**2. Materials and methods**

### **2.1 Preparation of plant extract**

*Syzygium cumini* leaves were thoroughly washed in running tap water for 15 min and then shade dried for two days at room temperature. Dry leaves were ground into fine powder in a mortar and pestle. The extract obtained was filtered through Whatman filter paper No.1. The filtrate was collected and stored at 4°C, which was further used for all experiments [24].

#### **2.2 Synthesis of AgNPs**

The well-grinded material was mixed with 100 mL of double distilled water and then transferred in 500 mL Erlenmeyer flask followed by continuous stirring on the magnetic stirrer for 10 min. The content was centrifuged at 10,000 rpm for 10 min for the removal of cell debris. 50 mL of aqueous silver nitrate (1 mM) was added to 10 mL of the leaf extract with continuous stirring. A color change from colorless to yellowish-brown, visually confirms the formation of AgNPs [25].

#### **2.3 Characterization**

The resulting solution was then diluted by using double distilled water and characterized using UV–Visible spectroscopy, X-ray diffraction, Energy dispersion spectroscopy, FT-IR and Transmission electron microscopy [26].

#### *2.3.1 UV-visible spectroscopy*

Silver nanoparticles were characterized by using Systronics UV–Vis spectrophotometer. The bio-reduction absorption spectra were monitored in 300–700 nm range.

#### *2.3.2 X-ray diffraction spectroscopy*

The biosynthesized AgNPs using *Syzygium cumini* leaf extract were lyophilized to a powder. The powdered or dried AgNPs were coated on the XRD grid, and the spectra were recorded using Rich Seifert p 300 instruments.

#### *2.3.3 Transmission electron microscopy and energy dispersive spectroscopy*

In order to know the morphology of the biosynthesized AgNPs, transmission electron microscopy (TEM) studies were carried out. The size and shape of the AgNPs were recorded by using the FEI (Netherland) model TECNAI-G2U twin operated at an accelerating voltage of 200 KV. EDS analysis was carried out at the same time by the EDS compatible with TEM.

#### *2.3.4 Fourier transform infrared spectroscopy (FTIR) analysis*

After the biosynthesis, AgNPs were centrifuged for 15 min at 10,000 rpm. The obtained pellet was re-dispersed in double distilled water to ensure the removal of any uncoordinated bio-molecules. In order to obtain the better separation of nanoparticles, the process of centrifugation was repeated twice. The purified pellet was then subjected to FTIR analysis (Shimadzu IR). AgNPs were mixed with KBr and subjected to IR source 500–4000 cm−1.

#### **2.4 Catalytic reduction of 4-nitrophenol**

The catalytic reaction was studied as mentioned by Ghosh et al. with slight modification. Briefly in standard quartz cuvette 1 mL of 0.1 mM aqueous NaBH4 solution mixed with 1.5 mL of 4-nitrophenol aqueous solution (0.25 mM). 100 μL of an aqueous suspension of AgNPs of *Syzygium cumini* (in double-distilled water) was added into the same and time-dependent absorption spectra were recorded every 5 min in the range of 260–520 nm at 28°C [27].

#### **2.5 Antimicrobial activity**

The antimicrobial activity of the biosynthesized AgNPs was tested against pathogenic bacteria such as *Serratia marcescens* (NCIM 2078), *Staphylococcus aureus* (NCIM 5021), *Pseudomonas aeruginosa* (NCIM 5029), *Salmonella typhimurium* (NCIM 2501) and *Klebsiella pneumonia* (NCIM 2957), etc. The organisms were collected from National Chemical Laboratory (NCL), Pune. Uniform spreading of bacterial cultures was carried out in the individual plates using a sterile glass spreader. Wells were made on the agar plates using a cork borer to about 10 mm diameter in nutrient agar medium. 100 μg of lyophilized AgNPs were added in 100 μL of distilled water. 50 μL dispersed solution was added to the well. The diameters of the inhibition zone surrounding the wells were measured in millimeters after 24 h. The antimicrobial effect of the biosynthesized AgNPs is directly proportional to the size of the spherical inhibition zone against microbial pathogens [28–30].

### **3. Result and discussion**

#### **3.1 Biosynthesis of AgNPs**

The reduction of Ag<sup>+</sup> to Ag0 NPs was carried out by using aqueous leaf extract of *Syzygium cumini*. The color change of the solution observed from colorless to yellowish-brown indicates that the synthesis of AgNPs shown in (**Figure 1(i)**). The UV–Vis absorption spectrum of the biosynthesized AgNPs demonstrated a characteristic absorption peak at 470 nm, which is a typical band for the silver shown in **Figure 1(ii)**. No other peak was observed in the spectrum, which confirmed that silver only [31]. The formation of AgNPs was further confirmed by using X-ray diffraction (XRD), FT-IR, EDS and transmission electron microscopy (TEM) analysis.

#### **3.2 Characterization**

#### *3.2.1 Transmission electron microscopy analysis*

The TEM image of the AgNPs is shown in **Figure 2**. TEM has been used to describe the size, shape and morphology of the biosynthesized AgNPs. From the figures, it is observed that the morphology of AgNPs is a hexagonal matrix. **Figure 2** shows the average particle size measured from the TEM image is 50 nm, which are in good agreement with the particle size calculated from XRD analysis.

Syzygium cumini *Mediated Green Synthesis of Silver Nanoparticles for Reduction... DOI: http://dx.doi.org/10.5772/intechopen.98473*

#### **Figure 1.**

*(i) Color change observed (a) before and (b) after formation of AgNPs. (ii)UV–visible spectra of the synthesized AgNPs.*

**Figure 2.** *TEM image of AgNPs.*

#### *3.2.2 X-ray diffraction analysis*

The presence of Ag crystal in the sample was confirmed by using an X-ray diffractometer. In XRD pattern, the Braggs reflections were observed at 2θ value 38.00 , 44.80 , 47.50 , 64.60 and 78.00 confirm the presence of AgNPs (**Figure 3**). A strong diffraction peak located at 38.00 was ascribed to the (111) facets of Ag. The XRD pattern thus clearly indicated that the AgNPs are in crystalline form. No impurities were observed in the XRD pattern.

**Figure 3.** *XRD pattern of synthesized AgNPs.*
