Light Spectroscopy (DLS), Scanning Electron Microscopy (SEM), UV-Vis spectra gave surface plasmon resonance (SPR) at 540 nm, Fourier Transform Infrared spectroscopy (FTIR) and X-ray diffraction (XRD). This revealed the reduction of gold ions (Au+) to gold metal (Au0) which indicated the formation of gold nanoparticles (AuNPs). Results: The antimicrobial action of biosynthesized AuNPs indicated effective activity against bacterial pathogens Aeromonas liquefaciens, Enterococcus fecalis, Micrococcus luteus, Salmonella typhimurium and fungal pathogens Candida albicans, Cryptococcus sp, Microsporum canis, Trichophyton rubrum. Conclusion: This revealed that gold nanoparticles could provide a safer alternative to conventional antimicrobial agents.


INTRODUCTION
Indian flora is the chief and cheap source of medicinal plants and plant products.For many decades, these medicinal plants have been widely utilized in Ayurveda.Nowadays, many such plants are gaining impor tance due to their unique bioactive constituents and their versatile applicability in various fields of research and development.The recent developments in nanotechnology have influenced all types of life.With the advancement of technologies a completely unique approach emulated for analysis and development within plant biology field, medicine and nanotechnology, such as development in using plants or their elements for the inexperienced synthesis of nanoparticles.Nano technology could be a staggeringly developing powerful technology that holds an enormous promise for the development of many novel products with its potential medical applications on early illness detection, treatment, and hindrance.New technologies often create new challenges to science in addition to their benefits, raise concerns about health and various environmental problems.Recent nanotechnology holds a promise and a broad aspect towards wide applications of nanoparticles in a multiple way of emerging fields of science and technology.Over last decades, nanotechnology has established as the great innova tion of science and technology.A number of methods are available for the fabrication of nanoparticles mainly chemical synthesis like reduction in solutions, physical synthesis like thermal decomposition, radiation assisted process and recently through green chemistry route or biological synthesis using plants, bacteria and fungi.Most of the chemical and physical methods of synthesis of nanoparticles can control the size and shape of nanoparticles.Therefore, a biological process with the ability to perform the same has been an exciting prospect.
Various microorganisms are exploited for the biosynthesis of nanopar ticles but recently a new trend has come to force i.e., the use of plants and plant products for the synthesis of nanoparticles because of its sponta neous, cost effective, environmentalfriendly protocol, suitable for large scale production. 1 The main mechanism considered for the synthesis of nanoparticles mediated by the plants is due to the presence of phyto chemicals.The major phytochemicals responsible for the spontaneous reduction of ions are flavonoids, terpenoids, carboxylic acids, quinones, aldehydes, ketones and amides.Many research works are available on the biosynthesis of nanoparticles using plants and plant leaves extract, such as Ficus benghalensis, 2 Rosa rugosa, 3 Stevia rebaudiana, 4 Chenopodium album, 5 Nicotiana tobaccum, 6 Trianthema decandra, 7 Polyalthia longifolia, 8 Cycas species, 9 Pinus desiflora, Diopyros kaki, Ginko biloba, Magnolia kobus, and Pllatanus orientalis, 10 Catharanthus roseus, 11 Pungamia pinnata, Hemidesmus indicus, Syzygium cumini, Allium cepa, and Pandaanus odor ifer, 12 Sesuvium portulacastrum L, 13 Acalypha indica, 14 Parthenium hys terophorous, 15 Capsicum annuum, 16 Piper longum, 17 Arbutus unedo, 18 Oci mum santum, 19 and mulberry species. 20On the basis of available scientific literature, this methodology was designed with a very simple, quick, efficient and environmental friendly method for gold nanoparticles synthesis at appropriate conditions using Azima tetracantha leaves ex tract.

Preparation of Azima tetracantha Leaves Extract
Fresh leaves of Azima tetracantha, Figure 1 were collected from Keela paluvur village (Latitude-11 0 02'27.56"N; Longitude-79 0 04'08.08"E), Ariyalur district of Tamil Nadu in India.No specific permissions were required for collecting these leaves as these plants grow commonly in our village as well as this field studies did not involve endangered or protected species.Fresh A. tetracantha leaves were individually collected, thoroughly washed with running tap water and then with double distilled water, up to 5 days shadedried and by using mechanical grinder a fine powder was prepared.20 g of the fine leaf powder was mixed with 200 ml of presterilized Milli Q water.The mixture was boile dat 60°C for 10 min to allow the conversion by boiling water bath.What man no.1 filter paper were used to filter the extract and stored in a refrigerator at 4°C for further studies.

Gold nanoparticles synthesis 21
Gold chloride was prepared at the 10 3 M concentration with presteril ized Milli Q water.10 ml of the leaf extract was mixed with 90 ml of 10 3 M gold chloride for the synthesis of gold nanoparticles.Gold chloride was taken in similar quantities without adding leaf extracts to main respective controls.The containers were covered tightly with aluminium foil to prevent photo reduction of gold ions, and thereby incubated under dark condition at room temperature and observations were recorded.The colour of the solution mixture of gold chloride and Azima tetracantha leaves extract changed from pale yellow colour to dark pink colour at 60°C and 10 min of reaction time.This indicated the reduction of gold metallic (Au + ) ions to gold (Au 0 ) nanoparticles.

Characterization of gold Nanoparticles
UV-Vis Spectroscopy 22 The optical property of biosynthesized gold nanoparticles samples were measured at room temperature by UVVis spectrophotometer (Perkin Elmer) operated at 1 nm between 200 and 800 nm range of resolution.

Fourier Transform Infrared Spectroscopy (FTIR)
The characterization of functional groups on the surface of AuNPs by leaf extracts were investigated by FTIR analysis (Shimadzu) and the spectra were scanned in the range of 4000-400 cm −1 ranges at a resolution of 4 cm −1 .The samples were prepared by dispersing the Au NPs uniformly in a matrix of dry KBr, compressed to form transparent disc.KBr was used as a standard to analyse the samples.

Scanning Electron Microscopy (SEM)
SEM analysis of gold nanoparticles was done using Jeol JSM5800 SEM machine.a very small amount of the sample was droppe on a carbon coated copper grid and thin films of the gold nanoparticles were prepared.The thin films were allowed to dry the sample was analysed.

X-ray diffraction measurements (XRD)
XRD measurements of the reduced Au NPs were recorded on Xray diffractometer (x'pert pananalytical) instrument operating at a current of 30 mA with Cu K (α) radiation voltage of 40 kV to determine the crystalline phase and material identification.The samples were taken in lids and put under instrument for analysis.All the data were collected in the angular range 30≤2θ≤500, under the same experimental conditions,

UV-Vis Spectral Study
Biosynthesized leaves extract mediated AuNPs particles were confirmed using UV-Vis spectrophotometer by analysing the excitation due to the applied electromagnetic field of surface plasmon resonance (SPR) at 540 nm and the peak was observed between 535550 nm. Figure 2 shows the UV absorption peaks of A. tetracantha.It clearly indicated the formation of AuNPs of the leaves extract of the plant.The change in colour is due to the excitation of surface plasmon vibration, which is indicated by the reduction of Au + ions to Au 0 ions at different time intervals.During each interval of time, the peak became clear, unique and rising.This unique peak clearly indicates the increase in synthesis of nanoparticles as the time increases.Similarly, the colour also became intensified as the time increases.Similar results were observed by some other researchers. 24,25ayaseelan et al, 2013 26 has reported that aqueous extract of Abelmoschus esculentus seeds showed the SPR at 536 nm.

FTIR spectrum of Azima tetracantha Leaves Extract mediated gold nanoparticles
To determine the functional groups of Azima tetracantha leaves extract, a FTIR analysis was done and the results were shown in Figure 3.The Azima tetracantha leaves extract exhibited a number of absorption peaks, reflecting its complexity in nature.FTIR analysis revealed the strong bands at 3306, 2124, and 1637 cm 1 .The band at 3306 cm 1 corre sponds to NHamine amino groups, 2124 alkane CH stretchinglipids, 1637 cm 1 corresponds to amide amino groups.FTIR analysis of the Azima tetracantha leaves extract indicated that the carboxyl groups (C=O), hydroxyl groups (OH) and amine (NH) groups of Azima tetracantha leaves extract are mainly involved in reduction of Au + to Au 0 nanoparticles.Same results were reported by other researchers and re ported that wave numbers signal stretching and vibrational bending of the peaks may be derived from compounds such as flavonoids, terpe noids, alkaloids and soluble proteins present in plants extracts and these may be responsible for the stabilization of gold nanoparticles. 27

Scanning Electron Microscopy (SEM)
SEM analysis of the products was recorded and the synthesised gold nanoparticles are found to be spherical in structure of about 80 nm in diameter (Figure 4).The SEM image showed the formation of gold nanoparticles using A. tetracantha leaves extract and confirmed the development of gold nanostructures.The same results were observed by SobczakKupiec et al. 28

X-ray diffraction measurements (XRD)
Gold nanoparticles were synthesized using leaves of A. tetracantha and the synthesis was confirmed by observable colour change in the mixture and also confirmed by UVVIS spectrum.Subsequently, XRD analysis    was used to analyse the phase distribution, crystallinity and purity of the synthesised nanoparticles.Xray diffraction (XRD) pattern of syn thesized particles were analysed and peak profile of relevant particles were found.
In Figure 5, a peak was observed at range of 2θ values of 38 which corre sponded to the Bragg's reflections such as 111, 200, 220 and 311.Similar peaks and lattice planes were observed by other researchers also. 24,25timicrobial Studies The antimicrobial activity of the antibiotics (Positive controlMethicilin and Itraconazole) and Azima tetracantha leaves extract mediated gold nanoparticles were challenged against various NCIM and MTCC microbes agar well diffusion assay.The test concentrations (15 and 30 μl/disc) produced zone of lysis on MHA and PDA plates for bacteria and fungi respectively.Azima tetracantha leaves extract mediated gold nanoparticles were most effective against Salmonella typhimurium NCIM 2501 (B4) while minimal effect was observed from Micrococcus luteus NCIM 2871 (B3) in the bacterial division.But in fungi, which was effective against Trichophyton rubrum MTCC 3272 (F4) produced a minimal smaller effect in Cryptococcus sp.MTCC 7076 (F2) (Figure 6 and Figure 7).The higher (30 μL/disc) concentration showed larger zone effect than the lower (15 μL/disc) concentration against certain micro organisms.All the microbial strains exhibited higher sensitivity to the higher concentration (30 μL) for the test sample when compared to the positive control except B3 (Table 1).Grace and Pandian 29 also reported a great bactericidal effect of gold nanoparticles and the welldeveloped chemical stability, appropriate smaller size make gold nanoparticles and surface chemistry easier to interact with the microorganisms.

CONCLUSION
This study advanced a fast, effective, convenient biological method and environmentalfriendly for the fabrication of stabilized gold nanopar ticles of average diameter of 80 nm using the leaves extract of Azima tetracantha.The formation of gold nanoparticles was confirmed and characterized by UVvis, FTIR, XRD and SEM analytical methods.The antimicrobial activity of biologically synthesized leaves mediated gold nanoparticles was evaluated against human bacterial pathogens Aeromonas liquefaciens, Enterococcus fecalis, Micrococcus luteus, Salmonella typhim urium and fungal pathogens Candida albicans, Cryptococcus sp, Micros porum canis, Trichophyton rubrum showing effective antimicrobial activity.