Silver nitrate (AgNO3) and all other chemicals were purchased from the Merck Chemicals, India. All the solvents used for this work were purchased from Finar limited, India. Silica gel (100-120 mesh) was purchased from silvery enterprises ltd., India. Clathria frondifera marine sponge was collected in mandapam sea area, Tamilnadu. The freshly collected marine sponge were washed several times with running tap water and then with double distilled water to remove excess salt from sea water. Deionized water was used for the preparation of all solutions. All the reagents were analytically pure (AR) and use as received without further purification.
Preparation of marine sponge extract
5g of the shadow dried marine sponge were weighed and boiled for 15 min in 100 ml double distilled water and the extracts are filtered through Whatman No.1 filter paper. The pure aqueous extracts were stored in a cool and dark place.
Another 5 g of shadow dried marine sponge were crushed manually in mortar and sieved to get uniform mesh. About 1 g of crushed sponge powder were directly soaked in 100 ml of methanol-deionized water mixed solvent of ratio 1:10 for 12 h, stirred for another 12 h and allowed to stand for 2 h. Then the supernatant was centrifuged, concentrated and stored in separate glass sample container in dark for further use.
Sample preparation for GC-MS
One portion of the both extracts were completely dried in rotary evaporator to obtain powder extracts and then all ethyl acetate soluble matter were completely separated out using column chromatography. This pure ethyl acetate extracts were concentrated and stored in a clean glass sample container for using GC-MS analysis.
Synthesis of Ag nanoparticles (AgNP) 1-4
100 mL of each extract (I & II) was taken in separate beakers and mixed with 100 mL of 0.05 M AgNO3 solution in 500 ml Erlenmeyer flask at room temperature. After 30 min of vigorous mixing, the flasks were kept aside for 24 h in cool and dark place. Every 2 h, the flask was monitored to check the colour of the solution. In the synthesis using extract I, the light yellow colour solution slowly becomes ruby red colour. In the synthesis using extract II, the solution changed into brownish within 4 h then there was further noticeable difference in the intensity upto 20 h, after that no change in the appearance of solution, which confirmed that the bio-reduction process is over within 24 h. After 24 h, both the synthesized AgNPs (1 & 3) were collected by centrifugation at 3000 rpm for 20 min and it was purified with double distilled water for three times. Then the AgNPs were allowed to dry at room temperature.
10 ml of concentrated extract I was mixed with 30 ml of 0.05 M AgNO3 solution with continuous 500 rpm magnetic stirring at room temperature in a glass beaker. The synthesis of AgNP3 using extract I was completed after 2 h magnetic stirring. Various stoichiometric ratios (1:1, 1:2, 2:1, 1:3 and 3:1) of 0.05 M AgNO3 and extract II solutions were taken for the synthesis of AgNP4. After the addition of extract II, within 10s the pale yellow colour changed into blackish brown. Then after 2 min stirring, the synthesized silver nanoparticles (AgNP4) were collected and washed several times with deionized water and dried in vacuum. Finally, all the synthesized silver nanoparticles were stored in a screw capped bottle for further characterization and were labelled as AgNP1-4 as shown in table 1.
FT-IR spectra of dry sponge powder was taken in Fourier Transform Infrared spectrophotometer. The marine sponge Clathria frondifera extracts I and II were analysed using GC-MS. The visual properties of the product were investigated by UV–visible absorption spectrometer at the wavelength range of 200-800 nm with acetone dispersed samples in quartz cuvette. Powder X-ray diffraction (XRD) spectral data were collected diffractometer. EDAX spectra of as synthesized AgNP and AFS were taken to confirm no more impurity or any other elements present in AgNP (Ag) and AFS (Ag, C, N and O). SEM images were recorded at 40,000× magnifications operating with 20.00 kV. TEM images of Ag NPs and AFS were taken Transmission electron microscope.
Evaluation of antimicrobial activity
The antibacterial study of Ag nano spheres was performed against the pure cultures of human pathogenic bacteria such as Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were isolated from clinical sample. It was confirmed by various biochemical tests. These were obtained from Microbiology Department, VHNSN College, Virudhunagar, India. Bacterial strains were cultured overnight at 37˚ C in Muller-Hinton broth for antimicrobial activity tests. Test strains were suspended in nutrient agar to give a final density of 5 X105 CFU/ml. The Minimum Inhibition Concentration (MIC) value was expressed as the lowest concentration inhibiting the bacterial growth. The method was recommended by the National committee for clinical Laboratory Standards (1). Nutrient agar (20 ml) was poured into each sterile Petri plates after inoculating culture (100 µl) of microorganisms and distributing medium in Petri plates homogenously. To make three wells on nutrient agar medium were added 25 µl and 50 µl of AFS sample. The plates were incubated at 37˚C for 24 h. Inhibition zones formed on the medium were evaluated in mm and the streptomycin antibiotic was used as the positive control.
In vitro cytotoxicity studies
HELA cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 oC in a humidified incubator containing 5% CO2. To check the cytotoxicity by MTT assay, HELA cells were seeded separately in a 96 well plate at a density of 4×103 cells per well and were incubated in the media containing marine sponge and AFS at various concentrations (20, 40, 60, 80, 100, 120 and 140 μg.mL−1) for 24 h. Cytotoxicity was measured by MTT assay and the absorbance was read at 595 nm.
HELA cells were cultured with and without marine sponge and AFS at various concentrations (80, 100 and 120 μg.mL−1). For all untreated and treated HELA cells, the images were viewed at 24 h and the images were captured usinga phase contrast microscope.
BSA-FITC coated silver nanoparticles were prepared by 1 mL of the AgNPs mixed with 0.5 mL BSA-FITC (0.15 μmol) and 1 mL NaOH (0.1 M). The mixture was stirred for 3days and FITC-AgNPs were separated by dialysis method. HELA cells were grown in a 6-well plate and FITC-AgNPs of 1 to 4 µM was added to the cells and visualized under fluorescence microscope.