2.1. Chemicals and Materials
ABTS Antioxidant Assay Kit (Sigma-Aldrich, USA # CS0790) and 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl were used to study antioxidant activity. Analytical standard Methanol (Fluka, # 34860) was used to solubilize the bioactive molecules of the crude extracts. N-acetyl L-cysteine [11] (Sigma Aldrich #A7250) was used to evaluate the antioxidant potential of the microalgae extracts. The 2’7’-dichlorofluorescin-diacetate (DCFH-DA) (Sigma Aldrich, #6883 SIGMA) was used to evaluate antioxidant scavenging capacity.
The standards used for the identification of the microalgae high value products are
(i) astaxanthin (Sigma Aldrich, P/N SML0982-50MG, Batch 86770), [1] beta-carotene (Sigma Aldrich, P/N C9760-5G, Lot MKCK2908), (iii) canthaxanthin (Sigma Aldrich, P/N 1175-1MG, Lot BCCB9917), and (iv) lutein (Sigma Aldrich, P/N 07168-1MG, Lot BCCC1688)
2.2. Culture of the microalgae strains
The strains used during this study belongs to Qatar university culture collection of cyano-bacteria and microalgae (QUCCCM). Two freshwater microalgae strains: A. quadricellulare QUCCCM10, N. conjuncta QUCCCM28, and two marine species Chlorocystis sp. QUCCCM60 and P. atomus QUCCCM130 were selected for the current study. These strains were subjected to investigation of their growth performance, metabolites content and antioxidant capacities. Marine and freshwater microalgae strains were cultured in f/2 [12] and BG11 [13] growth medium, respectively.
For assessment of the growth properties and metabolite content, the microalgae strains were cultured as follows: One single colony of each microalgae species was used to inoculate 10 ml of liquid growth medium prior to being incubated at 30 °C, under an agitation of 2 × g, a photon flux density of 100 µmol photons m-2 s-1 with 12h:2h dark: light cycles using an illuminated shaker Innova® 44R incubator shaker, New Brunswick Scientific, https://www.eppendorf.com/. After 7 days of cultivation, the cultures were scaled-up gradually to a volume of 100 ml and incubated under the same previously described conditions. Subsequently, an adequate volume was used to inoculate 1000 ml liquid growth medium with an initial optical density at 750 nm of 0.2 for the four strains. A daily assessment of the OD 750 nm was performed using a spectrophotometer Jenway 73100, http://www.jenway.com/. The biomass, harvested after 12 days of cultivation, was washed with water (freshwater algae) or ammonium formate 0.5 M (marine algae). It was then freeze dried, prior to metabolite extraction and quantification.
A different cultivation system targeting increase in producing high value products was applied. Cultures were scaled up gradually from 10 ml to 500 mL under the same previously described conditions. Then an adequate volume of this culture was used to inoculate a DASGIP parallel 1 L bioreactor system for phototrophic cultivation Eppendorf, https://www.eppendorf.com/ allowing higher light intensity than the shaker and better biomass productivity [14, 15]. This culture was grown for 20 days under a temperature of 30 °C, pH 8, an agitation of 2 × g, a continuous illu-mination of 400 μmol photons m−2 s−1 and 5% CO2, and using a depleted growth medium, (BG11 with 1/10 diluted nitrogen (NaNO3)). All cultures were performed in duplicate (n = 2). Samples were collected every 5 days (day 5, 10, 15 and 20), then washed and freeze dried prior to being subjected to maceration for high value-added product extraction.
2.3. Metabolites’ characterization of the microalgae isolates
For total protein, 25 mg of freeze-dried microalgae was dissolved in 5 mL sodium hydroxide (NaOH 0.1 M) and incubated overnight at 60 °C. The total protein content was determined for the supernatant using Folin-Ciocalteu reagent [16]. Serum albumin bovine was used as standard.
Total lipids were extracted from the algae biomass using the same method as [14]. The total lipid content was determined gravimetrically. Subsequently, the lipid content (%) was determined as described using the following equations:
% Lipid content = (Total Lipids (g)) / (Dry biomass (g)) * 100
Total carbohydrates were extracted from 25 mg of freeze-dried microalgae using phenol sulfuric acid reagent biomass according to [17]. Briefly, 25 mg was solubilized into glacial acetic acid, and incubated at 85 °C for 20 min to remove the chlorophyll. Then, pellets were hydrolyzed using hydrochloric acid (HCl 4 M) at 90 °C for 2 h. Finally, the supernatant was neutralized with water and subjected to calorimetric assay using phenol sulfuric acid.
2.4. Extraction and identification of Bioactive Molecules
The extraction of bioactive molecules was performed via maceration, using increasing polarity solvents such as hexane and acetone for both hydrophobic and hydrophilic metabolites, respectively. Briefly, 20 mg of lyophilized microalgae was dissolved in 2 ml of organic solvent and incubated at 20°C for 30 min after 10 min centrifuge at 2268 × g. The supernatant was stored, and the pellet was dissolved again in 2 ml of organic solvent and incubated for 30 min at room temperature. This extraction was carried out twice to recuperate the microalgae extract. Finally, the pellet will be dissolved in 1% DMSO then stored at 4°C in the obscurity for future use. The same protocol was used with both organic solvents.
For the identification of the high value products, the organic phase was separated, evaporated in a vacuum centrifuge, and reconstituted for injection on an ultra-high-pressure liquid chroma-tography system coupled to an Orbitrap Fusion Lumos Tribrid Mass Spectrometer via an ul-tra-high-pressure liquid chromatography system. The analysis was carried out individually for positive and negative polarities, as well as for identification and quality control samples. The identification of the Asterarcys quadricellulare pigments was carriecombination of the reconstituted organic phase of the 21 samples was used to make identification and quality control samples. The identification of the microalgae pigments was confirmed with co-injection of 5 standards of b-carotene, lutein, astaxanthin, Canthaxanthin, Fucoxanthin. The quantification of the pigments was estimated using different concentrations of the standards.
2.5. Evaluation of antioxidant activity
The level of antioxidant activity was determined for four selected strains extracted by acetone or hexane using the Trolox Equivalent Antioxidant Capacity assay (TEAC), utilizing the 2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS) antioxidant assay kit (Sigma-Aldrich kit, # CS0790, USA). The assay was performed as per the kit guidelines. Briefly, 6 concentrations of Trolox were used as a standard, and optic densities of 405 nm were determined via a synergy H4 hybrid multi-mode microplate reader Bio-Tek, https://www.biotek.com/. The decrease in absor-bency reflected the ABTS+ radical cation scavenging capacity and was plotted against the con-centration of the antioxidant. The TEAC value represents the ratio between the slope of this linear plot for scavenging of ABTS + radical cation by the extract compared to the slope of this plot for ABTS + radical cation scavenging by Trolox used as an antioxidant standard. TEAC was determined using the following formula:
Antioxidant capacity (mM relative to the concentration of the Trolox standard) = (Absorbance of the sample at 405nm – intercept) / (slope of standard curve) * dilution factor
Antioxidant capacity was determined of all the hexane and acetone extracts at different times of harvesting (day 5, day 10, day 15 and day 20).
The extract presenting the highest antioxidant activity was selected to study its ROS scav-enging potential in H9c2 cardiomyoblasts.
2.6. Cell Culture
H9c2 cardiomyoblasts, a clonal cell line derived from embryonic BD1 X rat ventricular heart tissue and known to exhibit many of the properties of skeletal muscle, were obtained from the American Type Culture Collection (ATCC). The H9c2 cell line was cultured in Dulbecco's Modified Eagle Medium F12 1:1 (DMEM, Lonza Bioscience, United States), supplemented with 10 % heat-inactivated Fetal Bovine Serum (FBS, Gibco, United States) and 1 % Penicillin-Streptomycin (Gibco) in a CO2 incubator (5 % CO2, 37 °C, Thermo Fisher Scientific, Waltham, MA, USA). To study the antioxidant activity, cells were treated with A. quadricellulare hexanoic extract (10 mg L-1) in the presence of H2O2 (100 mM). N-Acetylcysteine [11] (0.75 mM) and methanol were used as positive and negative controls, respectively.
2.7. Qualitative assessment of oxidative stress in H9c2 cardiomyoblasts treated with mi-croalgae extracts
Reactive oxygen species was directly visualized using H9c2 cardiomyoblasts stained with Dichloro-dihydro-fluorescein diacetate (DCFH-DA) and used as a qualitative assessment of the level of oxidative stress in H9c2 cardiomyoblasts which had been treated with the microalgae extracts presenting the highest antioxidant capacity. H9c2 cardiomyoblasts were plated in a microplate appropriate at a cell density of 500,000 cells and incubated for 24 h in a CO2 incubator Thermo Fisher Scientific, https://www.thermofisher.com/, 5 % CO2, 37 °C. The growth medium was replaced by starvation media (0.5 % FBS, 1 % P/S) and incubated for 24 h. Cells were treated as follows: (i) A. quadricellulare hexanoic extract (10 mg ml-1), [18] A. quadricellulare hexanoic extract (10 mg ml-1) + H2O2 (100 mM), (iii) NAC (0.75 mM) alone, (iv) NAC (0.75 mM) + H2O2 (100 mM) and (v) H2O2 (100 mM) alone. 100 mM H2O2 was added for 4 h. Following treatment, the medium was aspirated, prior to being replaced by PBS and 10 µM DCFH-DA for 30 min. Following incubation with DCFH-DA for 30 min, the PBS dye was aspirated and replaced by only PBS, and images were taken using a Carl Zeiss Axiovert Fluorescence Inverted Microscope 40 CFL, https://bostonmicroscopes.com /.Cells with only PBS were used as negative controls and cells with only H2O2 were used as positive controls.
2.8. Apoptosis analysis by western blotting
Western blotting was used to detect the protein expression of total caspase-3. H9c2 cardio-myoblasts were treated as described above. Cells were then lysed using Radio Immunoprecipitation assay (RIPA buffer). Protein concentration was determined using the DC Protein assay kit (Bio-Rad). An equal amount of protein (15 μg) was loaded into and run using 15 % SDS-PAGE and transferred into nitrocellulose membrane. Eventually, the membranes were blocked with 5 % Milk-1X TBST for 1 h, followed by overnight incubation at 4 °C with the following primary antibodies: caspase 3 (Santa Cruz, sc-271028). Anti-alpha tubulin (Abcam, ab4074) was used as the loading control. The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies. Visualization for immunoreactivity was completed using enhanced chemiluminescence (ECL) followed by imaging and quantification of bands using the FCM MultiFluor System, https://www.proteinsimple.com/fluorchem.html.
2. 9. Statistical analysis
Data was expressed as the mean of two independent parallel experiments with two repetitions per experiment (a total of four values per variable). ANOVA was used to assess the differences among the treatments. Standard error of the means was calculated at <0.05 level of significance.