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which remain stable for more than 3 months without any changes in the absorption spectrum. The reduction of pure Ag+ ions was monitored by measuring the UV-Vis spectrum of the reaction medium after diluting a small aliquot of the sample into distilled water. Aliquots of the reaction solution were measured using a UV- 1601 Schimadzu spectrophotometer operated at a resolution of 1nm. Fourier transform infrared spectroscopy The interaction between protein-silver nanoparticles were analyzed by Fourier transform infrared spectroscopy (FTIR) in the diffuse refluctance mode at a resolution of 4cm-1 in the KBr pellets and the spectra were recorded in the wavelength interval of 4000 to 400nm- 1. FTIR measurements were carried out to identify the possible biomolecules responsible for the reduction of the Ag+ ions and the capping of the bioreduced AgNPs synthesized by seaweed extract. For comparison, the seaweed filtrate was mixed with KBr powder and pelletized after drying properly and subjected to measurement. X-ray diffractometry X-ray diffraction (XRD) measurement of the seaweed reduced AgNPs was carried out using powder X-ray diffractometer instrument (PXRD-6000 SCHIMADZU) in the angle range of 10 ̊C-80 ̊C at 2θ, scan axis: 2:1 sym. The size of the AgNPs was calculated from the PXRD peak positions using Bragg’s law. Extended Dispersive analysis X-ray Spectroscopy The presence of elemental Silver was confirmed through EDS. Energy dispersive analysis X-ray spectrometer takes advantage of the photon nature of the light. In the X-ray range the energy of a single photon is just sufficient to produce a measurable pulse X ray; the output of an ultra low noise pre-amplifier connected to the low noise is a statistical measure of the corresponding quantum energy. A semiconductor material is used to detect the X-rays together with processing electronics to analyses the spectrum. The EDX observations were carried out in STIC, CUSAT, Kerala (JOEL Model JED-2300). SEM observation of silver nanoparticles The sample was prepared by placing a drop of AgNPs on carbon coated copper grid and subsequently drying in air, before transferring it to the microscope operated at an accelerated voltage of 120 KV (JOEL Model JSM-63 90 LV). In vitro assay for Cytotoxicity activity (MTT assay). Hep-2 (Human Epidermoid larynx carcinoma) cell lines were obtained from National centre for cell sciences Pune (NCCS). The cells were maintained in RPMI-1640 supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified atmosphere of 50 μg/ml CO2 at 37 °C. The Cytotoxicity of synthesized AgNPs on Human Epidermoid Larynx cancer cells was determined by the MTT assay (Mosmann et al., 1983). Cells (1 × 105/well) were plated in 100 μl of medium/well in 96-well plates. After 48 hours incubation the cell reaches the confluence. Then, cells were incubated in the presence of various concentrations of the samples in 0.1% DMSO for 48h at 37°C. After removal of the sample solution and washing with phosphate- buffered saline (pH 7.4), 20μl/well (5mg/ml) of 0.5% 3-(4, 5- dimethyl-2-thiazolyl)-2, 5-diphenyl--tetrazolium bromide cells (MTT) phosphate- buffered saline solution was added. After 4h incubation, 0.04M HCl/ isopropanol were added. Viable cells were determined by the absorbance at 570nm with reference at 655nm. Measurements were performed in 3 times, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The absorbance at 570 nm was measured with a UV spectrophotometer, using wells without sample containing cells as blanks. All experiments were performed in triplicate. The effect of the AgNPs on the proliferation of Human Epitheloid larynx cancer cells was expressed as the % cell viability, using the following formula: % cell viability = A570 of treated cells / A570 of control cells × 100%. Data analysis The IC50 values (concentration at which 50% of cells were death) are reported as mean± standard deviation of three independent experiments. The IC50 values against the Human laryngeal Hep-2 cancer cell lines were calculated for the synthesized AgNPs inhibiting at least 50% inhibition when tested at a concentration. One-way analysis of variance (ANOVA) and Student t-tests were used to compare data using Statistica version 5.0 software at a 95% confidence limit. RESULTS AND DISCUSSIONS Reduction of silver ion into Ag particles during exposure to the seaweed extract could be followed by color change. AgNPs exhibit dark yellowish-brown color in aqueous solution due to the surface Plasmon resonance phenomenon (Fig.1). UV-Vis spectrograph of Ag nanoparticles has been recorded as a function of time (Fig.2). Absorption spectra of Ag nanoparticles formed in the reaction media at 72 hrs has absorbance peak at 435 nm, broadening of peak. The position and the number of peaks in the absorption spectra are dependent on the shape of the particles: for an ellipsoidal particle there are two peaks whereas for spherical particle there is only one peak centered at 420 nm, indicating the formation of silver nanoparticles (13). The absorption maximum at 420 nm is attributed to the Mie scattering by silver metal (14). The appearance of the yellow color indicated the formation of AgNPs in the reaction mixture, as it is well-known that AgNPs exhibit striking colors (light yellow to brown) due to excitation of surface plasmon vibrations in the particles (15). It was reported that some amount of OH- groups tended to promote the reduction of silver ions in some chemical methods (16). FT-IR spectrum of AgNPs is shown in Fig.3. This spectrum shows the presence of bands at 3419, 1650, 1489, 1059 cm-1. The bands at 3419cm-1 corresponds to primary amine O-H band, 1650 cm-1 corresponds to primary amine N-H band, the band at 1489 cm-1 is assigned to methylene scissoring vibration from the protein in the solution, and the band at 1033 cm-1 were assigned to C-N stretching vibration of the proteins (17). The positions of these bands were close to that reported for native proteins (18). This evidence suggests that the protein molecules could possibly perform the function of the formation and stabilization of AgNPs in the aqueous medium. XRD pattern taken using powder X-ray diffractometer instrument (PXRD-6000 SCHIMADZU) in the angle range of 10 ̊C-80 ̊C of the AgNPs at 2θ, scan axis: 2:1 sym is shown in Fig.4. A number of Bragg reflections corresponding to (111), (200), (220), (311) sets of lattice planes are observed, which can be indexed to face-centered cubic silver. The peaks matches with the Joint Committee on Powder Diffraction Standards (file No. 04-0783), which further proves the formation of crystal AgNPs (19). The peaks were identified as AgNPs according to PCPDFWIN software (PDF#030921). The XRD pattern thus clearly shows that the AgNPs are crystalline in nature. Furthermore, the average diameter of the silver nanoparticles is calculated as 6.4nm by the Scherrer formula D = 0.89λ βcosθ Where D is mean grain size, λ is the wavelength for Cu target, β is the FWHM of diffraction peak and θ is the diffraction angle. Thus XRD is commonly used for determining the chemical composition and crystal structure of a material. The line broadening of the peaks is primarily due to small particle size. The X-ray diffraction results clearly show that the AgNPs formed by the reduction of silver ions by the extract of Gelidiella sp. are crystalline in nature (20). EDS micro-analysis is performed by measuring the energy and intensity distribution of X-ray signals generated by a focused electron beam on a specimen. Fig.5 shows the EDS spectrum recorded in the spot-profile mode. The optical absorption peak is observed at 3KeV, which is typical for the absorption of metallic AgNPs (21). Strong signals from the silver atoms are observed, while weaker signals from C, O, P and Cl atoms are also recorded. Those Bhimba et al. Int J Pharm Pharm Sci, Vol 4, Suppl 4, 710-715 711PDF Image | anticancer activity of silver nanoparticles extract of Gelidiella
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