Sample processing and extraction
A. mauritiana B. marmorata, H. atra, H. edulis, H. leucospilota, and H. polii belong to Order Aspidochirotida, and Family Holothuriidae were accumulated from different locations on Egyptian coasts and identified previously [25]. Samples were rinsed thoroughly, and their body fluids and interior organs were eliminated through an abdominal incision. Sample body walls were sliced into slightly pieces and then extracted at the ratio of 3:1 v/w with 96% ethanol solvent. The mixture was soaked, then the homogenization was extracted twice for one day at room temperature. The extract of sea cucumbers was eliminated after squeezing and filtration through filter paper of 0.45 μm. Afterward, the solvent was evaporated at low pressure using a rotary evaporator at 35°C. The obtained extracts were stored in the dark at 4° C until use. The purity of the crude extracts was tested on thin-layer chromatography plates, and the spots were seen under UV light and by adding sulfuric acid (10 %) up to the formation of maroon-dark purple spots [5], indicating the presence of saponin. Ultra Performance Liquid Chromatography-Mass Spectrometry was used previously to identify the metabolic compounds of each extract [16].
Hemolytic activity assay
The hemolysis assessment was investigated using blood obtained from a woman with an O+ blood group. The blood was accumulated in tubes containing an anticoagulant such as ethylenediaminetetraacetic acid (EDTA), then pelleted after centrifugation for 15 min at 800 × g to eliminate the plasma. The pellet, which contains erythrocytes, was cleansed in phosphate-buffered saline (PBS) (pH=7), and suspended in PBS to a final concentration of 3% [26]. PBS and standard saponin were used as negative and positive controls, respectively. The hemolytic activity was performed within 96-well plates in triplicate. Firstly, 100 μl of PBS was added to each well, then 100 μl of each extract. Finally, 100 μl of the blood suspension (3%) was added to each well. The absorbance of each sample was measured at 650 nm after four h of incubation at room temperature [19].
Cytotoxicity against BSA
Cytotoxicity of six Holothuroids (A. mauritiana, B. marmorata, H. atra, H. leucospilota, H. edulisand, H. polii) against biological organisms BSA was used to determine the most cytotoxic extracts according to Mashjoor and Yousefzadi [22]. Cysts of BSA hatched in artificial seawater (pH = 8.8 salinity = 35%) for 48h. The recently hatched nauplii larvae were collected with a pipette, and 20 nauplii were transferred to containers filled with artificial seawater (2 ml). We prepared four dilutions (1000, 500, 250, and 125 mg/ml) of every extract by dissolving sea cucumber extracts in distilled water integrated into the vials containing artificial seawater and BSA. After 24 h of exposure, the number of living nauplii was calculated. The percentage of mortality at each dosage and control seawater was defined by the lack of regulated forward motion during 30 seconds of examination. The mortality percentage was calculated using the following formulae:
% Mortality = Sum of dead nauplii/Original number of living nauplii × 100.
LC50 for each test dilutions was determined after 24 h via statistical probit analysis [27].
Cytotoxicity assay against human cell lines
The cytotoxic activity of the tested extracts was examined in Pharmcogency Laboratory, Faculty of Pharmacy, Mansoura University.
Cell line
Epitheliod Carcinoma (Hela), Epidermoid Carcinoma (HEP2), Colorectal carcinoma (HCT-116), and Human prostate cancer (PC3) are the tested human cancer cell lines. The cell line was purchased from American Type Culture Collection through the Holding company for biological products and vaccines, Cairo, Egypt.
Chemical reagents
Tetrazolium dye (MTT), Roswell Park Memorial Institute medium (RPMI-1640 medium), dimethyl sulfoxide (DMSO), and Doxorubicin (Dox) were obtained from sigma co., St. Louis, USA. Dox was used as a standard anticancer drug for comparison. Fetal Bovine serum was purchased from GIBCO, UK.
MTT assay
The cell lines were applied to assess the inhibitory effects of sea cucumbers on cell growth using the MTT assay,according to previous studies[5, 28].This colorimetrical test is based on changing tetrazolium bromide color from yellow to a purple formazan derivative in viable cells by mitochondrial succinate dehydrogenase. The cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum. The cells were cultured in a 96-well plate at a density of 1.0x104 cells/well for 48 h at 37°C under 5% Co2. One hundred units/ml of antibiotics penicillin and 100µg/ml streptomycin were added in a 5% Co2 incubator at 37° C. Following incubation, the cells were exposed to serial concentrations of each extract (100, 50, 25, 12.5, 6.25, 3.125, and 1.56 µg/ml), then incubated for 24h. After the treatment, 20 µl of MTT (5mg/ml) solution was added and incubated for four h. For dissolving the purple formazan, 100 µl of DMSO was added to each well. The colorimetric assay is measured at 570 nm absorbance using a plate reader (EXL 800, USA). The relative cell viability percentage was determined as (A570 of treated samples/A570 of the untreated sample) X 100.
Statistical analysis
A statistical analysis system, SPSS Version 17, was used to calculate mean values ± standard deviation (SD). All data were repeated three times. One-way analysis of variance (ANOVA) was utilized with the post hoc test to evaluate hemolytic activitybetween the studied groups and compare groups. Means < 0.001 was considered significant.