Extraction of silk sericin
Extraction of silk sericin by acid and alkali degradation
Silk sericin was extracted from cocoons using the modified protocol of Kurioka et al. (7). Briefly, the cocoons of mulberry (B. mori) silkworms were cut into small pieces and placed into a 1.25% citric acid (for acid degradation) or 0.5 M sodium carbonate (alkali degradation) solution (1 g of dry silk cocoon and 18 ml of citric acid solution) and boiled for 30 min. After that, insoluble fibers were removed by paper filtration. The obtained supernatant was filtered and immediately dialyzed in distilled water for 3 days using cellulose tubing [Cellusep T2; MWCO (molecular-mass cut-off) = 6000-8000; Sequin, TX, U.S.A.]. The protein solution was lyophilized using a Heto LL 3000 lyophilizer (HetoHolten A/S, Allerod, Denmark).
Extraction of silk sericin by high temperature under high pressure (autoclaving)
The cocoons of mulberry (B. mori) silkworms were cut into square pieces (approx. 5 mm2). Cocoon pieces were extracted in 70% ethanol three times for 24 h at room temperature (25°C). After drying, the residual cocoon shells (∼97% from initial cocoon weight) were autoclaved in purified water (1 g of dry silk cocoon and 30 ml of water) at 120°C and 15 lbf/in2 (1 lbf/in2 = 6.9 kPa) for 60 min (SS-320; Tomy Seiko, Tokyo, Japan). Centrifugation and filtration were performed to separate the silk fibroin along with other solid residues. After that, the protein solution was frozen and freeze-dried using a Heto LL 3000 lyophilizer (HetoHolten A/S, Allerod, Denmark).
Extraction of silk sericin by urea degradation
The silk sericin was isolated from the cocoons of mulberry (B. mori) silk using urea solution as described by Aramwit et al (13). Cocoon pieces (6 g) were soaked in 8 M urea (150 ml) for 30 min and then refluxed at 85°C for 30 min. All insoluble residues were removed by centrifugation and filtration. The obtained protein solution was dialyzed in distilled water using cellulose tubing (Cellusep T2; MWCO= 6000–8000; Sequin) for 3 days. The protein solution was lyophilized using a Heto LL 3000 lyophilizer (HetoHolten A/S, Allerod, Denmark).
Characterization of silk sericin
UV-vis absorption spectroscopy
UV-visible (UV-Vis) spectra were analyzed using a spectrophotometer (Thermo, Varioskan Flash, England) in the wavelength range of 200-500 nm.
Thermogravimetric analysis (TGA)
Thermogravimetric analysis was used to determine the thermal behavior. The composition silk sericin with different extraction methods was determined by thermogravimetric analysis (TGA) using a TGA Q50 instrument (TA Instrument, USA) with heating from 0 to 700°C at a rate of 10°C/min under nitrogen atmosphere.
Analysis of Amino Acid Composition
The amino acid compositions of silk sericin were measured using an amino acid analyzer (Hitachi L-8500A; Hitachi, Tokyo, Japan). The silk sericin was prepared for analysis by hydrolysis in methanesulfonic acid containing 0.2% 3-(2-aminoethyl) indole (Wako Pure Chemical Industries, Tokyo, Japan) at 100°C for 24 h under vacuum. All experiments were carried out three times in triplicate.
Cell culture
The B16F10 melanoma cell line (ATCC number CRL-6475) were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS; Gibco, USA), and 1% antibiotic-antimycotic solution (Gibco, USA). Cells were grown and maintained at 37°C with 5% CO2 in a humidified incubator.
Cytotoxic assay
The PrestoBlueTM reagent (Invitrogen, USA) was used to evaluate the influence of silk sericin on cell viability. Metabolically active cells are capable of reducing the PrestoBlue reagent, with colorimetric changes used as an indicator to quantify the viability of cells. B16F10 cells were seeded in 96 well plates at a density of 1x104 per well in cell culture medium and incubated for 24 h to allow cell adherence. Post incubation, cells were pretreated with different concentrations (10, 30 and 50 µg/ml) of sericin from various extraction methods for 24, 48, and 72 h. Following incubation, 10 µl PrestoBlue solution was added to each well, and then plates were placed back into the incubator for a further 30 min incubation. Fluorescence was measured using a microplate reader at 560 nm excitation and 590 nm emission (Thermo, Varioskan Flash, England). Morphology was examined using a light microscope.
Determination of intracellular antioxidant activity
The intracellular antioxidant activity of sericin against H2O2 was evaluated using the DCFH-DA assay (Invitrogen, USA). B16F10 cells were seeded into 96-black well plates at a density of 5 x 103 cells per well in cell culture medium and incubated for 24 h. Post incubation, fresh media was replaced with different concentrations (10, 30 and 50 µg/ml) of sericin and incubated for 48 h. Cells were washed with PBS and incubated with 0.1 µM of DCFH-DA at a volume of 100 µL/well for 30 minutes. After that, medium containing 2 mM H2O2 was added and incubated for 60 min. Post incubation, fluorescence was measured in a microplate reader at excitation and emission wavelengths of 485 and 528 nm (Thermo, Varioskan Flash, England).
Detection of intracellular reactive oxygen species (ROS) generation
The formation of intracellular ROS was measured by monitoring the changes in 2’,7’-dichlorofluorescein-diacetate (Invitrogen, USA) fluorescence. B16F10 cells were seeded into 96-black well plates at a density of 5 x 103 cells per well in cell culture medium and incubated for 24 h. Cells were washed with PBS and incubated with 0.1 µM of DCFH-DA at a volume of 100 µl/well for 30 minutes. After that, cells were washed with PBS again and treated with sericin for 48 h. Fluorescence was measured using a microplate reader at excitation and emission wavelengths of 485 and 528 nm (Thermo, Varioskan Flash, England).
Estimation of melanin content
Intracellular melanin was quantified in B16F10 cells cultured in DMEM with silk sericin. B16F10 cells were seeded into 12 well plates at a density of 1 x 105 cells per well in cell culture medium and incubated for 24 h to allow cell adherence. Post incubation, the media was replaced with fresh media containing sericin. The cells were collected after 48 h of incubation and the cell pellets were solubilized in 1 N NaOH for 1 h at 70°C to dissolve melanin. 200 µl of cell lysates were placed in 96 well plates and absorbance was recorded at 475 nm using a microplate reader (Thermo, Varioskan Flash, England).
Cellular tyrosinase activity
B16F10 cells were seeded into 24 well plates at a density of 5 x 104 cells per well in cell culture medium and incubated for 24 h. After incubation, the media was replaced with fresh media containing sericin and further incubated for 48 h. The cellular tyrosinase activity was measured using a commercial kit (Abcam, Cambridge, MA, USA). All the operating steps conducted according to the manufacturer’s instructions.
Statistical analyses
All experiments were carried out three times in triplicate. The results are expressed as mean± standard deviation for n=3. Statistical analysis was performed using one-way ANOVA and Tukey post-test using GraphPad Prism 5.0 (Graph-Pad Software Inc., CA, USA). Differences were considered significant when P < 0.05.