Green Synthesis and Characterization of Silver Nanoparticles Using Leaves Extracts of Neem ( Azadirachta indica ) and Bitter Leaf ( Vernonia amygdalina )

: Silver nanoparticles (AgNPs) were synthesized using Azadirachta indica and Vernonia amygdalina leaves extracts. Current nanotechnology research uses a lot of chemicals which are quite often toxic and flammable. In this research article, a simple and eco-friendly synthetic method for silver nanoparticles preparation was reported using the leaves extracts of Azadirachta indica (commonly called neem) and Vernonia amygdalina (commonly called bitter leaf) as reducing agents. The synthesis of AgNPs was monitored and confirmed with the use of UV-Visible spectrophotometer, Fourier Transform Infrared (FTIR) spectroscopy and Powder X-ray Diffraction (PXRD). The reduction process of Ag + to Ag 0 was observed by the change of color from yellow to brown for both leaves. The UV-Vis Spectra of AgNPs in aqueous solution showed absorbance peaks around 455 nm for Azadirachta indica and 460 nm for Vernonia amygdalina due to silver surface plasmon resonance. Crystallinity of the AgNPs was confirmed with PXRD. In addition, FTIR spectra showed that the AgNPs were capped with phytochemicals from the leaves extracts.

Nanotechnology is an important field of applied science and technology dealing with design, synthesis and manipulation of structure of particles ranging from approximately 1-100 nm. AgNPs are widely researched for diverse applications because of their fabulous superior characteristic features based on their properties such as size, morphology and other sizedependent properties (Smith et al., 2016). Nanoparticles have drawn the attention of researchers because of their extensive applications in areas such as mechanics, optics, biomedical sciences, chemical industry, electronics, space industries, drug gene delivery, energy science and catalysis (Schmid et al., 1992;Hoffman et al., 1992). Nanoparticles can be synthesized by various methods such as chemical and photochemical reactions in reverse micelles, thermal decomposition, electrochemical, sonochemical, microwave assisted process, and also by biological methods (Iravani et al., 2014). Among these methods, biological processes that are based on bacteria, fungi, bio-derived chemicals and plant extracts are considered as safe and economically viable for the nanomaterial fabrication (Valli and Vaseeharan, 2012). Plant extracts produce best capping material for the stabilization of silver nanoparticles. Researchers have discovered that phytochemicals present in plant extracts are responsible for metal ion reduction and capping of the newly formed particles during their growth processes (Smith et al., 2006;Wei et al., 2005). Alkaloids, flavonoids, terpenoids, ketones, amides, aldehydes, polyphenols and carboxylic acids present in plants are good promoters for bioreduction of metal ions into nanoparticles and also in supporting their subsequent stability. The objective of this work is to synthesize silver nanoparticles by simple, effective and eco-friendly method using leaves extracts of Azadirachta indica (commonly known as neem), a member of the Meliaceae family and Vernonia amygdalina (commonly known as bitter leaf) a tropical plant belonging to the family Compositae. The objective also includes the characterization of the as-synthesized nanoparticles. Both plants are known to be medicinal with various therapeutic benefits (Abay et al., 2015;Asante et al., 2016;Luo et al., 2011;Sinisi et al., 2016;Subarpriya, 2005). The synthesized silver nanoparticles were characterized by UV-Vis spectroscopy, FTIR and powder X-ray diffraction (PXRD).

MATERIALS AND METHODS
AgNO3 and 0.2 µm membrane filter paper are from VWR chemicals. Fresh leaf samples of Vernonia Amygdalina and Azadirachta Indica were purchased from Free Zone market at Aba, Abia State, Nigeria. UV-Visible spectra were taken on JASCO UV-Vis V-730 spectrophotometer with a resolution of 1 nm.
FTIR spectra analyses of the as-synthesized nanoparticles were taken on a Shimadzu FTIR spectrometer from 4000 cm -1 to 400 cm -1 at Redeemer University of Nigeria.
The fresh leaves were first dried under a shade until the moisture content was reduced. The plants were grounded to tiny particles and stored in polythene bags until used. Preparation of plant extracts: The leaf extract was prepared by taking 10 g of the grounded dried leaves into a 500 mL beaker with the addition 100 mL of distilled water and then stirred for about 15 minutes. The mixture was incubated in a cupboard for 30 minutes at 25 o C and was allowed to settle for another 30 minutes. Clear extract was collected by filtration using Whatman filter paper and then stored in the fridge at 4 o C for further use. This method was used for both leaves separately.
Synthesis of Silver Nanoparticles: Aqueous solution of silver nitrate (AgNO3) at concentration of 0.1 M was prepared and used for the synthesis of AgNPs. For the reduction of Ag + ions, 1 mL of A. indica extract was added into a clean test-tube and then 9 mL of 0.1 M aqueous AgNO3 solution was added into the extract. On addition of aqueous AgNO3 to the extract, colour change was noticed after about 30 minutes from yellow to brown. For the reduction of Ag + ions using V. amygdalina, 1 mL of the extract was added into a test-tube and then 9 mL of 0.1 M aqueous AgNO3 solution was added into the extract. On addition of AgNO3 to the extract, colour change was noticed after about 2 minutes from yellow to brown. Formation of AgNPs at different time intervals were monitored between 200 and 700 nm. Distilled water was used as blank. After the complete formation of the nanoparticles, the solution was then filtered using 0.2 μm membrane filter paper to separate out the nanosized particles from the suspension. The nanoparticles were then allowed to dry so as to be used for further analysis.

RESULTS AND DISCUSSION
Characterization of Silver Nanoparticles: UV-Vis Spectroscopy Analysis: The aqueous extracts of leaves of both neem and bitter leaf were used for the green synthesis of silver nanoparticles. The silver nanoparticles (AgNPs) appear brownish in colour in aqueous medium as a result of surface plasmon vibrations (Arya et al., 2017). As the different leaves extracts were added to aqueous silver nitrate solution, the colour of the extracts changed from yellow to brown for both Azadirachta indica and Vernonia amygdalina and finally with the formation of brownish precipitates indicating AgNP formation. Similar changes in colour have been observed in previous studies and hence confirmed the completion of reaction between leaf extract and AgNO3 (Arya et al., 2017). This was also confirmed by the UV-Vis spectrum of the precipitated silver nanoparticles which has been recorded as a function of time as shown in Figures 3 and 4. Generation of AgNPs exhibits unique and tunable optical properties resulting from its surface plasmon resonance (SPR) which is dependent on shape, size, and size distribution of the formed nanoparticles (Arya et al., 2017). Typical SPR of AgNPs appear near 440nm and is due to the collective oscillation or vibration of free electrons in the conduction band after excitation by incident light of particular wavelength. Excitation of surface plasmonic vibrations due to the reduction of Ag + ions was measured spectrophotometrically at different time intervals. The UV-Vis spectra recorded showed that the bioreduction of silver ions was achieved using neem and bitter leaf extracts as reducing agent. After 72 hours, it was observed that the formation of AgNPs with neem extracts was completed and after 24 hours, the formation of AgNPs synthesized with Bitter leaf extracts was completed.
The sharp bands of silver nanoparticles were observed around 455 nm in case of Azadirachta indica ( Figure  3), whereas the bands for Vernonia amygdalina were observed around 460 nm (Figure 4). From different literatures it was found that silver nanoparticles show SPR peak between 420 and 460 nm. The appearance of SPR at 455 nm and 460 nm and broadening of the peak indicated the formation of polydispersed large nanoparticles due to slow reduction rates (Arya et al., 2017).

NZEKEKWU, AK; ABOSEDE, OO
The medium bands at ca 1500 and 1380 cm -1 are attributed to -C=C-stretching mode and C-N stretching vibrations of aromatic amines respectively. bands at 1050 and 850-900 cm -1 are due to C-OH stretching of alcohols and C-O-C vibrations respectively. Therefore, FTIR analysis of the synthesized AgNPs using both neem and bitter leaves extracts respectively showed that these functional groups from the aqueous extracts of neem and bitter leaf were responsible for the reduction Ag + to Ag 0 , the AgNPs synthesis and stabilization, and are present on the surface of the synthesized nanoparticles. The Xray diffraction of the nanoparticles ( Figure 6) reveal that the nanoparticles are crystalline and also confirms that they are capped by biomolecular compounds which were responsible for the reduction of silver ions. Conclusion: Silver nanoparticles have been synthesized using leaves extracts of neem and bitter leaf showing absorption peaks at 455 nm and 460 nm respectively. FT-IR spectra also showed that the silver nanoparticles were capped by biomolecular compounds which were responsible for the reduction of silver ions. From the technological point of view, these obtained silver nanoparticles have potential applications in the biomedical field and this simple procedure has several advantages such as costeffectiveness, compatibility for medical and pharmaceutical applications.