Phospholipid extraction
Removal of oil by SC-CO2 extraction: For oil extraction, a supercritical fluid extraction method was applied. In particular, 100 g of freeze-dried, crushed fish bone was added to a 200 mL stainless steel extractor. Using SC-CO2 extraction at 45°C and 25 MPa pressure for 3 hours, the oil was extracted from the sample. Completion of oil extraction was ensured by repeated weighing of the extracted oil for 3 consecutive times at 5 min intervals which showed consistent weight. Around 35.15 ± 1.14% oil was recovered at this stage. Then, the residues were left in the extractor so that SNPL could be extracted using SC-CO2 and ethanol as a co-solvent. Organic solvent was utilized to extract SNPL from the de-oiled residue.
Extraction of phospholipid by SC-CO2 with ethanol as co-solvent: Phospholipid was extracted from the residue left behind after de-oiling fish bones using SC-CO2 with ethanol as the co-solvent. This process was carried out at 45°C and between 22.5 and 30 MPa, and varied CO2 densities were maintained throughout. Several studies have proposed the temperature and pressure required for phospholipid extraction. The flow rate of carbon dioxide was maintained at 27 g per minute during the whole 2.5 hours that it took to complete the extraction process. With the help of a solvent delivery pump, the flow rate of the co-solvent ethanol was pre-optimized and kept at 3 mL/min throughout the process (Model: M930, Young Lin Co., Korea). After using a rotary evaporator heated to 45 °C to remove the solvent from the ethanol–phospholipid combination, the SNPL that was extracted was kept at a temperature of -20 °C.
Cell culture
This study utilized SK-N-SH (ATCC, Manassas, VA, USA) and HUVEC (ATCC, Manassas, VA, USA) cell lines, which were maintained in a humidified environment containing 5% CO2 at 370C in MEM (Mediatech, Manassas, VA, USA) and EBM-2 medium, respectively. The cells were then subcultured until they had reached passage-3 and were ready for the experiment when they attained a confluence of around 80%. EBM-2 (Lonza, Walkersville, MD, USA) was supplemented with EGM-2 kit (Lonza, Walkersville, MD, USA) and MEM was supplied with 10% heat inactivated FBS and 1.1 % antibiotic antimycotic (Mediatech, Manassas, VA, USA).
Solution preparation
After dissolving 30 mg of SNPL in 150 µl of DMSO, the final concentration of the SNPL stock was brought up to 200 mg/ml. To treat SK-N-SH and HUVEC cells, stock was diluted in the appropriate medium and then prepared at a range of concentrations (600, 700, 800, and 900 µg/ml).
Cell viability assay
To assess the cytotoxicity of SNPL, control and experimental group cells were sown in a 96-well plate at a density of 1x104 cells/well; and blank wells containing 100µl of medium and no cells was incubated for 24 hours at 370C with 5% CO2. SK-N-SH and HUVEC experimental group cells were loaded with varying doses (600, 700, 800, and 900 µg/ml) of the SNPL and then incubated for another 24 hours. Next, media was changed and replaced with fresh media containing EZ-Cytox solution (10µl) in blank, control and experimental group wells, also protected from light and incubated for 2 hours at 370C 5%CO2. Next, the media in the blank, control, and experimental group wells were removed and replaced with new media containing EZ-Cytox solution (10µl). These wells were likewise shielded from light and incubated at 370 degrees Celsius with 5% CO2 for 2 hours. Using an ELISA plate reader at 460 nm, the cell viability was computed (Varioskan Lux, Vantaa, Finland). Cell viability calculated as [(experimental - blank) ÷ (control - blank)]x100. This experiment was conducted through repetition.
Protein extraction and Westernblot
A total of 20mL of MEM constituted SK-N-SH cells (adjusted: 1 x 105 cells/mL) was prepared, then 5ml were put into the 100mm cell culture plate and incubated at 370C with 5% CO2 for 24 h. The media were subsequently switched, and the control group received no treatment, while the experiment group received 600, 700, and 800 g/ml of SNPL and was incubated at 37 °C with 5% CO2 for 12 hours. After 12 hours, the cells were washed and scraped with cold PBS buffer and then transfer into 15ml tubes. Followed by 5 min of centrifugation at 1400 RPM. By adding 35 µL lysis buffer to the centrifuged cells, the cells are lysed (iNtRON BIOTECHNOLOGY, Cat. No. 17081, Korea). After 10 minutes of incubation on ice, lysates were obtained and cleared by centrifuging at 14000 rpm for 20 minutes at 40C. Using a 2 mg bovine albumin standard and Bradford reagent for an ELISA microplate reader at 595nm, protein concentration was determined. 12% SDS-PAGE was used to separate aliquots of whole cell lysates or cytosolic fractions, which were subsequently transferred to nitrocellulose membranes. PBST (PBS buffer with 0.5% Tween-20) with 5% skim milk was used to block the membranes. Following the blocking of any non-specific sites, the membranes were probed with primary antibodies (Cell Signaling Technology), washed three times in PBST, and then subjected to an incubation period of one hour with horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG as secondary antibodies (Cell Signaling Technology). The membranes were then rinsed with PBST and observed using an improved chemiluminescent detection solution (Abfrontier, Lot.QJN28, Seoul, Korea) and apparatus (Thermo Fisher Scientific, iBrightCL1000, Waltham. MA, USA). Adobe Photoshop CS6 was used to conduct the measurement of the band density.
RNA Extraction
After preparing 15 ml of MEM containing a constituted HeLa cell suspension (with an adjusted concentration of 1 x 105 cells/mL), a volume of 5 ml was plated onto each 100 mm cell culture dish, and the dishes were placed in an incubator at 370C containing 5% CO2 for 24 hours. The medium was then switched out, the control group did not get any of the extract, and the experimental groups were given 700 and 800 μg/ml of SNPL before being incubated at 370C with 5% CO2 for 12 hours. After a period of 24 hours, the media was discarded. Rinsing both the control and experimental groups (SNPL 700 and 800 μg/ml) with 1 ml of cold PBS in each plate, scraping, and collecting the cells in 1.5 ml Eppendorf tubes respectively were the next steps. 10 min were spent centrifuging at 8,000 RPM while the temperature was maintained at 40C. For extraction of RNA, the RNeasy® Mini Kit (50; QIAGEN, Hilden, Germany) was used.
Reverse Transcriptase PCR
cDNA was synthesized using extracted RNA using a PCR machine (SampliAmp Thermal cycler, Singapore) using a SuPrimeScript RT Premix (2X with oligo dT; GeNet Bio, Global Gene Network, Daejeon, Korea) with the appropriate amount of RNA and RNase-free water. PCR condition: (50 °C for 60 min, then 70 °C for 10 min) 1-cycle. Eventually maintained at 4 °C.
Real Time qPCR
The RT-qPCR method was utilized in order to ascertain the relative fold change in gene expression in HeLa cells. In each qPCR tube, 7 μl distilled water, 1 μl forward and 1 μl reverse primer (Table-1) (Bioneer, Daejeon, Korea), 1 μl cDNA, and 10 μl qPCR master-mix were mixed, then spun down. The experiment was conducted three times employing an RT-qPCR equipment (ExicyclerTM 96; Bioneer, Korea). The 2-∆∆CT approach(24) was used to determine the relative quantification result.
Table-1. List of designed primers used for RT-qPCR.
Genes
|
Product size
|
Sequences
|
Caspase-3
|
102 bp
|
Forward 5’-CTGTGAACCCTGCATTTGGC-3’
|
Reverse 5’-ACTTCGGAAGCTGAACCTGG-3’
|
Cytochrome-c
|
104 bp
|
Forward 5’-TGGCTTAATGTGTTCGCCCT-3’
|
Reverse 5’-AAGCCCAAGCAAAGAGGGAA-3’
|
Bax
|
102 bp
|
Forward 5’-ACGAGGGTGATAGGTGGTACA-3’
|
Reverse 5’-TGTTCTTCCCTTACCCACACG-3’
|
Apaf-1
|
101 bp
|
Forward 5’-TGGGTGACTGACCTTTGCTTT-3’
|
Reverse 5’-GTCTGTGAGGATTCCCCAGTG-3’
|
Caspase-9
|
105 bp
|
Forward 5’-GAAGAGACCTGGCCAGAACC-3’
|
Reverse 5’-ATTGCACAGCACGTTCACAC-3’
|
β-Actin
|
104 bp
|
Forward 5’-TCTTCCAGCCTTGCTTCCTG -3’
|
Reverse 5’-GGTGTACAGGTCTTTGCGGA-3’
|
DNA Fragmentation
15 mL of MEM containing a suspension of SK-N-SH cells (1 x 105 cells/mL) was made. Then, 5 mL of the suspension was put on each 100 mm culture dish and left to grow in the incubator for 24 hours. The medium was then changed, and the control and experimental groups containing 700 and 800 µg/ml SNPL were incubated for 48 hours. Kits (AccuPrep® Genomic DNA Extraction Kit; Bioneer, Korea) were used to extract genomic DNA from the control and experimental groups. An imaging device (Thermo Fisher Scientific, iBrightCL1000, USA) is used to assess for DNA fragmentation after gel electrophoresis.
Statistical analysis
The presentation and display of all data is done as Mean ± SE. Microsoft Excel was used for the statistical analysis, which included a comparison of each treatment group's mean value to that of the control group using an ANOVA and a T-test. The p-value was utilized to determine statistical significance.