Microalgae Cultivation Setup
The marine chlorophyte Tetraselmis chuii Butcher CCAP 66/21B and diatom Phaeodactylum tricornutum Bohlin CCAP 1052/1 were cultured in 5 L glass flasks, each containing 4 L of filtered-sterilised seawater. The seawater was enriched with Guillard f/2 medium (Guillard 1975; Mc Gee et al. 2018) and supplemented, only for P. tricornutum Bohlin, with silicate (30 mg L− 1). The freshwater glaucophyte Cyanophora paradoxa Korshikov CCAP 981/1 was grown in a 5 L glass bioreactor filled with 4 L of sterile Bold Basal Medium and Vitamins (BBM) (Bold 1949; Mc Gee et al. 2018). The cultures were maintained at a temperature of 20°C under a 14:10 light/dark cycle, with an illumination of 80 µmol photons m− 2 s− 1 provided by cool white fluorescent LEDs. Upon reaching the end of the exponential growth phase (30 days), the biomass was harvested by centrifugation (2000x g, 3 min) using an Eppendorf Centrifuge 5702. Desalting of the marine microalgal biomass was performed using 0.5 M ammonium formate. Subsequently, all biomass samples were freeze-dried and stored at -20°C until required for further processing.
Preparation of Microalgal Extract for Bioactivity Analysis
The microalgal extracts were prepared by extracting 100 mg of freeze-dried biomass in 30 mL of diethyl ether (Et2O) (Mc Gee et al. 2020). Samples were stored at 2–6⁰C for 24 h before centrifugation (2000x g, for 5 min) using an Eppendorf Centrifuge 5702. The supernatant was recovered and passed through a 0.22 µm PTFE syringe filter (Fisher Scientific). The supernatants of the extracts obtained were then placed in pre-weighed vials and evaporated to dryness. The dried extracts were resuspended in 100 µL of dimethyl sulfoxide (DMSO) and stored at -20ºC until needed. On the day of analysis, fresh working stocks were prepared using cell culture medium to achieve a DMSO concentration below 1%.
HPLC-UV analysis of Pigment Content
Pigments in the reconstituted extracts were separated at a constant room temperature on a Shimadzu Nexera LC-20AD system coupled with a Prominence Diode Array Detector SPD-M20A. The extracts were injected in a 20 µL loop and separated using a Phenomenex Onyx C18 100 × 4.6mm ID monolithic column. The gradient applied was that of Mc Gee et al. (2018), which made use of a stepped gradient solvent program involving a 10% B initial isocratic run for 0.10 min, a linear gradient to 65% B from 0.10-2.00 min, an isocratic hold at 65% B for 2 min, a linear gradient to 90% B for 1 min followed by a hold at 90% B for 1 min and a final re-equilibration step to the initial conditions from 6.01–7.50 min. The mobile phase was prepared using two solvent mixtures: A, consisting of 80:20 (v/v) methanol/0.5M ammonium acetate, and B, including 70:30 (v/v) acetone/acetonitrile. Pigment identification was attained by the comparison between the retention times and peak absorption spectrum of standards for zeaxanthin, chlorophyll a, chlorophyll b, β-carotene, lutein and fucoxanthin standards (Sigma and DHI).
Gas chromatography analysis (GCMS) of Fatty Acid content
FAME analysis of the reconstituted extracts was carried out by gas chromatography (GC) (Agilent Technologies 7890A GC System) coupled to mass spectrometry (MS) (Agilent Technologies 5975C inert Mass Selective Detector with Triple-Axis Detector) equipped with a DB-WAX 30 m column (0.25 mm internal diameter). Injections of extracts derivatised using TMSH were carried out in a splitless mode. The program was set up with an inlet temperature of 250°C and helium carrier gas at 20.9 psi. The oven gradient temperature was as follows: an initial hold at 50°C for 1 min followed by a 5°C/min ramp to 200°C and finally a 3°C/min ramp to 230°C for 18 min with a run time of 59 min. The mass spectrometry (MS) solvent delay was set to 5 min. Fatty acids were identified by comparing retention times to the Supelco® 37 Component FAME standards Mix and cross-referenced to the NIST MS Search 2.0 instrument’s internal library.
Antioxidant Capacity of the Microalgal Extracts
The antioxidant activity of the microalgal extracts at a concentration of 0.25 mg mL− 1 was determined using the Folin-Ciocalteu (FC) and Trolox Equivalent Antioxidant Capacity (TEAC) assays as previously described in Barone et al. (2021).
Determination of the Total Phenolic Content - Folin-Ciocalteu (FC) assay
The FC working solution (1:10 dilution) was generated by adding 1.5 mL of Folin-Ciocalteu reagent to 13.5 mL of ultrapure water. Sodium bicarbonate (Na2CO3) was prepared at a concentration of 37.5 g L − 1 in ultrapure water. Trolox, a water-soluble vitamin E analogue was used at a stock concentration of 1500 µM to generate a 10-point standard calibration curve in the range 0–30 µM. To determine the total phenolic content, 500 µL FC working stock and 400 µL Na2CO3 were added to 100 µL of microalgal extract or Trolox standard, and the absorbance was measured at 760 nm.
Evaluation of the antioxidant capacity - Trolox equivalent antioxidant capacity (TEAC) assay
For the TEAC assay, a radical cation solution was prepared using 7 mM of ABTS combined with 2.45 mM potassium persulfate. The ABTS + solution was diluted with ethanol (50%) to reach an absorbance of 0.700 ± 0.050 at 734 nm. For the analysis, 100 µL of the microalgal extract was mixed with 900 µL of diluted ABTS + solution and the samples were incubated for 15 min at room temperature. The absorbance of samples, standards and controls was immediately recorded at 734 nm.
Preparation of Human A549 Cells
The human lung adenocarcinoma cell line A549 (Sigma-Aldrich, UK) was grown in Ham's F-12 Nutrient Mix (BioSciences) supplemented with 10% fetal bovine serum (FBS) (Scientific Laboratory Supplies) and 1% Penicillin-Streptomycin (Pen-Strep) (Cytiva) in a 5% CO2 atmosphere at 37ºC based on Panja et al. (2016). Exponential phase cells were sub-cultured every two days, ensuring the use of actively proliferating cells throughout the experiments. Cell viability was determined using 0.4% trypan blue exclusion as described in Cooper et al. (2016).
Cell Viability Analysis – MTS assay
The effect of H2O2 and the extracts on cell viability was determined using the tetrazolium compound-based CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay (Promega) (Herbert et al. 2024). Initially, A549 lung cells (2x104 cells mL− 1) seeded in 96-well plates were treated with H2O2 (0.1 mM – 0.8 mM) for 24 h to induce oxidative stress, based on Yilmazer (2018). Prior to the microalgal extract treatment, A549 cells underwent a 24 h stress induction with H2O2 (0.5 mM). Subsequently, the H2O2 was removed, and cells were washed with PBS and treated with 200 µL of freshly prepared extract (150, 100 and 50 µg mL− 1) or medium only. DMSO (< 1%) treated cells and non-treated cells were included as vehicle and negative controls, respectively. Doxorubicin (5 µg mL− 1) was used as a positive control for cytotoxicity. Following treatment, an MTS cytotoxicity assay was performed in accordance with the manufacturer’s instructions. Briefly, the MTS reagent was added to each well and cells were incubated at 37⁰C for 3 h before measuring absorbance at 490 nm using a FLUOstar OPTIMA microplate reader. The absorbance recorded was analysed and the results were expressed as a percentage of cell viability of the extract-treated cells (at various concentrations) compared to the untreated control cells (without microalgal extract). Values were expressed as mean ± SD from three separate experiments, each performed in triplicate and samples were normalised using the vehicle control. The percentage of viable cells was calculated as follows:
% Cell viability\(=\frac{\text{A}\text{b}\text{s}\left(\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\right)-\text{A}\text{b}\text{s}\left(\text{b}\text{a}\text{c}\text{k}\text{g}\text{r}\text{o}\text{u}\text{n}\text{d}\right)}{\text{A}\text{b}\text{s}\left(\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)-\text{A}\text{b}\text{s}\left(\text{b}\text{a}\text{c}\text{k}\text{g}\text{r}\text{o}\text{u}\text{n}\text{d}\right)}\text{x} 100\)
Detection of Intracellular Levels of Reactive Oxygen Species (ROS)
The DCF-DA assay was used to evaluate the capacity of microalgal extracts to generate intracellular levels of reactive oxygen species (ROS) in the A549 lung cell line. The assay is based on the conversion rate of 2’, 7’-dichloro-dihydro-fluorescin diacetate (H2DCF-DA) into a fluorescent form 2’, 7’-dichlorofluorescein (DCF), as previously described in Nizioł-Łukaszewska et al. (2018) and Bujak et al. (2021). Cells were seeded in 96-well plates at a density of 5×104 cells mL− 1 and initially cultured for 24 h. Following this, 100 µL of H2DCF-DA (Cayman, VWR) at a concentration of 20 µM in serum-free medium (Ham’s F12) was added to the wells and the cells were incubated for 45 min at 37°C. The H2DCF-DA solution was then carefully removed and cells were washed twice with PBS before treatment with the stressor, hydrogen peroxide (H2O2), at a concentration of 0.5 mM for 24 h. After this incubation, cells were washed twice with PBS followed by the addition of varying concentrations (150, 100 and 50 µg mL− 1) of microalgal extracts for 24 and 48 h treatment periods. Once the incubation period was completed, the magnitude of fluorescence was measured using fluorescence spectroscopy with a microplate reader (FLUOstar OPTIMA) (excitation and emission wavelength 490/520nm, respectively). Values were expressed as mean ± SD of three separate experiments. Each experiment was performed in triplicate. The results were calculated as a percentage of untreated control against the extract-treated cells and were normalised with the vehicle control. The formula used for the percentage of reactive oxygen species (ROS) is as follows:
![](https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1717149717.png)
RNA extraction using Trizol reagent
A549 lung cells (3x105 cells mL− 1) were seeded in 6-well plates and incubated overnight before treatment with 0.5 mM H2O2 for 24 h to induce oxidative stress based on the method of Sansone et al. (2017). Subsequently, cells were treated with Et2O extracts at a range of concentrations (150, 100 and 50 µg mL− 1) for 24 and 48 h. After incubation, RNA was extracted using 400 µL Trizol reagent (Invitrogen ThermoFisher) per 1 x 107 cultured cells. This was followed by a 5 min room temperature incubation to permit complete dissociation of the nucleoprotein complex. Then 80 µL of chloroform was added to facilitate phase separation. The sample was then shaken gently for 15 s to create an emulsion and incubated at room temperature for 3 min. Followed by centrifugation at 12,000x g at 4ºC for 15 min (Hettich Mikro 200R). During centrifugation, three distinct layers were formed: a lower red phenol-chloroform layer, an interphase and an upper colourless aqueous phase. The RNA-containing aqueous phase was transferred to a fresh tube and RNA precipitation was achieved with the addition of 200 µL of ice-cold isopropanol, followed by a 10 min incubation at room temperature. The tubes were then centrifuged at 12,000x g at 4ºC for 10 min. The RNA precipitate was seen as a white gel-like pellet and the supernatant was carefully discarded. The RNA pellet was washed with 400 µL of 75% ethanol. The sample was vortexed and then centrifuged at 7500x g at 4ºC for 5 min. After discarding the supernatant, the RNA pellet was air-dried for 5–10 min and then re-suspended in 20 µL of RNase-free water. This was followed by heating at 60ºC and gentle mixing to ensure homogeneity before aliquoting. RNA was quantified using microvolume spectrophotometry (DeNovix DS-11). The purity of RNA was assessed using the 260 nm/280nm ratio, which was consistently within the range of 1.8-2.0, indicative of high purity. Following purification, the RNA samples were stored at -80ºC until required for analysis.
Reverse Transcription – Qualitative PCR analyses (RT-qPCR)
cDNA was prepared using 200 ng of each RNA sample using the QuantiTech® Reverse Transcription Kit (Qiagen) and following the manufacturing instructions. Subsequently, qPCR for amplification of cDNA was performed in a 10 µL reaction volume using the QuantiNova ™ SYBR® Green PCR kit (Qiagen, Cat. No. 208052) on the StepOnePlus Real-Time PCR System (Applied Biosystem) and based on the protocol of Sansone et al. (2017). The amplification program applied included an initial heat activation step at 95ºC for 10 min followed by 35 cycles at 95ºC for 15 s (denaturation) and 60ºC for 60 s (annealing/extension). Melting curves were performed to verify the specificity of the PCR assays (Table 1). Results were normalised to the endogenous control gene glyceraldehyde 3-phosphate dehydrogenase GADPH (F: 5’-GTCTCCTCTGACTTCAACAGCG-3’ and R:5’-ACCACCCTGTTGCTGTAGCCAA-3’) (Zhang et al. 2015). The relative gene expression level was analysed using the 2−∆∆Ct method (Livak and Schmittgen 2001). Cells treated with < 1% DMSO were used as a vehicle reference.
Statistical Analysis
Analysis of variance (ANOVA) was performed to identify significant differences between the antioxidant activity of microalgae extracts, cell viability, ROS production and gene expression in human lung cancer cells. The data matrix underwent principal component analysis (PCA) to evaluate the relationships between the various bioactivity variables measured for the three microalgal species. All statistical analyses were performed using the IBM SPSS Statistics 26 package.