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later exposed to infrared light (30 min) for solvent evaporation. TEM observations were performed on Transmission electron microscope (PHILIPS model CM 200) operated at an accelerating voltage of 200 kV with the resolution of 0.22 nm. 3.3. X-ray diffraction studies (XRD) Colloidal solution of phytosynthesized SNPs was layered on the glass holder and was allowed to dry. The diffraction pattern was recorded from diffraction angle range of 20o to 80o. The XRD studies were con- ducted using Rigaku mini flex bench top X-ray spectrophotometer. The XRD method is suitable to determine the crystal structures by analyzing the positions and intensities of diffraction peaks typically observed for well-crystallized material. 3.4. Fourier transform infra-red (FTIR) FTIR measurement was carried out to identify the possible biomole- cules responsible for surface capping and reducing agent for the Ag nanoparticles synthesized in E. scaber leaf extract. For FTIR analysis, 2–3 drops of colloidal solution of phytosynthesized AgNPs were thor- oughly mixed with KBr powder for moisture absorption in a clean mor- tar and pestle. Similarly, the plant extract was processed. FTIR spectra were recorded using Perkin Elmer FTIR Spectrum-100 model. 3.5. Anti-oxidant activity 3.5.1. DPPH radical scavenging assay The DPPH is a stable free radical and is widely used to assess the rad- ical scavenging activity of antioxidant component. DPPH scavenging ac- tivity was measured using the method described by Brand-Williams et al., (1995) with slight modifications. 1,1-diphenyl-2-picrylhydrazyl (DPPH) methanolic solution (0.002%) was used. DPPH concentration is reduced by the existence of an antioxidant at 515 nm and the absorp- tion gradually disappears with time. This method is based on the reduc- tion of DPPH in methanol solution in the presence of a hydrogen donating antioxidant due to the formation of the non radical form of DPPH. Briefly, 0.002% solution of DPPH in methanol was prepared, and 180 μl of this solution was added to 20 μl of the solution of all AgNP sam- ples at different concentrations. 5 mg AgNPs were dissolved in 1 ml of HPLC water to obtain the aliquot of AgNPs (50, 100, 150, 200 and 250 μg/ml). The diluted working solutions of the test AgNPs were prepared in HPLC water (sonicated for 10 min). The mixtures were kept at room temperature for 30 min. The analysis was performed using a Synergy H1-Multi plate reader (Biotek) instrument [15,16, and 17]. Ascorbic acid was used as a positive control (standard) and methanol as a blank. The experiments were performed in triplicate and percentage scavenging activity was calculated using following equation; [(Absorbance control − Absorbance sample)/Absorbance control] × 100. All experiments were repeated in triplicate and means with standard deviation were calculated. 4. Statistical analysis All the analysis was performed in triplicate and the data repre- sented as mean ± standard deviation. The statistical analysis was performed by non-linear regression (curve fit) using graph pad prism software. The P value was b0.05 regarded as significant. Fig. 4. a. UV–visible spectra of leaf extract of E. scaber. b. UV–visible spectra of synthesized nanoparticles in leaf extract of E. scaber (1 min to 10 min time course study). S.N. Kharat, V.D. Mendhulkar / Materials Science and Engineering C 62 (2016) 719–724 721 Fig. 3. Mechanism of the synthesis of AgNPs using E. scaber (Linn.) leaf extract.PDF Image | Antioxidant activity of Silver Nanoparticles using leaf extract
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