Antioxidant activity of Micractinium sp. (Chlorophyta) extracts against H2O2 induced oxidative stress in human breast adenocarcinoma cells

Antioxidant activity of Micractinium sp. (Chlorophyta) extracts against H2O2 induced oxidative stress in human breast adenocarcinoma cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antioxidant activity of Micractinium sp. (Chlorophyta) extracts against H2O2 induced oxidative stress in human breast adenocarcinoma cells Onur Bulut, Iskin Engin, Cagla Sonmez, Huseyin Avni Oktem This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4690459/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In response to the growing demand for high-value bioactive compounds, microalgae cultivation has gained a significant acceleration in recent years. Among these compounds, antioxidants have emerged as essential constituents in the food, pharmaceutical, and cosmetics industries. This study focuses on Micractinium sp. ME05, a green microalgal strain previously isolated from hot springs flora in our laboratory. Micractinium sp. cells were extracted using six different solvents, and their antioxidant capacity, as well as total phenolic, flavonoid, and carotenoid contents, were evaluated. The methanolic extracts demonstrated the highest antioxidant capacity, measuring 7.72 and 93.80 µmol trolox equivalents.g -1 dry weight (DW) according to the DPPH and FRAP assays, respectively. To further characterize the biochemical profile, reverse phase high-performance chromatography (RP-HPLC) was employed to quantify twelve different phenolics, including rutin, gallic acid, benzoic acid, cinnamic acid, and β-carotene, in the microalgal extracts. Notably, the acetone extracts of Micractinium sp. grown mixotrophically contained a high amount of gallic acid (469.21 ± 159.74 µg.g -1 DW), while 4-hydroxy benzoic acid (403.93 ± 20.98 µg.g -1 DW) was the main phenolic compound in the methanolic extracts under heterotrophic cultivation. Moreover, extracts from Micractinium sp. exhibited remarkable cytoprotective activity by effectively inhibiting hydrogen peroxide-induced oxidative stress and cell death in human breast adenocarcinoma (MCF-7) cells. In conclusion, with its diverse biochemical composition and adaptability to different growth regimens, Micractinium sp. emerges as a robust candidate for mass cultivation in nutraceutical and food applications. Microalgae antioxidants gallic acid 4-hydroxy benzoic acid oxidative stress cytoprotective effect Figures Figure 1 Highlights Antioxidant capacity of thermo-tolerant green microalga Micractinium sp. evaluated. Differential effects of two cultivation modes on antioxidants were compared. Mixotrophic cultures had higher antioxidant activity, phenolics and carotenoids. High amounts of phenolic compounds such as gallic acid and rutin detected by RP-HPLC. Micractinium sp. extracts inhibited oxidative stress in MCF-7 cells. 1. Introduction Oxidative stress is a biological phenomenon that occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to counteract their harmful effects. ROS can induce damage to cellular components, including proteins, lipids, and DNA, with prolonged exposure correlating strongly with various diseases. Antioxidants, encompassing vitamins, phenolic compounds, and carotenoids, constitute a vital component of the human diet, primarily sourced from fruits and vegetables (Forman and Zhang 2021 ). Carotenoids such as astaxanthin, lycopene, lutein and β-carotene, are a class of natural pigments abundant in yellow, orange and dark green leafy plants. β-carotene, in particular, functions as an antioxidant by scavenging various free radicals. Polyphenols, another crucial group of antioxidants, include phenolic acids, flavonoids, tannins, lignans, and stilbenes, and are predominantly found in vegetables, fruits, cereals, herbs, and spices (Zhang and Tsao 2016 ; Maoka 2020 ). Antioxidants act through diverse mechanisms, such as inhibiting enzymes like glutathione S-transferase, chelating trace metals involved in ROS production, and up-regulating antioxidant defense pathways. The documented anti-carcinogenic and anti-proliferative activities of phenolic compounds on various tumor cell lines highlight their potential therapeutic benefits (Dai and Mumper 2010 ; Abbaszadeh et al. 2019 ; Yuan et al. 2022 ). Furthermore, the positive cognitive outcomes in patients with Down syndrome associated with early developmental stage consumption of phenolic-rich dietary supplements add to the versatile spectrum of their effects, including antimicrobial, anti-inflammatory, and anti-viral activities (Vacca et al. 2016 ; Ambriz-Pérez et al. 2016 ; Parra-Riofrio et al. 2023 ; Lobiuc et al. 2023 ). Studies also reveal the efficacy of using dietary antioxidants in combination with chemo and radiation therapy in cancer patients to suppress the toxicity-related side effects of such treatments (Ferdous and Yusof 2021 ). In response to contemporary lifestyle challenges, where poor dietary habits and environmental factors contribute to oxidative stress, there is a growing global demand for antioxidants. In addition to their well-documented health benefits, antioxidants serve a crucial role in the preservation of packaged foods by inhibiting oxidation processes, thereby extending the shelf life of products. The food industry traditionally leaned towards synthetic antioxidants for this purpose; however, mounting concerns about their potential adverse health effects have prompted a shift in consumer preferences. There is now a strong inclination towards the use of natural antioxidants, driving researchers to explore new biological sources that are rich in these natural compounds (Franco et al. 2019 ; Poljsak et al. 2021 ). Microalgae emerge as promising natural sources of antioxidants due to their adaptability to adverse environmental conditions and their capacity to accumulate essential secondary metabolites, including phenolic compounds and carotenoids, with robust antioxidant capabilities. Notably, the antioxidant activity varies among microalgae species and cultivation conditions (Coulombier et al. 2021 ; Almendinger et al. 2021 ). Microalgal extracts have demonstrated cytotoxic effects on various human cancer cells including liver, colon, breast, lung, and brain, while some of them also exhibiting the ability to inhibit oxidative stress without causing cytotoxicity (Sansone et al. 2017 ; El-Baz et al. 2018 ; Abd El-Hack et al. 2019 ; Karakaş et al. 2019 ; Bulut et al. 2023 ). β-carotene from Dunaliella salina , polyunsaturated fatty acids from Nannochloropsis salina , sterols in N. oculate extracts and violaxanthin, a carotenoid compound, isolated from D. tertiolecta are examples of microalgal compounds with documented anti-cancer properties (Pasquet et al. 2011 ; Jayappriyan et al. 2013 ; Sanjeewa et al. 2016 ; Sayegh et al. 2016 ). Micractinium is a genus of green microalgae in the family Chlorellaceae (Chlorophyta). Different species of Micractinium are adapted to diverse geographical locations and exhibit high phenotypic plasticity (Krivina et al. 2023 ). Various strains of Micractinium showed promising biotechnological potential as feed supplement, biofuel, and in wastewater treatment (Abou-Shanab et al. 2014 ; Paliwal et al. 2016 ). Thermophilic/thermotolerant strains of Micractinium are advantageous in lowering the operational costs of cultivation in large scale bioreactors (Malavasi et al. 2020 ). Micractinium sp. METUNERGY05 (ME05), used in this study, was previously isolated in our laboratory from hot springs of Haymana, Turkey (Onay et al. 2014 ). It is a thermotolerant strain which is suitable for biodiesel production and can be cultivated both mixotrophically and heterotrophically using by-products of a sugar factory as sole carbon source, which reduces the operational costs (Engin et al. 2018a ; Engin et al. 2018b ). The aim of this study was to assess the antioxidant properties, along with the phenolic, flavonoid, and carotenoid contents of Micractinium sp. extracts cultivated under both mixotrophic and heterotrophic conditions. To achieve this, we employed six different solvents for the extraction of microalgal biomass. Utilizing RP-HPLC analysis, we explored the diverse profile of phenolics present in the extracts, providing valuable insights into the intricate biochemical composition of Micractinium sp. Subsequently, we evaluated the cytoprotective properties of the methanolic extracts of Micractinium sp. Specifically, we investigated their effectiveness against hydrogen peroxide (H 2 O 2 )-induced oxidative stress, shedding light on the potential bioactivity of these extracts in mitigating cellular damage. Our findings highlight Micractinium sp. as a promising natural source of antioxidants for nutraceutical applications and the food industry. The versatility of Micractinium sp. in adapting to different cultivation conditions further enhances its appeal as a bioresource with broad applications. 2. Materials and methods 2.1. Chemicals and reagents All chemicals and solvents used in this study were purchased from Sigma–Aldrich, AppliChem GmbH and Merck Company, and were analytical or HPLC grade. Molasses used during heterotrophic cultivation was obtained from Konya Sugar Factory, Turkey. 2.2 Microalgal culture and extract preparation 2.2.1. Growth and culture conditions of Micractinium sp. The green microalga Micractinium sp. cells were cultured in Tris-Acetate-Phosphate (TAP) growth medium (Sönmez et al. 2016 ). Detailed morphological, biochemical, and molecular characterization of Micractinium sp. ME05 strain was previously reported by Onay et al. ( 2014 ). For mixotrophic cultivation, Micractinium sp. was inoculated in 1 L TAP medium and grown at 25°C under photoperiod (16:8 h of light: dark) at 54 µmol.m − 2 .s − 1 light intensity with constant shaking until cells reached the stationary phase and were harvested. Heterotrophic cultivation was carried out by inoculating 2–3 x 10 5 cells.mL − 1 Micractinium sp. into 1 L Bold’s Basal Medium (BBM) supplemented with 19 g of molasses hydrolysate in 2-L Erlenmeyer flasks. The composition of BBM and molasses, preparation of molasses hydrolysate and the optimum molasses hydrolysate amount for the highest biomass concentration were previously described by Engin et al. ( 2018a ). Micractinium sp. cells were grown under complete darkness at 30°C with air supply at 0.5 L.min − 1 through an aquarium pump. The growth went by for 5 days until harvesting. 2.2.2. Preparation of microalgal extracts Micractinium sp. cells cultivated either mixotrophically or heterotrophically were centrifuged at 3600x g for 20 min. The cell pellet was lyophilized using a freeze-dryer and ground to a fine powder. Six different solvents, namely methanol, ethanol, acetone, hexane, ethyl acetate and water were used as extractants. 200 mg of lyophilized microalgae was extracted with 5 ml of solvent at room temperature by sonication in an ultrasonic water bath for 20 min followed by stirring on an orbital shaker for 1 h. The extract was centrifuged at 3800x g for 10 min and the aqueous phase was collected in separate bottles. The residual pellets were re-suspended in the same solvents and re-extracted as previously described in Bulut et al. ( 2019 ). The extracts were combined and filtered through a 0.45 µm pore size polytetrafluoroethylene (PTFE) syringe filter and dried using a rotary evaporator. The dried residues were weighed to calculate the extraction yields. Methanol was used as the vehicle solution for solubilization of the dried residues at a concentration of 20 mg.mL − 1 . The solubilized extracts were stored at -20°C until further analysis. 2.3. Antioxidant measurements 2.3.1 Measurement of the total phenolic content The Folin & Ciocalteu spectrophotometric method was used to measure the total phenolic content (TPC) of the microalgal extracts. The protocol previously reported by Bulut et al. ( 2019 ) was followed. 100 µL sample was mixed with 400 µL of Folin-Ciocalteu reagent (1:10 diluted in ultrapure water). The mixture was vortexed thoroughly and allowed to stand at room temperature for 5 min. 500 µL of 7.5% (w/v) NaNO 2 solution was added to the mixture and the tubes were incubated for 1.5 h in the dark at room temperature. Following the incubation, 200 µL of sample was transferred to a clear 96-well microplate and the absorbance of each well was measured at 760 nm using a UV–vis microplate reader. A standard curve prepared by serial dilution of gallic acid solutions ranging from 10 to 400 mg.L − 1 was used for calibration. TPC of the extracts was calculated as gallic acid equivalents using the regression equation of the standard curve. TPC was expressed as mg gallic acid equivalents per gram dry weight of sample (mg GAE.g − 1 DW). The analyses were performed as biological triplicates. 2.3.2 Measurement of the total flavonoid content The total flavonoid content (TFC) of the microalgal extracts was measured by the aluminum chloride method (Herald et al. 2012 ). One milliliter of extract was diluted 1:5 with ultrapure water; mixed with 0.3 mL of 5% (w/v) NaNO 2 and incubated at ambient temperature for 5 min. The sample was mixed with 0.3 mL of 10% (w/v) AlCl 3 in ethanol after incubation at ambient temperature for 5 min. This step was followed by another incubation at ambient temperature for 6 min. Then, 2 mL of 1 M NaOH was added to the mixture and the total volume was adjusted to 10 mL with ultrapure water. After a brief vortex, 200 µL of the sample was transferred to a clear 96-well microplate and the absorbance of each well was measured at 510 nm using a UV–vis microplate reader. A standard curve was prepared with serial dilution of quercetin solutions ranging from 10 to 400 mg.L − 1 . Total flavonoid content of the microalgal extracts was calculated using the regression equation of this standard curve. The results were expressed as mg quercetin equivalents per gram dry weight of sample (mg QE.g − 1 DW). 2.3.3. Measurement of the total carotenoid content Total carotenoid content of the microalgal extracts was calculated following the method by Lichtenthaler and Buschmann ( 2001 ) (Lichtenthaler and Buschmann 2001 ). Absorbance of the methanolic extracts was recorded at 470, 652 and 665 nm using a UV–vis spectrophotometer and total carotenoid content was calculated according to the Lichtenthaler equations as follows: c a (µg.mL − 1 ) = 16.72 A 665 – 9.16 A 652 c b (µg.mL − 1 ) = 34.09 A 652 – 15.28 A 665 c (x+c) (µg.mL − 1 ) = (1000 A 470 – 1.63 c a – 104.96 c b )/221 Where c a and c b are concentrations of chlorophyll a and b, respectively, and c (x+c) is the concentration of the total carotenoids. The results were expressed as mg carotenoid per gram dry weight of sample (mg carotenoid.g − 1 DW). 2.3.4. DPPH assay The DPPH radical scavenging activity of the microalgal extracts was measured according to Cheng et al. ( 2006 ). Briefly, 100 µL of microalgal extracts at concentrations ranging from 50 to 2000 µg.mL − 1 was mixed with 100 µL of 0.2 mM DPPH solution in a clear 96-well plate. The mixture was incubated in the dark at ambient temperature for 30 min, and the absorbance was recorded at 515 nm using a microplate reader with the trolox solution as the positive control and the DPPH solution as blank. The percentage of scavenged DPPH• radical was calculated according to the following equation: DPPH scavenging activity (%) = [1-( As - Asc )/ Ac ]*100 Where As is absorbance of the sample (100 µL of sample with 100 µL of DPPH• radical solution), Asc is absorbance of the sample control (100 µL of sample with 100 µL of methanol) and Ac is absorbance of the control (100 µL of methanol with 100 µL of DPPH• radical solution). A standard curve was prepared with serially diluted trolox solutions in the range of 2.5 to 80 µmol.L − 1 concentrations. Total antioxidant capacity of the microalgal extracts was calculated as trolox equivalents using the regression equation of the standard curve. The results were expressed as micromol of equivalent trolox per gram of dried weight (µmol TE.g − 1 DW) and (%) DPPH radical scavenging activity of the extract (Bulut et al. 2019 ). 2.3.5. FRAP assay The antioxidant capacity of the extracts was also evaluated by the FRAP assay through monitoring the reduction of Fe 3+ -TPTZ to blue-colored Fe 2+ -TPTZ (Firuzi et al. 2005 ). The working FRAP solution was freshly prepared by mixing ten volumes of acetate buffer, one volume of TPTZ solution and one volume of ferric chloride hexahydrate solution and warmed at 37°C in a water bath prior to use. 25 µL of the microalgal extract at concentrations between 50 to 2000 µg.mL − 1 was mixed with 175 µL of pre-warmed FRAP solution in a clear 96-well microplate. The microplate was allowed to stand at room temperature for 30 min in the dark. The absorbance of each sample was measured at 593 nm using a microplate reader. Trolox solutions ranging from 5 to 20 µmol.L − 1 were used for preparation of a standard curve. Total antioxidant capacity of the microalgal extracts was calculated as trolox equivalents using the regression equation of the standard graph. FRAP values were expressed as µmol trolox equivalents per gram dry weight of sample (µmol TE.g − 1 DW) (Bulut et al. 2019 ). 2.3.6. Reverse phase high performance liquid chromatography (RP-HPLC) analysis Twelve selected phenolic compounds; namely, gallic acid, benzoic acid, 4-hydroxy benzoic acid, vanillic acid, syringic acid, cinnamic acid, coumaric acid, caffeic acid, chlorogenic acid, rosmarinic acid, quercetin and rutin were identified in the microalgal extracts by reverse phase HPLC (Waters Alliance 2695, Waters Corporation, USA) coupled to a UV/Vis detector (Waters 2489 detector) as described by Bulut et al. ( 2019 ). The microalgal extract at a concentration of 1000 ppm was passed through a 0.45 µm PTFE syringe filter prior to injection. The chromatographic separation was performed in a C18 analytical column (ACE 5, AC Technologies, Scotland). Elution was carried out with a gradient pump mode involving three mobile phases; mobile phase A: 2% (v/v) acetic acid, mobile phase B: acetonitrile and 0.5% (v/v) acetic acid (1:1 v/v) and mobile phase C: acetonitrile. The gradient was set as following: 0–8 min: 95% A and 5% B; 8–10 min: 80% A and 20% B; 10–17 min: 78% A and 22% B; 17–19 min: 75% A and 25% B; 19–30 min: 73% A and 27% B; 30–35 min: 60% A and 40% B; 35–40 min: 55% A and 45% B; 40–45 min: 35% A and 65% B; 56 − 50 min: 10% B and 90% C; 50–52 min: 100% C; and 52–60 min: 95% A and 5% B. The flow rate was 1.2 mL.min − 1 , the injection volume was 20 µL and the column temperature was maintained at ambient temperature. Simultaneous monitoring was done via a UV/Vis detector with reference wavelength of 280 nm. Retention times and peak areas of both authentic standards and microalgal extracts were monitored automatically by Empower 3 Chromatography Data Software (Waters Corporation, USA). The concentration of individual phenolic compounds was quantified by comparison of the chromatographic peaks of the microalgal extracts to those of authentic standards. To identify the β-carotene content, a RP-HPLC system equipped with a Shimadzu LC-20AD pump (Shimazdu, Kyoto, Japan) and Shimazdu SPD-20A UV/Vis detector was used. An Inertsil ODS-2 C18 analytical column was used for the chromatographic runs (GL Sciences, Tokyo, Japan). The gradient pump mode consisting of two mobile phases (mobile phase A: 90% acetonitrile in water, mobile phase B: ethyl acetate) was used for elution at a flow rate of 1.0 mL.min − 1 . The peaks were detected at 450 nm. β-carotene concentration in the microalgal extracts was identified by comparison of the peak areas of the samples to the authentic standards. 2.4. Cell culture and assays 2.4.1. Inhibition of the H 2 O 2 -induced reactive oxygen species (ROS) generation in MCF-7 cells The human breast cancer cell line, MCF-7 (ATCC HTB-22), was obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 µg.mL − 1 ), and maintained in a CO 2 incubator with 5% CO 2 at 37°C. The effect of the methanolic microalgal extracts on inhibition of the intracellular H 2 O 2 -induced ROS generation in MCF-7 cells was evaluated by a fluorescence assay using the cell-permeant probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as described by Zhuang et al. ( 2017 ) with minor modifications. MCF-7 cells were pretreated with 50, 100, 200 or 400 µg.mL − 1 of the methanolic extracts, separately for 48 h in a 96-well black cell culture plate. Cells treated with the culture medium containing VS and ascorbic acid (8 µg.mL − 1 ) were used as the negative and the positive controls, respectively. After 48 h incubation with the methanolic extracts, the cells were exposed to 0.5 mM of H 2 O 2 for 6 h to induce intracellular oxidative stress via ROS generation. The cells were washed twice with PBS and incubated with serum-free medium containing DCFH-DA (20 µM) for 30 min in the dark at 37°C. Cells were immediately washed with PBS. The formation of the fluorescent 2',7'-dichlorofluorescein (DCF) due to oxidation of the non-fluorescent DCFH-DA by intracellular oxidative stress was detected by a fluorescence microplate reader with an excitation wavelength of 495 nm and an emission wavelength of 525 nm. 2.4.2. Apoptosis assay The cytoprotective effect of the methanolic extracts of mixotrophically grown Micractinium sp. on H 2 O 2 -induced apoptosis of MCF-7 cells was determined by an Annexin V-FITC and propidium iodide (PI) double-staining apoptosis assay kit (Takara Bio Inc., Japan) according to the manufacturer’s instructions. MCF-7 cells were firstly treated with the microalgal extracts and then, with H 2 O 2 as described in section 2.11. The cells were collected by trypsinization, washed with PBS twice, and resuspended in 200 µL of binding buffer containing 5 µL of Annexin V-FITC. After incubation of the cells for 15 min at room temperature in the dark, 10 µL of PI was added to cells and incubated for 10 min in an ice bath in the dark. Finally, the cells were analyzed using a flow cytometer. 2.5. Statistical analysis All experiments in this study were carried out in biological triplicates. Results were expressed as mean ± standard error. The analysis of the mean values was performed using the analysis of variance (ANOVA) test and Tukey’s post-hoc comparison test. A p -value < 0.001, < 0.01, and < 0.05 was considered as highly significant (***), very significant (**) and significant (*), respectively in statistical terms. Pearson’s correlation of determination ( R 2 ) was used to compute correlations among antioxidant assays, TPC and TFC under different growth conditions. The statistical analysis was conducted using R version 3.4.2. 3. Results 3.1. Extraction yields of Micractinium sp. in different solvents In this study, six different solvents with varying polarity, namely, methanol, ethanol, acetone, hexane, ethyl acetate and water were used to extract antioxidants from either mixotrophically or heterotrophically grown Micractinium sp. The extraction yields are given in Table 1 . The highest extraction yield of 30.40 ± 0.94% was obtained in methanol followed by 28.16 ± 1.08% in water ( p = 0.91) for mixotrophic growth. For heterotrophic growth, the highest yield was equal in methanol (38.23 ± 3.90%) and water (38.33 ± 0.34%) ( p = 1.0). The lowest extraction yield was in ethyl acetate (11.54 ± 1.47%) and acetone (8.29 ± 1.41%) for mixotrophic and heterotrophic cultivation, respectively. The difference in extraction yields of methanol and water with respect to acetone, ethyl acetate, ethanol and hexane was highly significant ( p < 0.001) for both mixotrophic and heterotrophic growth conditions. Table 1 Extraction yields of Micractinium sp. extracts prepared using different solvents and growth conditions. Extraction yield (%) Solvent Mixotrophic growth Heterotrophic growth Methanol 30.40 ± 0.94 b 38.23 ± 3.90 b Ethanol 14.85 ± 1.88 a 17.78 ± 4.78 a Acetone 16.46 ± 0.95 a 8.29 ± 1.41 a Hexane 12.70 ± 2.62 a 15.45 ± 2.00 a Ethyl acetate 11.54 ± 1.47 a 13.92 ± 1.31 a Water 28.16 ± 1.08 b 38.33 ± 0.34 b Results are expressed as mean ± standard error of three measurements ( n = 3). Means with different letters in the same column are statistically significant ( p < 0.05). 3.2. Antioxidant capacity of Micractinium sp. Measured by DPPH and FRAP assays Antioxidant capacity of Micractinium sp. extracts in six different solvents under two different growth regimens was measured by DPPH and FRAP assays. The results are given in Table 2 . DPPH assay results are expressed both as % DPPH radical scavenging activity of microalgal extracts at 1 mg.mL − 1 concentration and as micromoles trolox equivalent (TE) per gram dry weight (g − 1 DW) of microalgae. The former expression does not take into account the extraction yield of the samples in each solvent. For mixotrophic growth, the highest antioxidant capacity was measured in the methanolic extracts as 7.72 ± 0.95 and 93.80 ± 6.28 µmol TE.g − 1 DW followed by ethanol extracts as 6.41 ± 1.33 and 79.83 ± 7.56 µmol TE.g − 1 DW by DPPH and FRAP assays, respectively. Similarly, the highest antioxidant capacity in heterotrophically grown samples was recorded in the methanolic extracts as 6.82 ± 1.31 and 64.91 ± 4.28 µmol TE.g − 1 DW by DPPH and FRAP assays, respectively. The antioxidant capacities of mixotrophically grown microalgae were higher compared to heterotrophic samples. Particularly, the difference in antioxidant capacities measured by FRAP assay between mixotrophic and heterotrophic growth in methanol, ethanol and acetone extracts was statistically significant. The correlation between DPPH and FRAP assays was found to be highly significant ( p < 0.001) for both conditions, and the coefficient of determination ( R 2 ) values for these assays were calculated as 0.57 and 0.65 for mixotrophic and heterotrophic growth, respectively. The low correlation between two methods can be explained by the methodological differences in detection and measurement of the antioxidants (Bulut et al. 2019 ). Table 2 Antioxidant capacity of Micractinium sp. extracts in different solvent extracts determined by DPPH and FRAP assays. Mixotrophic growth Heterotrophic growth Solvent (%) DPPH Radical scavenging effect 1 DPPH (µmol TE.g − 1 DW) FRAP (µmol TE.g − 1 DW) (%) DPPH Radical scavenging effect 1 DPPH (µmol TE.g − 1 DW) FRAP (µmol TE.g − 1 DW) Methanol 39.61 ± 4.37 bc 7.72 ± 0.95 a 93.80 ± 6.28 c 28.10 ± 2.36 ab 6.82 ± 1.31 b 64.91 ± 4.28 d Ethanol 64.15 ± 5.24 b 6.41 ± 1.33 ac 79.83 ± 7.56 ac 35.75 ± 3.94 ac 3.82 ± 0.62 ab 50.43 ± 10.45 bd Acetone 46.48 ± 1.39 ab 4.97 ± 0.45 ab 68.88 ± 2.96 a 44.35 ± 9.44 a 2.32 ± 0.30 a 35.70 ± 1.85 ab Hexane 30.55 ± 19.09 ac 2.05 ± 0.28 b 15.70 ± 2.25 b 14.24 ± 5.28 bc 1.12 ± 0.47 a 11.73 ± 2.14 c Ethyl acetate 54.71 ± 1.42 ab 4.10 ± 0.45 ab 29.06 ± 6.32 b 31.30 ± 3.42 ab 2.79 ± 0.54 a 14.55 ± 3.33 ac Water 17.65 ± 2.91 c 2.93 ± 0.64 bc 7.69 ± 0.43 b 9.00 ± 2.01 b 1.68 ± 0.53 a 1.40 ± 0.50 c Results are mean ± standard error of three measurements ( n = 3). 1 Radical scavenging effects of algal extracts at 1 mg.mL − 1 concentration. Means with different letters in the same column are statistically significant (p < 0.05). 3.3. Total phenolic, flavonoid and carotenoid contents of Micractinium sp. extracts Total phenolic contents (TPC) of Micractinium sp. grown under two different growth conditions and extracted in six different solvents are given in Table 3 . The highest TPC was found in methanolic extracts as 18.11 ± 2.17 mg GAE.g − 1 DW and 11.47 ± 1.41 mg GAE.g − 1 DW for mixotrophic and heterotrophic growth, respectively. The difference between TPCs of two growth conditions in methanolic extracts is very significant ( p = 0.009). These results are consistent with a previous report in which, both Chlorella vulgaris and Scenedesmus obliquus had higher polyphenols in the mixotrophic culture compared to the heterotrophic culture (Shetty and Sibi 2015 ). The lowest TPCs of both mixotrophic and heterotrophic cultivation were measured in hexane extracts as 3.87 ± 0.83 mg GAE.g − 1 DW and 2.40 ± 0.19 mg GAE.g − 1 DW, respectively. The difference in results was not statistically significant. Table 3 Total phenolic, flavonoid and carotenoids of Micractinium sp. extracts prepared using different solvents and growth conditions. Mixotrophic growth Heterotrophic growth Solvent Total phenolic content (mg GAE.g − 1 DW) Total flavonoid content (mg QE.g − 1 DW) Carotenoid content (mg.g − 1 DW) Total phenolic content (mg GAE.g − 1 DW) Total flavonoid content (mg QE.g − 1 DW) Carotenoid content (mg.g − 1 DW) Methanol 18.11 ± 2.17 b 5.72 ± 0.26 a 2.27 ± 0.18 b 11.47 ± 1.41 c 3.22 ± 0.27 b 1.65 ± 0.01 b Ethanol 7.40 ± 1.46 a 5.21 ± 1.70 a NT 6.67 ± 1.07 b 1.89 ± 0.35 a NT Acetone 7.37 ± 0.83 a 4.21 ± 0.68 ab 3.02 ± 0.11 a 4.77 ± 0.35 ab 1.40 ± 0.16 a 0.32 ± 0.05 a Hexane 3.87 ± 0.83 a 1.07 ± 0.17 b NT 2.40 ± 0.19 a 0.86 ± 0.07 a NT Ethyl acetate 8.02 ± 0.52 a 2.39 ± 0.08 ab 3.17 ± 0.21 a 4.76 ± 0.09 ab 2.09 ± 0.45 ab 0.32 ± 0.06 a Water 6.65 ± 1.32 a 1.15 ± 0.07 b NT 7.07 ± 0.66 b 1.47 ± 0.02 a NT Results are mean ± standard error of three measurements ( n = 3). NT: Not Tested. Means with different letters in the same column are statistically significant (p < 0.05). The highest total flavonoid content (TFC) of mixotrophic Micractinium sp. was detected in the methanolic extracts (5.72 ± 0.26 mg QE.g − 1 DW) followed by the ethanol extracts (5.21 ± 1.70 mg QE.g − 1 DW) (Table 3 ). TFC of heterotrophically grown cell extracts was the highest in methanol with a concentration of 3.22 ± 0.27 mg.QE g − 1 DW. The difference in TFC of the methanolic extracts between mixotrophic and heterotrophic cultivation was not statistically significant. However, there was a significant reduction in TFC of ethanol extracts from heterotrophic samples (1.89 ± 0.35 mg QE.g − 1 DW) compared to ethanol extracts of the mixotrophic culture (5.21 ± 1.70 mg QE.g − 1 DW). The total carotenoid content (TCC) of Micractinium sp. was calculated in methanol, acetone and ethyl acetate extracts as 2.27 ± 0.18 mg.g − 1 DW, 3.02 ± 0.11 mg.g − 1 DW and 3.17 ± 0.21 mg.g − 1 DW, respectively under mixotrophic cultivation (Table 3 ). TCC of heterotrophically grown microalgae was recorded as 1.65 ± 0.01 mg.g − 1 DW in methanol, 0.32 ± 0.05 mg.g − 1 DW in acetone and 0.32 ± 0.06 mg.g − 1 DW in the ethyl acetate extracts. The difference in carotenoid content between mixotrophic and heterotrophic growth was statistically significant for ethyl acetate extracts ( p < 0.05). 3.4. Correlation of the antioxidant capacity with the phenolic, flavonoid, and carotenoid contents The correlation of determination ( R 2 ) values between the antioxidant capacity and the total phenolics, flavonoids and carotenoids of Micractinium sp. in different solvent extracts were calculated both for mixotrophic and heterotrophic growth conditions. The R 2 value between the DPPH assay and TPC in ethanol extracts of mixotrophically cultivated microalgae was 0.99 ( p = 0.01). This result is consistent with the strong correlation between TPC of Chlorella vulgaris and Scenedesmus obliquus and the DPPH assay reported by Shetty and Sibi ( 2015 ). In the same study, it was shown that the contribution of phenolics to the antioxidant potential was irrespective of the cultivation mode (Shetty and Sibi 2015 ). In the present study, other R 2 values of 0.90 or higher were obtained between DPPH or FRAP assay and TPC, TFC and TCC in various solvent extracts of Micractinium sp. both under mixotrophic and heterotrophic growth conditions; however, none of them were found to be statistically significant ( p > 0.05) (Supplementary Tables 1 and 2). The statistically significant positive correlation between DPPH assay and TPC in the ethanol extracts suggests that polyphenols that are highly soluble in ethanol greatly contribute to the antioxidant activity of microalgae. Ethanol is also advantageous as a solvent as it is safe for human consumption (Dai and Mumper 2010 ). 3.5. Identification of selected phenolic compounds in Micractinium sp. extracts by RP-HPLC Twelve different phenolic compounds that fall in three categories, namely, flavonols; rutin and quercetin, benzoic acid derivatives; 4-hydroxy benzoic acid, benzoic acid, gallic acid, syringic acid and vanillic acid and cinnamic acid and derivatives; caffeic acid, rosmarinic acid, coumaric acid and chlorogenic acid were quantified by RP-HPLC in methanol, acetone and ethyl acetate extracts of the mixotrophically and heterotrophically grown Micractinium sp. (Table 4 ). Gallic acid (469.21 ± 159.74 µg.g − 1 DW) in the acetone extracts of mixotrophic microalgae was the highest phenolic compound detected. Under heterotrophic growth, 4-hydroxy benzoic acid (403.93 ± 20.98 µg.g − 1 DW) in the methanolic extracts was the most abundant phenolic compound. Strikingly, the amount of the same compound in the methanolic extracts of mixotrophic Micractinium sp. was only 1.98 ± 0.91 µg.g − 1 DW. Acetone is a powerful solvent of flavonols and consistently rutin concentration in acetone extracts (212.09 ± 122.46 µg.g − 1 DW in mixotrophic samples) was significantly higher than the other solvents. Overall, there were considerable differences in the amounts of phenolic compounds between mixotrophic and heterotrophic microalgal extracts. Table 4 The phenolic compounds in different solvent extracts of Micractinium sp. identified by RP-HPLC. Amount 1 (µg.g − 1 DW) Mixotrophic growth Heterotrophic growth Phenolic compound Methanol Acetone Ethyl acetate Methanol Acetone Ethyl acetate Benzoic acid derivatives Gallic acid 129.08 ± 2.65 a 469.21 ± 159.74 a ND 125.06 ± 11.68 a 12.93 ± 3.93 a ND Benzoic acid 13.62 ± 2.63 a 37.84 ± 2.20 a 8.77 ± 2.55 a 107.20 ± 16.85 b 28.09 ± 14.36 a 18.50 ± 3.56 a 4-Hydroxy Benzoic acid 1.98 ± 0.91 a 0.95 ± 0.12 a 1.10 ± 0.63 a 403.93 ± 20.98 b 40.48 ± 4.10 a 4.75 ± 0.60 a Vanillic acid 13.37 ± 7.72 a 5.37 ± 1.54 a ND 47.66 ± 2.44 b 11.91 ± 1.19 a 4.46 ± 0.18 a Syringic acid 27.99 ± 6.87 ab 9.32 ± 0.58 a 5.01 ± 0.61 a 56.42 ± 13.04 b 5.70 ± 0.53 a 6.77 ± 0.88 a Cinnamic acid derivatives Cinnamic acid 10.34 ± 6.86 b 18.06 ± 0.77 b ND 196.44 ± 6.70 c 51.88 ± 2.26 a 38.24 ± 3.61 ab Coumaric acid 19.36 ± 15.25 a 4.41 ± 1.46 a 9.58 ± 1.29 a 10.85 ± 1.49 a 0.55 ± 0.43 a 5.94 ± 0.02 a Caffeic acid 16.46 ± 9.50 a 4.13 ± 0.99 a ND 3.87 ± 0.86 a 4.51 ± 0.30 a ND Chlorogenic acid 11.27 ± 5.61 a 2.30 ± 0.10 a 1.55 ± 0.89 a 2.78 ± 1.05 a 0.79 ± 0.55 a 1.86 ± 0.08 a Rosmarinic acid 34.84 ± 2.89 a 1.83 ± 0.51 a 1.98 ± 1.15 a 18.45 ± 10.60 a ND 4.22 ± 0.59 a Flavonols Quercetin 65.63 ± 0.49 a 2.70 ± 0.94 b 30.02 ± 13.28 ab 37.09 ± 8.67 ab 28.20 ± 5.81 ab 14.24 ± 1.66 b Rutin 53.91 ± 0.58 a 212.09 ± 122.46 a ND 39.89 ± 0.10 a 80.33 ± 38.33 a ND 1 Results are mean ± standard error of two measurements. ND: Not Detected. Means with different letters in the same row are statistically significant (p < 0.05). The amount of the carotenoid, β-carotene was quantified in the methanolic extracts of the mixotrophically or the heterotrophically cultivated Micractinium sp. by RP-HPLC (Table 5 ). β-carotene content under mixotrophic growth (52.28 ± 1.45 µg.g − 1 DW) was slightly higher than the β-carotene amount under heterotrophic growth (45.99 ± 3.46 µg.g − 1 DW). Although different detection and calculation methods have been used, β-carotene accounts for approximately 2% of the total carotenoids in methanolic extracts of Micractinium sp. Table 5 β-carotene amounts in methanolic extracts of Micractinium sp. identified by RP-HPLC. Growth β-carotene amount 1 (µg.g − 1 DW) Mixotrophic 52.28 ± 1.45 a Heterotrophic 45.99 ± 3.46 a 1 Results are mean ± standard error of two measurements. Means with different letters in the same column are statistically significant (p < 0.05) 3.6. Inhibitory effect of Micractinium sp. extracts on intracellular ROS generation Micractinium sp. methanolic extract with the highest antioxidant activity under mixotrophic cultivation was tested for its ability to inhibit intracellular oxidative stress induced by H 2 O 2 in MCF-7 cells by the DCFH-DA assay. This method is based on the oxidation of non-fluorescent DCFH-DA into fluorescent DCF by intracellular ROS. Therefore, the intensity of intracellular fluorescent signal is proportional to the amount of intracellular ROS (Oparka et al. 2016 ). H 2 O 2 was used to promote oxidative stress as it is a relatively stable ROS generator and can result in the accumulation of ROS within the cell at elevated concentrations leading to cell damage and death (Oparka et al. 2016 ; Zhuang et al. 2017 ). Pre-incubation of MCF-7 cells with the microalgal extracts for 48 h significantly changed the levels of intracellular ROS (Fig. 1 A). The inhibitory effect of the microalgal extracts on the intracellular ROS levels occurred in a concentration-dependent manner. Pre-treatment with the methanolic extract reduced intracellular ROS in MCF-7 cells by 23.80% and 72.60% at the lowest (50 µg.mL − 1 ) and the highest concentration (400 µg.mL − 1 ), respectively. Concentration-dependent inhibitory effect of the microalgal extracts was highly significant compared to the control cells treated with the vehicle solution (methanol) only ( p < 0.001). 3.7. Cytoprotective activity of Micractinium sp. extracts against H 2 O 2 -induced cell apoptosis The methanolic extract, which exhibited the highest antioxidant activity in the mixotrophically cultivated Micractinium sp. was evaluated for its ability to protect MCF-7 cells against H 2 O 2 -induced apoptosis. Upon treatment with 1 mM H 2 O 2 , the percentage of viable, necrotic, and apoptotic MCF-7 cells were calculated as 54.83 ± 3.87, 34.56 ± 2.92, and 10.60 ± 0.95%, respectively (Fig. 1 B). The percentage of viable MCF-7 cells pre-treated with the methanolic extract of Micractinium sp. increased to 74.00 ± 2.32% ( p < 0.001). Pre-treatment with the methanolic microalgal extract led to a two-fold decrease in the number of necrotic cells (17.36 ± 1.78%) and caused a slight reduction in the apoptosis rate (8.60 ± 1.45%). Discussion The pursuit of finding a single solvent capable of solubilizing all target compounds during extraction is challenging. Phenolic compounds, characterized by diverse chemical structures, often form attachments to sugars or proteins in vivo, impacting their solubility across various solvents (Farvin and Jacobsen 2013 ; Monteiro et al. 2020 ). Consequently, we assessed the extraction yields of antioxidants from Micractinium sp. under both mixotrophic and heterotrophic conditions using six different solvents with varying polarities. The selection of different solvents aimed to encompass a wide range of polarities, thereby enhancing our understanding of solvent-specific extraction efficiencies and the nature of the compounds being extracted. In our study, methanol and water emerged as the most efficient solvents, yielding the highest extraction percentages under both growth conditions. This finding aligns with previous studies demonstrating the efficacy of methanol and water in extracting bioactive compounds from microalgae (Wang et al. 2009 ; Jerez-Martel et al. 2017 ). These results underscore the importance of solvent selection in optimizing extraction efficiency and yield. Furthermore, the significant disparity in extraction yields between methanol and water compared to other solvents highlights the critical influence of solvent polarity on the extraction of bioactive compounds. The antioxidant capacities of various macroalgae and microalgae have been extensively studied, with notable variations observed across different species and strains (Goiris et al. 2012 ; Farvin and Jacobsen 2013 ; Machu et al. 2015 ). Notably, our study is the first to evaluate the antioxidant capacity of a Micractinium species. Methanolic extracts exhibited the highest antioxidant capacity under both growth conditions, as evidenced by both DPPH and FRAP assays. This observation aligns with prior research highlighting the superior antioxidant potential of methanolic extracts from various microalgae species (Monteiro et al. 2020 ). The strong correlation between the DPPH and FRAP assays suggests their complementary nature in assessing antioxidant capacity, despite methodological differences (Munteanu and Apetrei 2021 ). The observed moderate correlation coefficients indicate that while both assays provide valuable insights into the antioxidant potential of microalgal extracts, they may capture different aspects of antioxidant activity. Comparisons of antioxidant capacities across studies are challenging due to variations in laboratory conditions and methodologies. However, two thermo-tolerant strains, Scenedesmus sp. ME02 and Hindakia tetrachotoma ME03, isolated from the same thermal waters as Micractinium sp., were recently evaluated for their antioxidant capacity (Bulut et al. 2019 ; Bulut et al. 2023 ). Our results demonstrated that ethyl acetate and water extracts of Micractinium sp. exhibited higher antioxidant capacity than Scenedesmus sp. ME02 but lower than H. tetrachotoma ME03. The observed differences in antioxidant capacity among the thermo-tolerant strains can be attributed to genetic variability, which influences the production and composition of antioxidant compounds. Each strain has unique metabolic pathways that determine the types and amounts of antioxidants synthesized, affected by enzyme activities and metabolic fluxes. Additionally, strain-specific adaptations to their thermal environments may result in the production of unique antioxidants that confer thermal stress protection. The presence and concentration of secondary metabolites, which act as antioxidants and are often species-specific, further contribute to the variations in antioxidant capacity (Gauthier et al. 2020 ; Coulombier et al. 2021 ). Microalgae cultivation methods, including autotrophy, heterotrophy, and mixotrophy, significantly influence their biochemical content. In this study, we evaluated the antioxidant activity of Micractinium sp. cells grown under mixotrophic and heterotrophic conditions. The notable contrast in antioxidant capacities between these samples underscores the impact of cultivation mode on the biochemical composition and subsequent antioxidant properties of Micractinium sp. Heterotrophic cultivation, particularly when utilizing molasses and vinasse as carbon sources, proved advantageous for achieving higher biomass and lower costs. Micractinium sp. demonstrated adaptability to various growth and temperature regimens, further influencing its biochemical content (Onay et al. 2014 ; Engin et al. 2018c ). Surprisingly, limited information exists on how heterotrophic growth affects the antioxidant capacity of microalgae compared to other cultivation conditions. In a previous study, the antioxidant activities of Chlorella vulgaris and Scenedesmus obliquus across autotrophic, mixotrophic, and heterotrophic conditions were compared (Shetty and Sibi 2015 ). Consistent with our findings, methanolic extracts of both microalgae exhibited reduced antioxidant potential during heterotrophic growth compared to autotrophic and mixotrophic conditions. This decline in antioxidant activity under heterotrophic conditions aligns with the understanding that environmental factors, including light and ultraviolet exposure, along with internal processes such as photosynthesis, generate ROS. To counteract oxidative damage from ROS, microalgae produce antioxidants as a defense mechanism. Importantly, microalgae cultivated under mixotrophic conditions are exposed to light and rely on both photosynthesis and an additional carbon source in the culture medium for energy. Consequently, it is plausible to expect a higher antioxidant capacity in mixotrophic cultures compared to heterotrophic growth conditions due to the increased antioxidant activity prompted by the combined effects of photosynthesis and light exposure. This discovery emphasizes the complex relationship between microalgae cultivation methods, environmental influences, and their antioxidant responses, providing insight into the subtle changes in their biochemical processes across different growth environments (Engin et al. 2018b ; Gauthier et al. 2020 ; Abreu et al. 2022 ). Studies on other microalgae, such as Tetraselmis suecica and Hindakia tetrachotoma ME03, have highlighted their antioxidant activity and potential applications in the cosmetic and biotechnological industries (Sansone et al. 2017 ; Bulut et al. 2023 ). The present investigation into Micractinium sp. ME05 extracts expands the limited knowledge of the in vitro antioxidant activity of green microalgae, further supporting their potential for biotechnological applications. The evaluation of total phenolic contents (TPC) in Micractinium sp., cultivated under varied growth conditions and extracted using different solvents, provided insights into the phenolic composition of the microalgal biomass. Consistent with prior research, Micractinium sp. extracted with polar solvents exhibited higher concentrations of phenolic compounds compared to nonpolar hexane extracts, regardless of the cultivation method (Goiris et al. 2012 ; Bulut et al. 2019 ). Methanolic extracts showed the highest TPC, aligning with previous studies suggesting the effectiveness of methanol in extracting phenolic compounds from microalgae (Safafar et al. 2015 ). Interestingly, hexane extracts displayed the lowest TPC across both growth conditions, indicating the limited capacity of this solvent to extract phenolic compounds from Micractinium sp. Additionally, our study identified a higher phenolic content in Micractinium sp. compared to Scenedesmus sp. ME02 in ethyl acetate and water extracts, revealing differences in the composition of these two freshwater strains isolated from the same thermal flora (Bulut et al. 2019 ). These findings underscore the critical role of solvent selection in optimizing phenolic extraction efficiency. The health-promoting properties of flavonoids, commonly found in fruits and vegetables, are extensively documented in scientific literature, yet research on flavonoid content in microalgae remains relatively scarce (Kozłowska and Szostak-Węgierek 2019 ). However, insights from previous studies have illuminated the presence of a diverse array of flavonoids across different classes of microalgae, albeit in smaller quantities compared to terrestrial plants (Goiris et al. 2014 ). Methanolic extracts emerged as the most efficient solvent for extracting flavonoids from Micractinium sp., consistent with its effectiveness in extracting other bioactive compounds. Importantly, our findings reveal that Micractinium sp. exhibits a higher total flavonoid content compared to several other microalgae species, as demonstrated by Bulut et al. ( 2019 ) and Safafar et al. ( 2015 ). This observation underscores the potential of Micractinium sp. as a promising natural source of flavonoids, suggesting its suitability as a potential substitute for synthetic antioxidants in the industry. The relatively higher flavonoid content in Micractinium sp. extracts, particularly when cultivated under mixotrophic conditions, highlights the importance of cultivation strategies in modulating the biochemical composition and potential applications of microalgae-derived products. The total carotenoid content (TCC) in Micractinium sp. exhibits significant variation across different cultivation modes, with higher concentrations observed in mixotrophic cultures compared to heterotrophic cultures. This study represents the first attempt to compare the TCC of microalgae under different cultivation modes, exploring the dynamics of carotenoid accumulation in response to varied growth conditions. In a previous study, two Micractinium sp. strains, designated as CCNM 1006 and CCNM 1041, were evaluated for their total carotenoid contents. Both strains displayed slightly higher quantities of total carotenoids compared to Micractinium sp. As part of a broader study encompassing 57 distinct microalgae strains, Micractinium sp. fell within the medium range concerning its TCC (Paliwal et al. 2016 ). The pivotal role of carotenoids in microalgae involves safeguarding chlorophylls from the detrimental effects of light exposure by scavenging ROS (Sathasivam and Ki 2018 ). Our findings indicate a higher accumulation of carotenoids in mixotrophic microalgae compared to cultures grown heterotrophically. This underscores the vital role of carotenoids in responding to light exposure, a phenomenon crucial for mitigating oxidative stress through ROS scavenging. Importantly, our study marks the first attempt to compare the TCC of microalgae grown under distinct cultivation modes, offering valuable insights into the dynamics of carotenoid accumulation in response to varied growth conditions. Despite the well-established role of phenolic compounds in plant antioxidant capacity, their contribution to microalgal antioxidant potential remains debated (Li et al. 2007 ; Shetty and Sibi 2015 ; Bulut et al. 2019 ; Bulut et al. 2023 ). The diverse nature of microalgal antioxidants collectively contributes to their overall antioxidant capacity. Therefore, we investigated the profile of phenolics, and β-carotene present in the extracts. This study stands out as the first to quantify individual phenolic compounds in a Micractinium species and compare their relative quantities under distinct cultivation modes. Twelve phenolic compounds, categorized into flavonols, benzoic acid derivatives, and cinnamic acid derivatives, were quantified in methanol, acetone, and ethyl acetate extracts from both mixotrophically and heterotrophically grown microalgae. In this comparative exploration, Chlorella pyrenoidosa , another freshwater green microalga, exhibited considerably lower concentrations of gallic acid compared to Micractinium sp., showcasing the distinct composition of these two species (Machu et al. 2015 ). 4-hydroxy benzoic acid content (20 µg.g − 1 sample) was higher in C. pyrenoidosa compared to mixotrophic Micractinium sp. (approximately 2 µg.g − 1 DW in methanol extract) but much lower than in heterotrophically grown culture (approximately 400 µg.g − 1 DW in methanolic extract). The significant increase in 4-hydroxy benzoic acid content under heterotrophic growth in Micractinium sp. raises questions about the underlying mechanisms governing these variations, especially considering its known antimicrobial properties used in various industries (food, pharmaceutical, and cosmetics). Interestingly, despite Micractinium sp. displaying a higher total flavonoid content than Scenedesmus sp. ME02, specific flavonoids like quercetin and rutin were found to be significantly lower in Micractinium sp. This suggests the possible presence of other, unexplored flavonoids in Micractinium sp., hinting at the complexity and diversity of its biochemical profile (Bulut et al. 2019 ). Additionally, the comparison of cinnamic acid derivatives, chlorogenic, and caffeic acid concentrations between Scenedesmus sp. and Micractinium sp. adds another layer to the variations in flavonoid composition within different microalgal species collected from the same geothermal flora (Goiris et al. 2014 ). In emphasizing the importance of specific compounds within microalgae, it is crucial to consider the diverse range of metabolites and their potential applications. Major carotenoid groups, including carotenes (such as β-carotene and lycopene) and xanthophylls (like lutein, astaxanthin, and fucoxanthin), each serve distinct roles. The prevalence of β-carotene in green microalgae like Dunaliella salina and Spirulina maxima underscores their nutritional significance, while Haematococcus pluvialis stands out as a key source of astaxanthin—a commercially valuable product renowned for its various health benefits (Sathasivam and Ki 2018 ; Maoka 2020 ; Zheng et al. 2023 ). In our study, we also quantified β-carotene in methanolic extracts of both mixotrophically and heterotrophically cultivated Micractinium sp., noting slightly higher β-carotene content in mixotrophic cultures compared to heterotrophic ones. Despite this minor difference, β-carotene accounted for approximately 2% of the total carotenoids in methanolic extracts of Micractinium sp., highlighting its substantial presence. These findings underscore the influence of cultivation conditions on carotenoid biosynthesis, with light exposure likely boosting β-carotene production in mixotrophic cultures due to its role in photoprotection and light harvesting. The methanolic extract of Micractinium sp., which demonstrated the highest antioxidant activity under mixotrophic cultivation, underwent further evaluation for its potential to mitigate intracellular oxidative stress and apoptosis induced by H 2 O 2 in MCF-7 cells. Utilizing the DCFH-DA assay, a well-established method for measuring intracellular ROS levels, our study revealed a significant reduction in ROS in a concentration-dependent manner following pre-incubation with the microalgal extract (Oparka et al. 2016 ). This substantial decrease in ROS levels is particularly noteworthy as it highlights the potent antioxidant capacity of extracts in protecting cells from oxidative damage induced by H 2 O 2 , a stable ROS generator known to cause significant cellular damage at elevated concentrations (Zhuang et al. 2017 ). The concentration-dependent response observed in this study aligns with the notion that higher concentrations of antioxidants can more effectively neutralize ROS, thereby providing greater protection against oxidative stress. Apoptosis, or programmed cell death, represents a fundamental cellular process crucial for maintaining tissue homeostasis and eliminating damaged or aberrant cells. Dysregulation of apoptotic pathways is closely associated with various pathological conditions, including cancer (Vitale et al. 2023 ). In our study, we explored the potential of Micractinium sp. extracts in modulating apoptotic responses induced by H 2 O 2 , a potent oxidizing agent known to trigger apoptotic cascades in cancer cells (Zhuang et al. 2017 ). Our findings reveal a significant reduction in apoptotic rates in MCF-7 breast adenocarcinoma cells pre-treated with Micractinium sp. methanolic extracts, suggesting a cytoprotective effect against H 2 O 2 -induced apoptosis. This finding suggests that the methanolic extract of Micractinium sp. not only scavenges ROS effectively but also enhances cell survival under oxidative stress conditions. The significant improvement in cell viability and prevention of necrotic and apoptotic cell death pathways underscore the therapeutic potential of Micractinium sp. extracts in combating oxidative stress-related cellular damage. These findings align with prior research indicating that microalgal extracts possess robust antioxidant properties and effectively alleviate oxidative stress in diverse cell lines (Sansone et al. 2017 ; Vahdati et al. 2020; Bulut et al. 2023 ). In a previous study, Bechelli et al. ( 2011 ) investigated the cytotoxic effects of algae, including Dunaliella salina extracts, on normal hematopoietic and leukemia cells by Annexin staining, demonstrating a significant reduction in cell viability induced by D. salina ethanolic extracts. Similarly, Karakaş et al. ( 2019 ) demonstrated that the cytotoxic effects of extracts from Chlorella protothecoides and Nannochloropsis oculate on human brain glioblastoma and colon colorectal carcinoma cell lines. To the best of our knowledge, the current study marks the first demonstration of in vitro cytoprotective activity in cell extracts from a Micractinium species. Furthermore, while other studies have explored the cytotoxic effects of various algae extracts on different cell lines, this study uniquely demonstrates the in vitro cytoprotective activity of Micractinium species, opening avenues for further investigations into specific bioactive compounds. The ability of Micractinium sp. extracts to modulate cell death pathways and enhance cellular viability in the face of oxidative stress holds significant implications for biomedical applications. While our study provides valuable insights into the cytoprotective effects of Micractinium sp. extracts against H 2 O 2 -induced oxidative stress in breast adenocarcinoma cells, several avenues for future research warrant exploration. Further elucidation of the underlying molecular mechanisms governing the cytoprotective activity of Micractinium sp. extracts, including their impact on apoptotic signaling pathways and cellular redox balance, is essential for fully harnessing their therapeutic potential. In addition to whole cell extracts, specific bioactive compounds derived from microalgae have been examined for their antioxidant activity on cell lines. For instance, β-carotene extracted from D. salina strongly reduced cell viability of prostate cancer cells (Jayappriyan et al. 2013 ). Another carotenoid, violaxanthin isolated from D. tertiolecta showed anti-cancer activity on MCF-7 cells (Pasquet et al. 2011 ). Polyunsaturated fatty acids extracted from Nannochloropsis salina also exhibited in vitro anti-proliferative effect on MCF-7 cells (Sayegh et al. 2016 ). While these studies highlight the potential of individual compounds, the use of crude extracts is also important. Crude extracts contain a complex mixture of various bioactive compounds that can work synergistically, potentially enhancing their overall antioxidant and cytotoxic effects. This synergism can lead to a more effective mitigation of oxidative stress and inhibition of cancer cell proliferation compared to isolated compounds. Therefore, exploring the bioactivity of crude extracts provides a holistic understanding of their therapeutic potential and can uncover interactions that may be missed when studying single compounds. Micractinium sp. contains a rich profile of fatty acids, which may collectively contribute to its antioxidant activity. The versatile characteristics of Micractinium sp., including its adaptability to both mixotrophic and heterotrophic conditions, wide temperature range (16°C-50°C), and diverse biochemical composition, position it as an ideal candidate for mass cultivation with promising applications in the nutraceutical and food industries. Our study represents the first attempt to quantify specific phenolic compounds in a Micractinium species and compare their concentrations under different cultivation methods. Significantly, the antioxidant-rich extracts of Micractinium sp. exhibited a notable inhibitory effect on ROS production and apoptosis induced by H 2 O 2 in MCF-7 cells. This discovery provides valuable insights into the relatively unexplored field of in vitro antioxidant activity of green microalgae for potential biotechnological applications. Future investigations focusing on the identification and characterization of specific bioactive compounds derived from Micractinium sp. can further enhance our understanding of its antioxidant activity, both in vitro and in vivo, thus contributing to the advancement of microalgal biotechnology. Conclusion In this study, the evaluation of antioxidant activity in mixotrophically and heterotrophically grown Micractinium sp. cells using six different solvents for extraction has yielded significant insights. Among these solvents, methanol emerged as particularly effective, with Micractinium sp. methanolic extracts demonstrating the highest antioxidant activity. The notable reduction in oxidative stress and the observed cytoprotective effects on MCF-7 cells underscore the therapeutic potential of Micractinium sp., particularly in addressing oxidative stress-related disorders. A comprehensive comparative analysis revealed intriguing distinctions between mixotrophically and heterotrophically grown microalgal extracts. Overall, mixotrophic samples exhibited a superior antioxidant capacity, accompanied by higher levels of total phenolics, flavonoids, and carotenoids. This suggests that the cultivation method has a significant impact on the biochemical composition of Micractinium sp., influencing its potential health-promoting attributes. Specifically, mixotrophic samples displayed elevated concentrations of gallic acid and rutin, compounds associated with various health benefits. In contrast, heterotrophic samples showcased substantial accumulations of 4-hydroxy benzoic acid and cinnamic acid, indicating a distinct biochemical profile under these growth conditions. This study breaks new ground by quantifying the amounts of these phenolic compounds in a Micractinium species for the first time. Moreover, it pioneers the documentation of the antioxidant and cytoprotective activities of Micractinium sp., expanding the understanding of its potential applications in microalgal biotechnology. Future investigations could focus on the targeted extraction of specific bioactive compounds from Micractinium sp. This approach would allow for a more detailed exploration of the in vitro and in vivo antioxidant activities, both in isolation and in conjunction with whole cell extracts. Such focused studies will undoubtedly contribute to unraveling the therapeutic potential and specific health benefits associated with Micractinium sp. ME05. Declarations Acknowledgements We would like to thank Konya Food and Agriculture University (KFAU) Strategic Products Research and Development Center (SARGEM) for technical support with the HPLC analysis and KFAU Research and Development Center for Diagnostic Kits (KIT-ARGEM) for the use of the facilities. The MCF-7 cells are a kind gift of Hasan Huseyin Kazan. We especially appreciate Assoc. Prof. Dr. Okan Bulut from the University of Alberta for his help and guidance with statistical analysis. Funding: This work was supported by the Konya Food and Agriculture University Research Project BAP-2019/0040. Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Onur Bulut and Işkın Engin. The first draft of the manuscript was written by Çağla Sönmez and Onur Bulut, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethics approval: Not applicable. References Abbaszadeh H, Keikhaei B, Mottaghi S (2019) A review of molecular mechanisms involved in anticancer and antiangiogenic effects of natural polyphenolic compounds. Phytother Res 33:2002-2014. https://doi.org/10.1002/ptr.6403 Abd El-Hack ME, Abdelnour S, Alagawany M, Abdo M, Sakr MA, Khafaga AF, Mahgoub SA, Elnesr SS, Gebriel MG (2019) Microalgae in modern cancer therapy: Current knowledge. Biomed Pharmacother 111:42-50. https://doi.org/10.1016/j.biopha.2018.12.069 Abou-Shanab RA, El-Dalatony MM, El-Sheekh MM, Ji MK, Salama ES, Kabra AN, Jeon BH (2014) Cultivation of a new microalga, Micractinium reisseri , in municipal wastewater for nutrient removal, biomass, lipid, and fatty acid production. Biotechnol Bioprocess Eng 19:510-518. https://doi.org/10.1007/s12257-013-0485-z Abreu AP, Morais RC, Teixeira JA, Nunes J (2022) A comparison between microalgal autotrophic growth and metabolite accumulation with heterotrophic, mixotrophic and photoheterotrophic cultivation modes. Renew Sustain Energy Rev 159:112247. https://doi.org/10.1016/j.rser.2022.112247 Almendinger M, Saalfrank F, Rohn S, Kurth E, Springer M, Pleissner D (2021) Characterization of selected microalgae and cyanobacteria as sources of compounds with antioxidant capacity. Algal Res 53:102168. https://doi.org/10.1016/j.algal.2020.102168 Ambriz-Pérez DL, Leyva-López N, Gutierrez-Grijalva EP, Heredia, JB (2016) Phenolic compounds: Natural alternative in inflammation treatment. A Review. Cogent Food & Agriculture, 2(1), 1131412. https://doi.org/10.1080/23311932.2015.1131412 Bechelli J, Coppage M, Rosell K, Liesveld J (2011) Cytotoxicity of algae extracts on normal and malignant cells. Leuk Res Treat 2011:1-6. https://doi.org/10.4061/2011/373519 Brewer MS (2011) Natural antioxidants: sources, compounds, mechanisms of action, and potential applications. Compr Rev Food Sci Food Saf 10:221-247. https://doi.org/10.1111/j.1541-4337.2011.00156.x Bulut O, Akın D, Sönmez Ç, Öktem A, Yücel M, Öktem HA (2019) Phenolic compounds, carotenoids, and antioxidant capacities of a thermo-tolerant Scenedesmus sp. (Chlorophyta) extracted with different solvents. J Appl Phycol 31:1675-1683. https://doi.org/10.1007/s10811-018-1726-5 Bulut O, Sönmez Ç, Öktem HA (2023) Hindakia tetrachotoma ME03 (Chlorophyta) has high phenolic content, antioxidant capacity, and attenuates H 2 O 2 -induced oxidative stress and apoptosis in human cells. Phycologia 62(4), 293-302. https://doi.org/10.1080/00318884.2023.2202520 Cheng Z, Moore J, Yu L (2006) High-throughput relative DPPH radical scavenging capacity assay. J Agric Food Chem 54:7429-7436. https://doi.org/10.1021/jf0611668 Coulombier N, Jauffrais T, Lebouvier N (2021) Antioxidant compounds from microalgae: A review. Mar Drugs 19:549. https://doi.org/10.3390/md19100549 Dai J, Mumper RJ (2010) Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 15:7313-7352. https://doi.org/10.3390/molecules15107313 El-Baz FK, Hussein RA, Mahmoud K, Abdo SM (2018) Cytotoxic activity of carotenoid-rich fractions from Haematococcus pluvialis and Dunaliella salina microalgae and the identification of the phytoconstituents using LC-DAD/ESI-MS. Phytother Res 32:298-304. https://doi.org/10.1002/ptr.5976 Engin IK, Cekmecelioglu D, Yücel AM, Oktem HA (2018a) Heterotrophic growth and oil production from Micractinium sp. ME05 using molasses. J Appl Phycol 30:3483-3492. https://doi.org/10.1007/s10811-018-1486-2 Engin IK, Cekmecelioglu D, Yücel AM, Oktem HA (2018b) Evaluation of heterotrophic and mixotrophic cultivation of novel Micractinium sp. ME05 on vinasse and its scale up for biodiesel production. Bioresour Technol 251:128-134. https://doi.org/10.1016/j.biortech.2017.12.023 Engin IK, Cekmecelioglu D, Yücel AM, Oktem HA (2018c) Enhancement of heterotrophic biomass production by Micractinium sp. ME05. Waste Biomass Valorization 9:811-820. https://doi.org/10.1007/s12649-017-9846-8 Farvin KS, Jacobsen C (2013) Phenolic compounds and antioxidant activities of selected species of seaweeds from Danish coast. Food Chem 138:1670-1681. https://doi.org/10.1016/j.foodchem.2012.10.078 Ferdous UT, Yusof ZNB (2021) Medicinal prospects of antioxidants from algal sources in cancer therapy. Front Pharmacol 12:593116. https://doi.org/10.3389/fphar.2021.593116 Firuzi O, Lacanna A, Petrucci R, Marrosu G, Saso L (2005) Evaluation of the antioxidant activity of flavonoids by “ferric reducing antioxidant power” assay and cyclic voltammetry. Biochim Biophys Acta Gen Subj 1721:174-184. https://doi.org/10.1016/j.bbagen.2004.11.001 Forman HJ, Zhang H (2021) Targeting oxidative stress in disease: Promise and limitations of antioxidant therapy. Nat Rev Drug Discov 20:689-709. https://doi.org/10.1038/s41573-021-00233-1 Franco R, Navarro G, Martínez-Pinilla E (2019) Antioxidants versus food antioxidant additives and food preservatives. Antioxidants 8:542. https://doi.org/10.3390/antiox8110542 Gauthier MR, Senhorinho GNA, Scott JA (2020) Microalgae under environmental stress as a source of antioxidants. Algal Res 52:102104. https://doi.org/10.1016/j.algal.2020.102104 Goiris K, Muylaert K, Fraeye I, Foubert I, De Brabanter J, De Cooman L (2012) Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. J Appl Phycol 24:1477-1486. https://doi.org/10.1007/s10811-012-9804-6 Goiris K, Muylaert K, Voorspoels S, Noten B, De Paepe D, Baart GJ, De Cooman L (2014) Detection of flavonoids in microalgae from different evolutionary lineages. J Phycol 50:483-492. https://doi.org/10.1111/jpy.12180 Herald TJ, Gadgil P, Tilley M (2012) High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour. J Sci Food Agric 92:2326-2331. https://doi.org/10.1002/jsfa.5633 Jayappriyan KR, Rajkumar R, Venkatakrishnan V, Nagaraj S, Rengasamy R (2013) In vitro anticancer activity of natural β-carotene from Dunaliella salina EU5891199 in PC-3 cells. Biomed Prev Nutr 3:99-105. https://doi.org/10.1016/j.bionut.2012.08.003 Jerez-Martel I, García-Poza S, Rodríguez-Martel G, Rico M, Afonso-Olivares C, Gómez-Pinchetti JL (2017) Phenolic profile and antioxidant activity of crude extracts from microalgae and cyanobacteria strains. J Food Qual 2017:1-10. https://doi.org/10.1155/2017/2924508 Karakaş CY, Tekarslan Şahin H, Inan B, Özçimen D, Erginer YÖ (2019) In vitro cytotoxic activity of microalgal extracts loaded nano-micro particles produced via electrospraying and microemulsion methods. Biotechnol Prog 35. https://doi.org/10.1002/btpr.2876 Kozłowska A, Szostak-Węgierek D (2019) Flavonoids – Food sources, health benefits, and mechanisms involved. In: Mérillon JM, Ramawat K (eds) Bioactive molecules in food. Reference Series in Phytochemistry. Springer, Cham, pp 1-20. https://doi.org/10.1007/978-3-319-54528-8_54-1 Krivina E, Sinetova M, Savchenko T, Degtyaryov E, Tebina E, Temraleeva A (2023) Micractinium lacustre and M. thermotolerans spp. nov. (Trebouxiophyceae, Chlorophyta): Taxonomy, temperature-dependent growth, photosynthetic characteristics and fatty acid composition. Algal Res 71:103042. https://doi.org/10.1016/j.algal.2023.103042 Li HB, Cheng KW, Wong CC, Fan KW, Chen F, Jiang Y (2007) Evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae. Food Chem 102:771-776. https://doi.org/10.1016/j.foodchem.2006.06.022 Lichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: Measurement and characterization by UV-VIS spectroscopy. Curr Protoc Food Anal Chem, 1(1), F4-3. https://doi.org/10.1002/0471142913.faf0403s01 Lobiuc A, Pavăl NE, Mangalagiu II, Gheorghiță R, Teliban GC, Amăriucăi-Mantu D, Stoleru V (2023) Future antimicrobials: Natural and functionalized phenolics. Molecules 28:1114. https://doi.org/10.3390/molecules28031114 Machu L, Misurcova L, Vavra Ambrozova J, Orsavova J, Mlcek J, Sochor J, Jurikova T (2015) Phenolic content and antioxidant capacity in algal food products. Molecules 20:1118-1133. https://doi.org/10.3390/molecules20011118 Malavasi V, Soru S, Cao G (2020) Extremophile microalgae: the potential for biotechnological application. J Phycol 56:559-573. https://doi.org/10.1111/jpy.12965 Maoka T (2020) Carotenoids as natural functional pigments. J Nat Med 74:1-16. https://doi.org/10.1007/s11418-019-01364-x Monteiro M, Santos RA, Iglesias P, Couto A, Serra CR, Gouvinhas I, Barros A, Olivia-Teles A, Enes P, Díaz-Rosales P (2020) Effect of extraction method and solvent system on the phenolic content and antioxidant activity of selected macro- and microalgae extracts. J Appl Phycol 32:349-362. https://doi.org/10.1007/s10811-019-01927-1 Munteanu IG, Apetrei C (2021) Analytical methods used in determining antioxidant activity: A review. Int J Mol Sci 22:3380. https://doi.org/10.3390/ijms22073380 Onay M, Sonmez C, Oktem HA, Yucel AM (2014) Thermo-resistant green microalgae for effective biodiesel production: isolation and characterization of unialgal species from geothermal flora of Central Anatolia. Bioresour Technol 169:62-71. https://doi.org/10.1016/j.biortech.2014.06.078 Oparka M, Walczak J, Malinska D, van Oppen LM, Szczepanowska J, Koopman WJ, Wieckowski MR (2016) Quantifying ROS levels using CM-H 2 DCFDA and HyPer. Methods 109:3-11. https://doi.org/10.1016/j.ymeth.2016.06.008 Paliwal C, Ghosh T, George B, Pancha I, Maurya R, Chokshi K, Ghosh A, Mishra S (2016) Microalgal carotenoids: Potential nutraceutical compounds with chemotaxonomic importance. Algal Res 15:24-31. https://doi.org/10.1016/j.algal.2016.01.017 Parra-Riofrio G, Moreno P, García-Rosado E, Alonso MC, Uribe-Tapia E, Abdala-Diaz RT, Bejar J (2023) Tetraselmis suecica and Porphyridium cruentum exopolysaccharides show anti-VHSV activity on RTG-2 cells. Aquac Int 31(6):3145-3157. https://doi.org/10.1007/s10499-023-01202-8 Pasquet V, Morisset P, Ihammouine S, Chepied A, Aumailley L, Berard JB, Serive B, Kaas R, Lanneluc I, Thiery V, Lafferriere M, Picot JM, Patrice T, Cadoret JP, Picot L (2011) Antiproliferative activity of violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts. Mar Drugs 9:819-831. https://doi.org/10.3390/md9050819 Poljsak B, Kovač V, Milisav I (2021) Antioxidants, food processing and health. Antioxidants 10:433. https://doi.org/10.3390/antiox10030433 Safafar H, Van Wagenen J, Møller P, Jacobsen C (2015) Carotenoids, phenolic compounds and tocopherols contribute to the antioxidative properties of some microalgae species grown on industrial wastewater. Mar Drugs 13:7339-7356. https://doi.org/10.3390/md13127069 Sanjeewa KKA, Fernando IPS, Samarakoon KW, Lakmal HH, Kim EA, Kwon ON, Dilshara MG, Lee JB, Jeon YJ (2016) Anti-inflammatory and anti-cancer activities of sterol-rich fraction of cultured marine microalga Nannochloropsis oculata . Algae 31:277-287. https://doi.org/10.4490/algae.2016.31.6.29 Sansone C, Galasso C, Orefice I, Nuzzo G, Luongo E, Cutignano A, Romano G, Brunet C, Fontana A, Esposito F, Ianora A (2017) The green microalga Tetraselmis suecica reduces oxidative stress and induces repairing mechanisms in human cells. Sci Rep 7:1-12. https://doi.org/10.1038/srep41215 Sathasivam R, Ki JS (2018) A review of the biological activities of microalgal carotenoids and their potential use in healthcare and cosmetic industries. Mar Drugs 16(1):26. https://doi.org/10.3390/md16010026 Sayegh F, Elazzazy A, Bellou S, Moustogianni A, Elkady AI, Baeshen MN, Aggelis G (2016) Production of polyunsaturated single cell oils possessing antimicrobial and anticancer properties. Ann Microbiol 66:937-948. https://doi.org/10.1007/s13213-015-1176-0 Shetty V, Sibi G (2015) Relationship between total phenolics content and antioxidant activities of microalgae under autotrophic, heterotrophic and mixotrophic growth. J Food Res Sci 4:1-9. https://doi.org/10.3923/jfrs.2015.1.9 Sönmez Ç, Elçin E, Akın D, Öktem HA, Yücel M (2016) Evaluation of novel thermo-resistant Micractinium and Scenedesmus sp. for efficient biomass and lipid production under different temperature and nutrient regimes. Bioresour Technol 211:422-428. https://doi.org/10.1016/j.biortech.2016.03.125 Vacca RA, Valenti D, Caccamese S, Daglia M, Braidy N, Nabavi SM (2016) Plant polyphenols as natural drugs for the management of Down syndrome and related disorders. Neurosci Biobehav Rev 71:865-877. https://doi.org/10.1016/j.neubiorev.2016.10.023 Vahdati SN, Lashkari A, Navasatli SA, Ardestani SK, Safavi M (2022) Butylated hydroxyl-toluene, 2,4-Di-tert-butylphenol, and phytol of Chlorella sp. protect the PC12 cell line against H 2 O 2 -induced neurotoxicity. Biomed Pharmacother 145:112415. https://doi.org/10.1016/j.biopha.2021.112415 Vitale I, Pietrocola F, Guilbaud E, Aaronson SA, Abrams JM, Adam D, ... Pasparakis M (2023) Apoptotic cell death in disease—Current understanding of the NCCD 2023. Cell Death Differ 30:1097-1154. https://doi.org/10.1038/s41418-023-01153-w Wang T, Jonsdottir R, Ólafsdóttir G (2009) Total phenolic compounds, radical scavenging and metal chelation of extracts from Icelandic seaweeds. Food Chem 116:240-248. https://doi.org/10.1016/j.foodchem.2009.02.041 Yuan M, Zhang G, Bai W, Han X, Li C, Bian S (2022) The role of bioactive compounds in natural products extracted from plants in cancer treatment and their mechanisms related to anticancer effects. Oxid Med Cell Longev 2022. https://doi.org/10.1155/2022/1429869 Zhang H, Tsao R (2016) Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr Opin Food Sci 8:33-42. https://doi.org/10.1016/j.cofs.2016.02.002 Zheng X, Shao C, Zhu J, Zhang L, Huang Q (2023) Study of the antioxidant capacity of astaxanthin in cells against radiation-induced strong oxidative stress. Aquac Int 31(5):2705-2725. https://doi.org/10.1007/s10499-023-01105-8 Zhuang Y, Ma Q, Guo Y, Sun L (2017) Protective effects of rambutan (Nephelium lappaceum) peel phenolics on H 2 O 2 -induced oxidative damages in HepG2 cells and d-galactose-induced aging mice. Food Chem Toxicol 108:554-562. https://doi.org/10.1016/j.fct.2017.01.022 Additional Declarations No competing interests reported. Supplementary Files BulutSupplementarydata.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4690459","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330334434,"identity":"d090b00f-6a9e-47b5-85f5-76ade2a2f0b2","order_by":0,"name":"Onur Bulut","email":"","orcid":"","institution":"Konya Food and Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Onur","middleName":"","lastName":"Bulut","suffix":""},{"id":330334435,"identity":"9fafc0a2-9ec0-4249-b201-746a48d8008d","order_by":1,"name":"Iskin Engin","email":"","orcid":"","institution":"Middle East Technical University","correspondingAuthor":false,"prefix":"","firstName":"Iskin","middleName":"","lastName":"Engin","suffix":""},{"id":330334436,"identity":"df50a916-b4d2-45a2-8681-c9ba8bc7d181","order_by":2,"name":"Cagla Sonmez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIie3RIQvCQBTA8TcObuXh6g2nn2EyMOpXGQiunCJYlAWDwSL4bS5PBq4YjILFaV1ZEBQM3i0Yb9oE7x8eb3A/2G0AJtNvZiVyeGo5y4GNT4wiiADEVwv9hlCmnmqJs8oOyR162Gej3fzGex4Fkl+OGsL2fLJdwwCRjYenlhjIF6NBwDXEBx4mCEQS3j25gkiCtKklThFun7CoyNQViw8Ii5IUIa2IVYq0nrBjAannZ4j7ImhaIkNKau7ibKI8L2Zx217xTvkQcd+xl/lVR+Qf8dVHqCJYTe1xlX1+r9a99rTJZDL9Yy930T+AeI9dVwAAAABJRU5ErkJggg==","orcid":"","institution":"Atilim University","correspondingAuthor":true,"prefix":"","firstName":"Cagla","middleName":"","lastName":"Sonmez","suffix":""},{"id":330334437,"identity":"a6e129d6-99d2-4ee8-a6ba-1b1e582fa7e4","order_by":3,"name":"Huseyin Avni Oktem","email":"","orcid":"","institution":"Middle East Technical University","correspondingAuthor":false,"prefix":"","firstName":"Huseyin","middleName":"Avni","lastName":"Oktem","suffix":""}],"badges":[],"createdAt":"2024-07-05 07:36:55","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4690459/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4690459/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60962594,"identity":"98734070-bac8-49b0-9dcb-b34801e6efc4","added_by":"auto","created_at":"2024-07-24 05:20:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99990,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Inhibitory effects of methanolic extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp. and ascorbic acid (AA) on intracellular ROS generation in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced MCF-7 cells. (B) Cytoprotective effects of the methanolic extract of\u003cem\u003e Micractinium\u003c/em\u003e sp. on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced MCF-7 cell apoptosis and necrosis. (***) indicates the significance at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4690459/v1/3264a6dcf90204e9bd1e2109.png"},{"id":60963067,"identity":"c9bcc8bb-4ac6-4f52-8521-7ac0161c11cc","added_by":"auto","created_at":"2024-07-24 05:28:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1362484,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4690459/v1/d17abd54-93db-4b09-9828-a7453a62b009.pdf"},{"id":60962595,"identity":"12947d0d-8b3f-4836-ae3c-b6cc0fee934d","added_by":"auto","created_at":"2024-07-24 05:20:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19346,"visible":true,"origin":"","legend":"","description":"","filename":"BulutSupplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-4690459/v1/9da8a1b8fa5569274265998d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antioxidant activity of Micractinium sp. (Chlorophyta) extracts against H2O2 induced oxidative stress in human breast adenocarcinoma cells","fulltext":[{"header":"Highlights","content":"\u003cp\u003eAntioxidant capacity of thermo-tolerant green microalga \u003cem\u003eMicractinium\u003c/em\u003e sp. evaluated.\u003c/p\u003e\n\u003cp\u003eDifferential effects of two cultivation modes on antioxidants were compared.\u003c/p\u003e\n\u003cp\u003eMixotrophic cultures had higher antioxidant activity, phenolics and carotenoids.\u003c/p\u003e\n\u003cp\u003eHigh amounts of phenolic compounds such as gallic acid and rutin detected by RP-HPLC.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicractinium\u003c/em\u003e sp. extracts inhibited oxidative stress in MCF-7 cells.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eOxidative stress is a biological phenomenon that occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to counteract their harmful effects. ROS can induce damage to cellular components, including proteins, lipids, and DNA, with prolonged exposure correlating strongly with various diseases. Antioxidants, encompassing vitamins, phenolic compounds, and carotenoids, constitute a vital component of the human diet, primarily sourced from fruits and vegetables (Forman and Zhang \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Carotenoids such as astaxanthin, lycopene, lutein and β-carotene, are a class of natural pigments abundant in yellow, orange and dark green leafy plants. β-carotene, in particular, functions as an antioxidant by scavenging various free radicals. Polyphenols, another crucial group of antioxidants, include phenolic acids, flavonoids, tannins, lignans, and stilbenes, and are predominantly found in vegetables, fruits, cereals, herbs, and spices (Zhang and Tsao \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Maoka \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAntioxidants act through diverse mechanisms, such as inhibiting enzymes like glutathione S-transferase, chelating trace metals involved in ROS production, and up-regulating antioxidant defense pathways. The documented anti-carcinogenic and anti-proliferative activities of phenolic compounds on various tumor cell lines highlight their potential therapeutic benefits (Dai and Mumper \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Abbaszadeh et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yuan et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, the positive cognitive outcomes in patients with Down syndrome associated with early developmental stage consumption of phenolic-rich dietary supplements add to the versatile spectrum of their effects, including antimicrobial, anti-inflammatory, and anti-viral activities (Vacca et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ambriz-P\u0026eacute;rez et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Parra-Riofrio et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lobiuc et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Studies also reveal the efficacy of using dietary antioxidants in combination with chemo and radiation therapy in cancer patients to suppress the toxicity-related side effects of such treatments (Ferdous and Yusof \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn response to contemporary lifestyle challenges, where poor dietary habits and environmental factors contribute to oxidative stress, there is a growing global demand for antioxidants. In addition to their well-documented health benefits, antioxidants serve a crucial role in the preservation of packaged foods by inhibiting oxidation processes, thereby extending the shelf life of products. The food industry traditionally leaned towards synthetic antioxidants for this purpose; however, mounting concerns about their potential adverse health effects have prompted a shift in consumer preferences. There is now a strong inclination towards the use of natural antioxidants, driving researchers to explore new biological sources that are rich in these natural compounds (Franco et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Poljsak et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicroalgae emerge as promising natural sources of antioxidants due to their adaptability to adverse environmental conditions and their capacity to accumulate essential secondary metabolites, including phenolic compounds and carotenoids, with robust antioxidant capabilities. Notably, the antioxidant activity varies among microalgae species and cultivation conditions (Coulombier et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Almendinger et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicroalgal extracts have demonstrated cytotoxic effects on various human cancer cells including liver, colon, breast, lung, and brain, while some of them also exhibiting the ability to inhibit oxidative stress without causing cytotoxicity (Sansone et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; El-Baz et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Abd El-Hack et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Karakaş et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bulut et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). β-carotene from \u003cem\u003eDunaliella salina\u003c/em\u003e, polyunsaturated fatty acids from \u003cem\u003eNannochloropsis salina\u003c/em\u003e, sterols in \u003cem\u003eN. oculate\u003c/em\u003e extracts and violaxanthin, a carotenoid compound, isolated from \u003cem\u003eD. tertiolecta\u003c/em\u003e are examples of microalgal compounds with documented anti-cancer properties (Pasquet et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Jayappriyan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sanjeewa et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sayegh et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMicractinium\u003c/em\u003e is a genus of green microalgae in the family \u003cem\u003eChlorellaceae\u003c/em\u003e (Chlorophyta). Different species of \u003cem\u003eMicractinium\u003c/em\u003e are adapted to diverse geographical locations and exhibit high phenotypic plasticity (Krivina et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Various strains of \u003cem\u003eMicractinium\u003c/em\u003e showed promising biotechnological potential as feed supplement, biofuel, and in wastewater treatment (Abou-Shanab et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Paliwal et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thermophilic/thermotolerant strains of \u003cem\u003eMicractinium\u003c/em\u003e are advantageous in lowering the operational costs of cultivation in large scale bioreactors (Malavasi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eMicractinium\u003c/em\u003e sp. METUNERGY05 (ME05), used in this study, was previously isolated in our laboratory from hot springs of Haymana, Turkey (Onay et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It is a thermotolerant strain which is suitable for biodiesel production and can be cultivated both mixotrophically and heterotrophically using by-products of a sugar factory as sole carbon source, which reduces the operational costs (Engin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Engin et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of this study was to assess the antioxidant properties, along with the phenolic, flavonoid, and carotenoid contents of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts cultivated under both mixotrophic and heterotrophic conditions. To achieve this, we employed six different solvents for the extraction of microalgal biomass. Utilizing RP-HPLC analysis, we explored the diverse profile of phenolics present in the extracts, providing valuable insights into the intricate biochemical composition of \u003cem\u003eMicractinium\u003c/em\u003e sp. Subsequently, we evaluated the cytoprotective properties of the methanolic extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp. Specifically, we investigated their effectiveness against hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)-induced oxidative stress, shedding light on the potential bioactivity of these extracts in mitigating cellular damage. Our findings highlight \u003cem\u003eMicractinium\u003c/em\u003e sp. as a promising natural source of antioxidants for nutraceutical applications and the food industry. The versatility of \u003cem\u003eMicractinium\u003c/em\u003e sp. in adapting to different cultivation conditions further enhances its appeal as a bioresource with broad applications.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and reagents\u003c/h2\u003e \u003cp\u003eAll chemicals and solvents used in this study were purchased from Sigma\u0026ndash;Aldrich, AppliChem GmbH and Merck Company, and were analytical or HPLC grade. Molasses used during heterotrophic cultivation was obtained from Konya Sugar Factory, Turkey.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microalgal culture and extract preparation\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Growth and culture conditions of \u003cem\u003eMicractinium\u003c/em\u003e sp.\u003c/h2\u003e \u003cp\u003eThe green microalga \u003cem\u003eMicractinium\u003c/em\u003e sp. cells were cultured in Tris-Acetate-Phosphate (TAP) growth medium (S\u0026ouml;nmez et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Detailed morphological, biochemical, and molecular characterization of \u003cem\u003eMicractinium\u003c/em\u003e sp. ME05 strain was previously reported by Onay et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor mixotrophic cultivation, \u003cem\u003eMicractinium\u003c/em\u003e sp. was inoculated in 1 L TAP medium and grown at 25\u0026deg;C under photoperiod (16:8 h of light: dark) at 54 \u0026micro;mol.m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity with constant shaking until cells reached the stationary phase and were harvested. Heterotrophic cultivation was carried out by inoculating 2\u0026ndash;3 x 10\u003csup\u003e5\u003c/sup\u003e cells.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003eMicractinium\u003c/em\u003e sp. into 1 L Bold\u0026rsquo;s Basal Medium (BBM) supplemented with 19 g of molasses hydrolysate in 2-L Erlenmeyer flasks. The composition of BBM and molasses, preparation of molasses hydrolysate and the optimum molasses hydrolysate amount for the highest biomass concentration were previously described by Engin et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). \u003cem\u003eMicractinium\u003c/em\u003e sp. cells were grown under complete darkness at 30\u0026deg;C with air supply at 0.5 L.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e through an aquarium pump. The growth went by for 5 days until harvesting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Preparation of microalgal extracts\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMicractinium\u003c/em\u003e sp. cells cultivated either mixotrophically or heterotrophically were centrifuged at 3600x g for 20 min. The cell pellet was lyophilized using a freeze-dryer and ground to a fine powder. Six different solvents, namely methanol, ethanol, acetone, hexane, ethyl acetate and water were used as extractants. 200 mg of lyophilized microalgae was extracted with 5 ml of solvent at room temperature by sonication in an ultrasonic water bath for 20 min followed by stirring on an orbital shaker for 1 h. The extract was centrifuged at 3800x g for 10 min and the aqueous phase was collected in separate bottles. The residual pellets were re-suspended in the same solvents and re-extracted as previously described in Bulut et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The extracts were combined and filtered through a 0.45 \u0026micro;m pore size polytetrafluoroethylene (PTFE) syringe filter and dried using a rotary evaporator. The dried residues were weighed to calculate the extraction yields. Methanol was used as the vehicle solution for solubilization of the dried residues at a concentration of 20 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The solubilized extracts were stored at -20\u0026deg;C until further analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Antioxidant measurements\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Measurement of the total phenolic content\u003c/h2\u003e \u003cp\u003eThe Folin \u0026amp; Ciocalteu spectrophotometric method was used to measure the total phenolic content (TPC) of the microalgal extracts. The protocol previously reported by Bulut et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) was followed. 100 \u0026micro;L sample was mixed with 400 \u0026micro;L of Folin-Ciocalteu reagent (1:10 diluted in ultrapure water). The mixture was vortexed thoroughly and allowed to stand at room temperature for 5 min. 500 \u0026micro;L of 7.5% (w/v) NaNO\u003csub\u003e2\u003c/sub\u003e solution was added to the mixture and the tubes were incubated for 1.5 h in the dark at room temperature. Following the incubation, 200 \u0026micro;L of sample was transferred to a clear 96-well microplate and the absorbance of each well was measured at 760 nm using a UV\u0026ndash;vis microplate reader. A standard curve prepared by serial dilution of gallic acid solutions ranging from 10 to 400 mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was used for calibration. TPC of the extracts was calculated as gallic acid equivalents using the regression equation of the standard curve. TPC was expressed as mg gallic acid equivalents per gram dry weight of sample (mg GAE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW). The analyses were performed as biological triplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Measurement of the total flavonoid content\u003c/h2\u003e \u003cp\u003eThe total flavonoid content (TFC) of the microalgal extracts was measured by the aluminum chloride method (Herald et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). One milliliter of extract was diluted 1:5 with ultrapure water; mixed with 0.3 mL of 5% (w/v) NaNO\u003csub\u003e2\u003c/sub\u003e and incubated at ambient temperature for 5 min. The sample was mixed with 0.3 mL of 10% (w/v) AlCl\u003csub\u003e3\u003c/sub\u003e in ethanol after incubation at ambient temperature for 5 min. This step was followed by another incubation at ambient temperature for 6 min. Then, 2 mL of 1 M NaOH was added to the mixture and the total volume was adjusted to 10 mL with ultrapure water. After a brief vortex, 200 \u0026micro;L of the sample was transferred to a clear 96-well microplate and the absorbance of each well was measured at 510 nm using a UV\u0026ndash;vis microplate reader. A standard curve was prepared with serial dilution of quercetin solutions ranging from 10 to 400 mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Total flavonoid content of the microalgal extracts was calculated using the regression equation of this standard curve. The results were expressed as mg quercetin equivalents per gram dry weight of sample (mg QE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Measurement of the total carotenoid content\u003c/h2\u003e \u003cp\u003eTotal carotenoid content of the microalgal extracts was calculated following the method by Lichtenthaler and Buschmann (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) (Lichtenthaler and Buschmann \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Absorbance of the methanolic extracts was recorded at 470, 652 and 665 nm using a UV\u0026ndash;vis spectrophotometer and total carotenoid content was calculated according to the Lichtenthaler equations as follows:\u003c/p\u003e \u003cp\u003e \u003cem\u003ec\u003c/em\u003e \u003csub\u003e \u003cem\u003ea\u003c/em\u003e \u003c/sub\u003e (\u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;16.72 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e665\u003c/em\u003e\u003c/sub\u003e \u0026ndash; 9.16 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e652\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ec\u003c/em\u003e \u003csub\u003e \u003cem\u003eb\u003c/em\u003e \u003c/sub\u003e (\u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;34.09 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e652\u003c/em\u003e\u003c/sub\u003e \u0026ndash; 15.28 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e665\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ec\u003c/em\u003e \u003csub\u003e \u003cem\u003e(x+c)\u003c/em\u003e \u003c/sub\u003e (\u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) = (1000 \u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e470\u003c/em\u003e\u003c/sub\u003e \u0026ndash; 1.63 \u003cem\u003ec\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e \u0026ndash; 104.96 \u003cem\u003ec\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e)/221\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003ec\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ec\u003c/em\u003e\u003csub\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sub\u003e are concentrations of chlorophyll a and b, respectively, and \u003cem\u003ec\u003c/em\u003e\u003csub\u003e\u003cem\u003e(x+c)\u003c/em\u003e\u003c/sub\u003e is the concentration of the total carotenoids. The results were expressed as mg carotenoid per gram dry weight of sample (mg carotenoid.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. DPPH assay\u003c/h2\u003e \u003cp\u003eThe DPPH radical scavenging activity of the microalgal extracts was measured according to Cheng et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Briefly, 100 \u0026micro;L of microalgal extracts at concentrations ranging from 50 to 2000 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was mixed with 100 \u0026micro;L of 0.2 mM DPPH solution in a clear 96-well plate. The mixture was incubated in the dark at ambient temperature for 30 min, and the absorbance was recorded at 515 nm using a microplate reader with the trolox solution as the positive control and the DPPH solution as blank. The percentage of scavenged DPPH\u0026bull; radical was calculated according to the following equation:\u003c/p\u003e \u003cp\u003eDPPH scavenging activity (%) = [1-(\u003cem\u003eAs\u003c/em\u003e-\u003cem\u003eAsc\u003c/em\u003e)/\u003cem\u003eAc\u003c/em\u003e]*100\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eAs\u003c/em\u003e is absorbance of the sample (100 \u0026micro;L of sample with 100 \u0026micro;L of DPPH\u0026bull; radical solution), \u003cem\u003eAsc\u003c/em\u003e is absorbance of the sample control (100 \u0026micro;L of sample with 100 \u0026micro;L of methanol) and \u003cem\u003eAc\u003c/em\u003e is absorbance of the control (100 \u0026micro;L of methanol with 100 \u0026micro;L of DPPH\u0026bull; radical solution). A standard curve was prepared with serially diluted trolox solutions in the range of 2.5 to 80 \u0026micro;mol.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentrations. Total antioxidant capacity of the microalgal extracts was calculated as trolox equivalents using the regression equation of the standard curve. The results were expressed as micromol of equivalent trolox per gram of dried weight (\u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) and (%) DPPH radical scavenging activity of the extract (Bulut et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5. FRAP assay\u003c/h2\u003e \u003cp\u003eThe antioxidant capacity of the extracts was also evaluated by the FRAP assay through monitoring the reduction of Fe\u003csup\u003e3+\u003c/sup\u003e-TPTZ to blue-colored Fe\u003csup\u003e2+\u003c/sup\u003e-TPTZ (Firuzi et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The working FRAP solution was freshly prepared by mixing ten volumes of acetate buffer, one volume of TPTZ solution and one volume of ferric chloride hexahydrate solution and warmed at 37\u0026deg;C in a water bath prior to use. 25 \u0026micro;L of the microalgal extract at concentrations between 50 to 2000 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was mixed with 175 \u0026micro;L of pre-warmed FRAP solution in a clear 96-well microplate. The microplate was allowed to stand at room temperature for 30 min in the dark. The absorbance of each sample was measured at 593 nm using a microplate reader. Trolox solutions ranging from 5 to 20 \u0026micro;mol.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were used for preparation of a standard curve. Total antioxidant capacity of the microalgal extracts was calculated as trolox equivalents using the regression equation of the standard graph. FRAP values were expressed as \u0026micro;mol trolox equivalents per gram dry weight of sample (\u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) (Bulut et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.3.6. Reverse phase high performance liquid chromatography (RP-HPLC) analysis\u003c/h2\u003e \u003cp\u003eTwelve selected phenolic compounds; namely, gallic acid, benzoic acid, 4-hydroxy benzoic acid, vanillic acid, syringic acid, cinnamic acid, coumaric acid, caffeic acid, chlorogenic acid, rosmarinic acid, quercetin and rutin were identified in the microalgal extracts by reverse phase HPLC (Waters Alliance 2695, Waters Corporation, USA) coupled to a UV/Vis detector (Waters 2489 detector) as described by Bulut et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The microalgal extract at a concentration of 1000 ppm was passed through a 0.45 \u0026micro;m PTFE syringe filter prior to injection. The chromatographic separation was performed in a C18 analytical column (ACE 5, AC Technologies, Scotland). Elution was carried out with a gradient pump mode involving three mobile phases; mobile phase A: 2% (v/v) acetic acid, mobile phase B: acetonitrile and 0.5% (v/v) acetic acid (1:1 v/v) and mobile phase C: acetonitrile. The gradient was set as following: 0\u0026ndash;8 min: 95% A and 5% B; 8\u0026ndash;10 min: 80% A and 20% B; 10\u0026ndash;17 min: 78% A and 22% B; 17\u0026ndash;19 min: 75% A and 25% B; 19\u0026ndash;30 min: 73% A and 27% B; 30\u0026ndash;35 min: 60% A and 40% B; 35\u0026ndash;40 min: 55% A and 45% B; 40\u0026ndash;45 min: 35% A and 65% B; 56\u0026thinsp;\u0026minus;\u0026thinsp;50 min: 10% B and 90% C; 50\u0026ndash;52 min: 100% C; and 52\u0026ndash;60 min: 95% A and 5% B. The flow rate was 1.2 mL.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the injection volume was 20 \u0026micro;L and the column temperature was maintained at ambient temperature. Simultaneous monitoring was done via a UV/Vis detector with reference wavelength of 280 nm. Retention times and peak areas of both authentic standards and microalgal extracts were monitored automatically by Empower 3 Chromatography Data Software (Waters Corporation, USA). The concentration of individual phenolic compounds was quantified by comparison of the chromatographic peaks of the microalgal extracts to those of authentic standards.\u003c/p\u003e \u003cp\u003eTo identify the β-carotene content, a RP-HPLC system equipped with a Shimadzu LC-20AD pump (Shimazdu, Kyoto, Japan) and Shimazdu SPD-20A UV/Vis detector was used. An Inertsil ODS-2 C18 analytical column was used for the chromatographic runs (GL Sciences, Tokyo, Japan). The gradient pump mode consisting of two mobile phases (mobile phase A: 90% acetonitrile in water, mobile phase B: ethyl acetate) was used for elution at a flow rate of 1.0 mL.min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The peaks were detected at 450 nm. β-carotene concentration in the microalgal extracts was identified by comparison of the peak areas of the samples to the authentic standards.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell culture and assays\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Inhibition of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced reactive oxygen species (ROS) generation in MCF-7 cells\u003c/h2\u003e \u003cp\u003eThe human breast cancer cell line, MCF-7 (ATCC HTB-22), was obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and maintained in a CO\u003csub\u003e2\u003c/sub\u003e incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe effect of the methanolic microalgal extracts on inhibition of the intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced ROS generation in MCF-7 cells was evaluated by a fluorescence assay using the cell-permeant probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) as described by Zhuang et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) with minor modifications. MCF-7 cells were pretreated with 50, 100, 200 or 400 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the methanolic extracts, separately for 48 h in a 96-well black cell culture plate. Cells treated with the culture medium containing VS and ascorbic acid (8 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were used as the negative and the positive controls, respectively. After 48 h incubation with the methanolic extracts, the cells were exposed to 0.5 mM of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 6 h to induce intracellular oxidative stress via ROS generation. The cells were washed twice with PBS and incubated with serum-free medium containing DCFH-DA (20 \u0026micro;M) for 30 min in the dark at 37\u0026deg;C. Cells were immediately washed with PBS. The formation of the fluorescent 2',7'-dichlorofluorescein (DCF) due to oxidation of the non-fluorescent DCFH-DA by intracellular oxidative stress was detected by a fluorescence microplate reader with an excitation wavelength of 495 nm and an emission wavelength of 525 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Apoptosis assay\u003c/h2\u003e \u003cp\u003eThe cytoprotective effect of the methanolic extracts of mixotrophically grown \u003cem\u003eMicractinium\u003c/em\u003e sp. on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis of MCF-7 cells was determined by an Annexin V-FITC and propidium iodide (PI) double-staining apoptosis assay kit (Takara Bio Inc., Japan) according to the manufacturer\u0026rsquo;s instructions. MCF-7 cells were firstly treated with the microalgal extracts and then, with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as described in section 2.11. The cells were collected by trypsinization, washed with PBS twice, and resuspended in 200 \u0026micro;L of binding buffer containing 5 \u0026micro;L of Annexin V-FITC. After incubation of the cells for 15 min at room temperature in the dark, 10 \u0026micro;L of PI was added to cells and incubated for 10 min in an ice bath in the dark. Finally, the cells were analyzed using a flow cytometer.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments in this study were carried out in biological triplicates. Results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. The analysis of the mean values was performed using the analysis of variance (ANOVA) test and Tukey\u0026rsquo;s post-hoc comparison test. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u0026lt; 0.01, and \u0026lt;\u0026thinsp;0.05 was considered as highly significant (***), very significant (**) and significant (*), respectively in statistical terms. Pearson\u0026rsquo;s correlation of determination (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e) was used to compute correlations among antioxidant assays, TPC and TFC under different growth conditions. The statistical analysis was conducted using R version 3.4.2.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Extraction yields of \u003cem\u003eMicractinium\u003c/em\u003e sp. in different solvents\u003c/h2\u003e\n \u003cp\u003eIn this study, six different solvents with varying polarity, namely, methanol, ethanol, acetone, hexane, ethyl acetate and water were used to extract antioxidants from either mixotrophically or heterotrophically grown \u003cem\u003eMicractinium\u003c/em\u003e sp. The extraction yields are given in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The highest extraction yield of 30.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94% was obtained in methanol followed by 28.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08% in water (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.91) for mixotrophic growth. For heterotrophic growth, the highest yield was equal in methanol (38.23\u0026thinsp;\u0026plusmn;\u0026thinsp;3.90%) and water (38.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.0). The lowest extraction yield was in ethyl acetate (11.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47%) and acetone (8.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41%) for mixotrophic and heterotrophic cultivation, respectively. The difference in extraction yields of methanol and water with respect to acetone, ethyl acetate, ethanol and hexane was highly significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for both mixotrophic and heterotrophic growth conditions.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eExtraction yields of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts prepared using different solvents and growth conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eExtraction yield (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMixotrophic growth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHeterotrophic growth\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.23\u0026thinsp;\u0026plusmn;\u0026thinsp;3.90 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.78 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHexane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthyl acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of three measurements (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). Means with different letters in the same column are statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Antioxidant capacity of \u003cem\u003eMicractinium\u003c/em\u003e sp. Measured by DPPH and FRAP assays\u003c/h2\u003e\n \u003cp\u003eAntioxidant capacity of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts in six different solvents under two different growth regimens was measured by DPPH and FRAP assays. The results are given in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. DPPH assay results are expressed both as % DPPH radical scavenging activity of microalgal extracts at 1 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentration and as micromoles trolox equivalent (TE) per gram dry weight (g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) of microalgae. The former expression does not take into account the extraction yield of the samples in each solvent. For mixotrophic growth, the highest antioxidant capacity was measured in the methanolic extracts as 7.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 and 93.80\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28 \u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW followed by ethanol extracts as 6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 and 79.83\u0026thinsp;\u0026plusmn;\u0026thinsp;7.56 \u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW by DPPH and FRAP assays, respectively. Similarly, the highest antioxidant capacity in heterotrophically grown samples was recorded in the methanolic extracts as 6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31 and 64.91\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28 \u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW by DPPH and FRAP assays, respectively. The antioxidant capacities of mixotrophically grown microalgae were higher compared to heterotrophic samples. Particularly, the difference in antioxidant capacities measured by FRAP assay between mixotrophic and heterotrophic growth in methanol, ethanol and acetone extracts was statistically significant. The correlation between DPPH and FRAP assays was found to be highly significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) for both conditions, and the coefficient of determination (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e) values for these assays were calculated as 0.57 and 0.65 for mixotrophic and heterotrophic growth, respectively. The low correlation between two methods can be explained by the methodological differences in detection and measurement of the antioxidants (Bulut et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAntioxidant capacity of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts in different solvent extracts determined by DPPH and FRAP assays.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eMixotrophic growth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eHeterotrophic growth\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(%) DPPH Radical scavenging effect\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDPPH\u003c/p\u003e\n \u003cp\u003e(\u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFRAP\u003c/p\u003e\n \u003cp\u003e(\u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(%) DPPH Radical scavenging effect\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDPPH\u003c/p\u003e\n \u003cp\u003e(\u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFRAP\u003c/p\u003e\n \u003cp\u003e(\u0026micro;mol TE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.61\u0026thinsp;\u0026plusmn;\u0026thinsp;4.37 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e93.80\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.91\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28 d\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.15\u0026thinsp;\u0026plusmn;\u0026thinsp;5.24 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e79.83\u0026thinsp;\u0026plusmn;\u0026thinsp;7.56 ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94 ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.43\u0026thinsp;\u0026plusmn;\u0026thinsp;10.45 bd\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.96 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.35\u0026thinsp;\u0026plusmn;\u0026thinsp;9.44 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHexane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.55\u0026thinsp;\u0026plusmn;\u0026thinsp;19.09 ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.24\u0026thinsp;\u0026plusmn;\u0026thinsp;5.28 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthyl acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.06\u0026thinsp;\u0026plusmn;\u0026thinsp;6.32 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33 ac\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.65\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 bc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eResults are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of three measurements (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). \u003csup\u003e1\u003c/sup\u003e Radical scavenging effects of algal extracts at 1 mg.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e concentration. Means with different letters in the same column are statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Total phenolic, flavonoid and carotenoid contents of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts\u003c/h2\u003e\n \u003cp\u003eTotal phenolic contents (TPC) of \u003cem\u003eMicractinium\u003c/em\u003e sp. grown under two different growth conditions and extracted in six different solvents are given in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. The highest TPC was found in methanolic extracts as 18.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17 mg GAE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW and 11.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 mg GAE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW for mixotrophic and heterotrophic growth, respectively. The difference between TPCs of two growth conditions in methanolic extracts is very significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009). These results are consistent with a previous report in which, both \u003cem\u003eChlorella vulgaris\u003c/em\u003e and \u003cem\u003eScenedesmus obliquus\u003c/em\u003e had higher polyphenols in the mixotrophic culture compared to the heterotrophic culture (Shetty and Sibi \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). The lowest TPCs of both mixotrophic and heterotrophic cultivation were measured in hexane extracts as 3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 mg GAE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW and 2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 mg GAE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW, respectively. The difference in results was not statistically significant.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTotal phenolic, flavonoid and carotenoids of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts prepared using different solvents and growth conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eMixotrophic growth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eHeterotrophic growth\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSolvent\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal phenolic content\u003c/p\u003e\n \u003cp\u003e(mg GAE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal flavonoid content\u003c/p\u003e\n \u003cp\u003e(mg QE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCarotenoid content\u003c/p\u003e\n \u003cp\u003e(mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal phenolic content\u003c/p\u003e\n \u003cp\u003e(mg GAE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal flavonoid content\u003c/p\u003e\n \u003cp\u003e(mg QE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCarotenoid content\u003c/p\u003e\n \u003cp\u003e(mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHexane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthyl acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eResults are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of three measurements (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). NT: Not Tested. Means with different letters in the same column are statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe highest total flavonoid content (TFC) of mixotrophic \u003cem\u003eMicractinium\u003c/em\u003e sp. was detected in the methanolic extracts (5.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 mg QE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) followed by the ethanol extracts (5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 mg QE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). TFC of heterotrophically grown cell extracts was the highest in methanol with a concentration of 3.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 mg.QE g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW. The difference in TFC of the methanolic extracts between mixotrophic and heterotrophic cultivation was not statistically significant. However, there was a significant reduction in TFC of ethanol extracts from heterotrophic samples (1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 mg QE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) compared to ethanol extracts of the mixotrophic culture (5.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70 mg QE.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW).\u003c/p\u003e\n \u003cp\u003eThe total carotenoid content (TCC) of \u003cem\u003eMicractinium\u003c/em\u003e sp. was calculated in methanol, acetone and ethyl acetate extracts as 2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW, 3.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW and 3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW, respectively under mixotrophic cultivation (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). TCC of heterotrophically grown microalgae was recorded as 1.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW in methanol, 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW in acetone and 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mg.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW in the ethyl acetate extracts. The difference in carotenoid content between mixotrophic and heterotrophic growth was statistically significant for ethyl acetate extracts (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Correlation of the antioxidant capacity with the phenolic, flavonoid, and carotenoid contents\u003c/h2\u003e\n \u003cp\u003eThe correlation of determination (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e) values between the antioxidant capacity and the total phenolics, flavonoids and carotenoids of \u003cem\u003eMicractinium\u003c/em\u003e sp. in different solvent extracts were calculated both for mixotrophic and heterotrophic growth conditions. The \u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e value between the DPPH assay and TPC in ethanol extracts of mixotrophically cultivated microalgae was 0.99 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01). This result is consistent with the strong correlation between TPC of \u003cem\u003eChlorella vulgaris\u003c/em\u003e and \u003cem\u003eScenedesmus obliquus\u003c/em\u003e and the DPPH assay reported by Shetty and Sibi (\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the same study, it was shown that the contribution of phenolics to the antioxidant potential was irrespective of the cultivation mode (Shetty and Sibi \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the present study, other \u003cem\u003eR\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e values of 0.90 or higher were obtained between DPPH or FRAP assay and TPC, TFC and TCC in various solvent extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp. both under mixotrophic and heterotrophic growth conditions; however, none of them were found to be statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Supplementary Tables\u0026nbsp;1 and 2). The statistically significant positive correlation between DPPH assay and TPC in the ethanol extracts suggests that polyphenols that are highly soluble in ethanol greatly contribute to the antioxidant activity of microalgae. Ethanol is also advantageous as a solvent as it is safe for human consumption (Dai and Mumper \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Identification of selected phenolic compounds in \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts by RP-HPLC\u003c/h2\u003e\n \u003cp\u003eTwelve different phenolic compounds that fall in three categories, namely, flavonols; rutin and quercetin, benzoic acid derivatives; 4-hydroxy benzoic acid, benzoic acid, gallic acid, syringic acid and vanillic acid and cinnamic acid and derivatives; caffeic acid, rosmarinic acid, coumaric acid and chlorogenic acid were quantified by RP-HPLC in methanol, acetone and ethyl acetate extracts of the mixotrophically and heterotrophically grown \u003cem\u003eMicractinium\u003c/em\u003e sp. (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Gallic acid (469.21\u0026thinsp;\u0026plusmn;\u0026thinsp;159.74 \u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) in the acetone extracts of mixotrophic microalgae was the highest phenolic compound detected. Under heterotrophic growth, 4-hydroxy benzoic acid (403.93\u0026thinsp;\u0026plusmn;\u0026thinsp;20.98 \u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) in the methanolic extracts was the most abundant phenolic compound. Strikingly, the amount of the same compound in the methanolic extracts of mixotrophic \u003cem\u003eMicractinium\u003c/em\u003e sp. was only 1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91 \u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW. Acetone is a powerful solvent of flavonols and consistently rutin concentration in acetone extracts (212.09\u0026thinsp;\u0026plusmn;\u0026thinsp;122.46 \u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW in mixotrophic samples) was significantly higher than the other solvents. Overall, there were considerable differences in the amounts of phenolic compounds between mixotrophic and heterotrophic microalgal extracts.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe phenolic compounds in different solvent extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp. identified by RP-HPLC.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eAmount\u003csup\u003e1\u003c/sup\u003e (\u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eMixotrophic growth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eHeterotrophic growth\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhenolic compound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEthyl acetate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAcetone\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEthyl acetate\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBenzoic acid derivatives\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGallic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e469.21\u0026thinsp;\u0026plusmn;\u0026thinsp;159.74 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125.06\u0026thinsp;\u0026plusmn;\u0026thinsp;11.68 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBenzoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.63 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e107.20\u0026thinsp;\u0026plusmn;\u0026thinsp;16.85 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.09\u0026thinsp;\u0026plusmn;\u0026thinsp;14.36 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-Hydroxy Benzoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e403.93\u0026thinsp;\u0026plusmn;\u0026thinsp;20.98 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.48\u0026thinsp;\u0026plusmn;\u0026thinsp;4.10 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVanillic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.37\u0026thinsp;\u0026plusmn;\u0026thinsp;7.72 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSyringic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.99\u0026thinsp;\u0026plusmn;\u0026thinsp;6.87 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56.42\u0026thinsp;\u0026plusmn;\u0026thinsp;13.04 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCinnamic acid derivatives\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCinnamic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.34\u0026thinsp;\u0026plusmn;\u0026thinsp;6.86 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e196.44\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70 c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoumaric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.36\u0026thinsp;\u0026plusmn;\u0026thinsp;15.25 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaffeic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.46\u0026thinsp;\u0026plusmn;\u0026thinsp;9.50 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChlorogenic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.27\u0026thinsp;\u0026plusmn;\u0026thinsp;5.61 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRosmarinic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.45\u0026thinsp;\u0026plusmn;\u0026thinsp;10.60 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFlavonols\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.02\u0026thinsp;\u0026plusmn;\u0026thinsp;13.28 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.09\u0026thinsp;\u0026plusmn;\u0026thinsp;8.67 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.81 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRutin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e212.09\u0026thinsp;\u0026plusmn;\u0026thinsp;122.46 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.33\u0026thinsp;\u0026plusmn;\u0026thinsp;38.33 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003e1\u003c/sup\u003e Results are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of two measurements. ND: Not Detected. Means with different letters in the same row are statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe amount of the carotenoid, \u0026beta;-carotene was quantified in the methanolic extracts of the mixotrophically or the heterotrophically cultivated \u003cem\u003eMicractinium\u003c/em\u003e sp. by RP-HPLC (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). \u0026beta;-carotene content under mixotrophic growth (52.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 \u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW) was slightly higher than the \u0026beta;-carotene amount under heterotrophic growth (45.99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46 \u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW). Although different detection and calculation methods have been used, \u0026beta;-carotene accounts for approximately 2% of the total carotenoids in methanolic extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u0026beta;-carotene amounts in methanolic extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp. identified by RP-HPLC.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGrowth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026beta;-carotene amount \u003csup\u003e1\u003c/sup\u003e (\u0026micro;g.g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMixotrophic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHeterotrophic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.99\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\"\u003e\u003csup\u003e1\u003c/sup\u003e Results are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of two measurements. Means with different letters in the same column are statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. Inhibitory effect of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts on intracellular ROS generation\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eMicractinium\u003c/em\u003e sp. methanolic extract with the highest antioxidant activity under mixotrophic cultivation was tested for its ability to inhibit intracellular oxidative stress induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in MCF-7 cells by the DCFH-DA assay. This method is based on the oxidation of non-fluorescent DCFH-DA into fluorescent DCF by intracellular ROS. Therefore, the intensity of intracellular fluorescent signal is proportional to the amount of intracellular ROS (Oparka et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used to promote oxidative stress as it is a relatively stable ROS generator and can result in the accumulation of ROS within the cell at elevated concentrations leading to cell damage and death (Oparka et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhuang et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003ePre-incubation of MCF-7 cells with the microalgal extracts for 48 h significantly changed the levels of intracellular ROS (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The inhibitory effect of the microalgal extracts on the intracellular ROS levels occurred in a concentration-dependent manner. Pre-treatment with the methanolic extract reduced intracellular ROS in MCF-7 cells by 23.80% and 72.60% at the lowest (50 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and the highest concentration (400 \u0026micro;g.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), respectively. Concentration-dependent inhibitory effect of the microalgal extracts was highly significant compared to the control cells treated with the vehicle solution (methanol) only (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7. Cytoprotective activity of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cell apoptosis\u003c/h2\u003e\n \u003cp\u003eThe methanolic extract, which exhibited the highest antioxidant activity in the mixotrophically cultivated \u003cem\u003eMicractinium\u003c/em\u003e sp. was evaluated for its ability to protect MCF-7 cells against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis. Upon treatment with 1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the percentage of viable, necrotic, and apoptotic MCF-7 cells were calculated as 54.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87, 34.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92, and 10.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95%, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). The percentage of viable MCF-7 cells pre-treated with the methanolic extract of \u003cem\u003eMicractinium\u003c/em\u003e sp. increased to 74.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pre-treatment with the methanolic microalgal extract led to a two-fold decrease in the number of necrotic cells (17.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78%) and caused a slight reduction in the apoptosis rate (8.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45%).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pursuit of finding a single solvent capable of solubilizing all target compounds during extraction is challenging. Phenolic compounds, characterized by diverse chemical structures, often form attachments to sugars or proteins in vivo, impacting their solubility across various solvents (Farvin and Jacobsen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Monteiro et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consequently, we assessed the extraction yields of antioxidants from \u003cem\u003eMicractinium\u003c/em\u003e sp. under both mixotrophic and heterotrophic conditions using six different solvents with varying polarities. The selection of different solvents aimed to encompass a wide range of polarities, thereby enhancing our understanding of solvent-specific extraction efficiencies and the nature of the compounds being extracted. In our study, methanol and water emerged as the most efficient solvents, yielding the highest extraction percentages under both growth conditions. This finding aligns with previous studies demonstrating the efficacy of methanol and water in extracting bioactive compounds from microalgae (Wang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Jerez-Martel et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These results underscore the importance of solvent selection in optimizing extraction efficiency and yield. Furthermore, the significant disparity in extraction yields between methanol and water compared to other solvents highlights the critical influence of solvent polarity on the extraction of bioactive compounds.\u003c/p\u003e\u003cp\u003eThe antioxidant capacities of various macroalgae and microalgae have been extensively studied, with notable variations observed across different species and strains (Goiris et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Farvin and Jacobsen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Machu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Notably, our study is the first to evaluate the antioxidant capacity of a \u003cem\u003eMicractinium\u003c/em\u003e species. Methanolic extracts exhibited the highest antioxidant capacity under both growth conditions, as evidenced by both DPPH and FRAP assays. This observation aligns with prior research highlighting the superior antioxidant potential of methanolic extracts from various microalgae species (Monteiro et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The strong correlation between the DPPH and FRAP assays suggests their complementary nature in assessing antioxidant capacity, despite methodological differences (Munteanu and Apetrei \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The observed moderate correlation coefficients indicate that while both assays provide valuable insights into the antioxidant potential of microalgal extracts, they may capture different aspects of antioxidant activity.\u003c/p\u003e\u003cp\u003eComparisons of antioxidant capacities across studies are challenging due to variations in laboratory conditions and methodologies. However, two thermo-tolerant strains, \u003cem\u003eScenedesmus\u003c/em\u003e sp. ME02 and \u003cem\u003eHindakia tetrachotoma\u003c/em\u003e ME03, isolated from the same thermal waters as \u003cem\u003eMicractinium\u003c/em\u003e sp., were recently evaluated for their antioxidant capacity (Bulut et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bulut et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our results demonstrated that ethyl acetate and water extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp. exhibited higher antioxidant capacity than \u003cem\u003eScenedesmus\u003c/em\u003e sp. ME02 but lower than \u003cem\u003eH. tetrachotoma\u003c/em\u003e ME03. The observed differences in antioxidant capacity among the thermo-tolerant strains can be attributed to genetic variability, which influences the production and composition of antioxidant compounds. Each strain has unique metabolic pathways that determine the types and amounts of antioxidants synthesized, affected by enzyme activities and metabolic fluxes. Additionally, strain-specific adaptations to their thermal environments may result in the production of unique antioxidants that confer thermal stress protection. The presence and concentration of secondary metabolites, which act as antioxidants and are often species-specific, further contribute to the variations in antioxidant capacity (Gauthier et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Coulombier et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroalgae cultivation methods, including autotrophy, heterotrophy, and mixotrophy, significantly influence their biochemical content. In this study, we evaluated the antioxidant activity of \u003cem\u003eMicractinium\u003c/em\u003e sp. cells grown under mixotrophic and heterotrophic conditions. The notable contrast in antioxidant capacities between these samples underscores the impact of cultivation mode on the biochemical composition and subsequent antioxidant properties of \u003cem\u003eMicractinium\u003c/em\u003e sp. Heterotrophic cultivation, particularly when utilizing molasses and vinasse as carbon sources, proved advantageous for achieving higher biomass and lower costs. Micractinium sp. demonstrated adaptability to various growth and temperature regimens, further influencing its biochemical content (Onay et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Engin et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018c\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSurprisingly, limited information exists on how heterotrophic growth affects the antioxidant capacity of microalgae compared to other cultivation conditions. In a previous study, the antioxidant activities of \u003cem\u003eChlorella vulgaris\u003c/em\u003e and \u003cem\u003eScenedesmus obliquus\u003c/em\u003e across autotrophic, mixotrophic, and heterotrophic conditions were compared (Shetty and Sibi \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consistent with our findings, methanolic extracts of both microalgae exhibited reduced antioxidant potential during heterotrophic growth compared to autotrophic and mixotrophic conditions. This decline in antioxidant activity under heterotrophic conditions aligns with the understanding that environmental factors, including light and ultraviolet exposure, along with internal processes such as photosynthesis, generate ROS. To counteract oxidative damage from ROS, microalgae produce antioxidants as a defense mechanism. Importantly, microalgae cultivated under mixotrophic conditions are exposed to light and rely on both photosynthesis and an additional carbon source in the culture medium for energy. Consequently, it is plausible to expect a higher antioxidant capacity in mixotrophic cultures compared to heterotrophic growth conditions due to the increased antioxidant activity prompted by the combined effects of photosynthesis and light exposure. This discovery emphasizes the complex relationship between microalgae cultivation methods, environmental influences, and their antioxidant responses, providing insight into the subtle changes in their biochemical processes across different growth environments (Engin et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e; Gauthier et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Abreu et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eStudies on other microalgae, such as \u003cem\u003eTetraselmis suecica\u003c/em\u003e and \u003cem\u003eHindakia tetrachotoma\u003c/em\u003e ME03, have highlighted their antioxidant activity and potential applications in the cosmetic and biotechnological industries (Sansone et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bulut et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The present investigation into \u003cem\u003eMicractinium\u003c/em\u003e sp. ME05 extracts expands the limited knowledge of the in vitro antioxidant activity of green microalgae, further supporting their potential for biotechnological applications.\u003c/p\u003e\u003cp\u003eThe evaluation of total phenolic contents (TPC) in \u003cem\u003eMicractinium\u003c/em\u003e sp., cultivated under varied growth conditions and extracted using different solvents, provided insights into the phenolic composition of the microalgal biomass. Consistent with prior research, \u003cem\u003eMicractinium\u003c/em\u003e sp. extracted with polar solvents exhibited higher concentrations of phenolic compounds compared to nonpolar hexane extracts, regardless of the cultivation method (Goiris et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Bulut et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Methanolic extracts showed the highest TPC, aligning with previous studies suggesting the effectiveness of methanol in extracting phenolic compounds from microalgae (Safafar et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Interestingly, hexane extracts displayed the lowest TPC across both growth conditions, indicating the limited capacity of this solvent to extract phenolic compounds from \u003cem\u003eMicractinium\u003c/em\u003e sp. Additionally, our study identified a higher phenolic content in \u003cem\u003eMicractinium\u003c/em\u003e sp. compared to \u003cem\u003eScenedesmus\u003c/em\u003e sp. ME02 in ethyl acetate and water extracts, revealing differences in the composition of these two freshwater strains isolated from the same thermal flora (Bulut et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These findings underscore the critical role of solvent selection in optimizing phenolic extraction efficiency.\u003c/p\u003e\u003cp\u003eThe health-promoting properties of flavonoids, commonly found in fruits and vegetables, are extensively documented in scientific literature, yet research on flavonoid content in microalgae remains relatively scarce (Kozłowska and Szostak-Węgierek \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, insights from previous studies have illuminated the presence of a diverse array of flavonoids across different classes of microalgae, albeit in smaller quantities compared to terrestrial plants (Goiris et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Methanolic extracts emerged as the most efficient solvent for extracting flavonoids from \u003cem\u003eMicractinium\u003c/em\u003e sp., consistent with its effectiveness in extracting other bioactive compounds. Importantly, our findings reveal that \u003cem\u003eMicractinium\u003c/em\u003e sp. exhibits a higher total flavonoid content compared to several other microalgae species, as demonstrated by Bulut et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Safafar et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This observation underscores the potential of \u003cem\u003eMicractinium\u003c/em\u003e sp. as a promising natural source of flavonoids, suggesting its suitability as a potential substitute for synthetic antioxidants in the industry. The relatively higher flavonoid content in \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts, particularly when cultivated under mixotrophic conditions, highlights the importance of cultivation strategies in modulating the biochemical composition and potential applications of microalgae-derived products.\u003c/p\u003e\u003cp\u003eThe total carotenoid content (TCC) in \u003cem\u003eMicractinium\u003c/em\u003e sp. exhibits significant variation across different cultivation modes, with higher concentrations observed in mixotrophic cultures compared to heterotrophic cultures. This study represents the first attempt to compare the TCC of microalgae under different cultivation modes, exploring the dynamics of carotenoid accumulation in response to varied growth conditions. In a previous study, two \u003cem\u003eMicractinium\u003c/em\u003e sp. strains, designated as CCNM 1006 and CCNM 1041, were evaluated for their total carotenoid contents. Both strains displayed slightly higher quantities of total carotenoids compared to \u003cem\u003eMicractinium\u003c/em\u003e sp. As part of a broader study encompassing 57 distinct microalgae strains, \u003cem\u003eMicractinium\u003c/em\u003e sp. fell within the medium range concerning its TCC (Paliwal et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The pivotal role of carotenoids in microalgae involves safeguarding chlorophylls from the detrimental effects of light exposure by scavenging ROS (Sathasivam and Ki \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our findings indicate a higher accumulation of carotenoids in mixotrophic microalgae compared to cultures grown heterotrophically. This underscores the vital role of carotenoids in responding to light exposure, a phenomenon crucial for mitigating oxidative stress through ROS scavenging. Importantly, our study marks the first attempt to compare the TCC of microalgae grown under distinct cultivation modes, offering valuable insights into the dynamics of carotenoid accumulation in response to varied growth conditions.\u003c/p\u003e\u003cp\u003eDespite the well-established role of phenolic compounds in plant antioxidant capacity, their contribution to microalgal antioxidant potential remains debated (Li et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Shetty and Sibi \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bulut et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bulut et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The diverse nature of microalgal antioxidants collectively contributes to their overall antioxidant capacity. Therefore, we investigated the profile of phenolics, and β-carotene present in the extracts. This study stands out as the first to quantify individual phenolic compounds in a \u003cem\u003eMicractinium\u003c/em\u003e species and compare their relative quantities under distinct cultivation modes. Twelve phenolic compounds, categorized into flavonols, benzoic acid derivatives, and cinnamic acid derivatives, were quantified in methanol, acetone, and ethyl acetate extracts from both mixotrophically and heterotrophically grown microalgae.\u003c/p\u003e\u003cp\u003eIn this comparative exploration, \u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e, another freshwater green microalga, exhibited considerably lower concentrations of gallic acid compared to \u003cem\u003eMicractinium\u003c/em\u003e sp., showcasing the distinct composition of these two species (Machu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). 4-hydroxy benzoic acid content (20 µg.g\u003csup\u003e− 1\u003c/sup\u003e sample) was higher in \u003cem\u003eC. pyrenoidosa\u003c/em\u003e compared to mixotrophic \u003cem\u003eMicractinium\u003c/em\u003e sp. (approximately 2 µg.g\u003csup\u003e− 1\u003c/sup\u003e DW in methanol extract) but much lower than in heterotrophically grown culture (approximately 400 µg.g\u003csup\u003e− 1\u003c/sup\u003e DW in methanolic extract). The significant increase in 4-hydroxy benzoic acid content under heterotrophic growth in \u003cem\u003eMicractinium\u003c/em\u003e sp. raises questions about the underlying mechanisms governing these variations, especially considering its known antimicrobial properties used in various industries (food, pharmaceutical, and cosmetics).\u003c/p\u003e\u003cp\u003eInterestingly, despite \u003cem\u003eMicractinium\u003c/em\u003e sp. displaying a higher total flavonoid content than \u003cem\u003eScenedesmus\u003c/em\u003e sp. ME02, specific flavonoids like quercetin and rutin were found to be significantly lower in \u003cem\u003eMicractinium\u003c/em\u003e sp. This suggests the possible presence of other, unexplored flavonoids in \u003cem\u003eMicractinium\u003c/em\u003e sp., hinting at the complexity and diversity of its biochemical profile (Bulut et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, the comparison of cinnamic acid derivatives, chlorogenic, and caffeic acid concentrations between \u003cem\u003eScenedesmus\u003c/em\u003e sp. and \u003cem\u003eMicractinium\u003c/em\u003e sp. adds another layer to the variations in flavonoid composition within different microalgal species collected from the same geothermal flora (Goiris et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn emphasizing the importance of specific compounds within microalgae, it is crucial to consider the diverse range of metabolites and their potential applications. Major carotenoid groups, including carotenes (such as β-carotene and lycopene) and xanthophylls (like lutein, astaxanthin, and fucoxanthin), each serve distinct roles. The prevalence of β-carotene in green microalgae like \u003cem\u003eDunaliella salina\u003c/em\u003e and \u003cem\u003eSpirulina maxima\u003c/em\u003e underscores their nutritional significance, while \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e stands out as a key source of astaxanthin—a commercially valuable product renowned for its various health benefits (Sathasivam and Ki \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Maoka \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In our study, we also quantified β-carotene in methanolic extracts of both mixotrophically and heterotrophically cultivated \u003cem\u003eMicractinium\u003c/em\u003e sp., noting slightly higher β-carotene content in mixotrophic cultures compared to heterotrophic ones. Despite this minor difference, β-carotene accounted for approximately 2% of the total carotenoids in methanolic extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp., highlighting its substantial presence. These findings underscore the influence of cultivation conditions on carotenoid biosynthesis, with light exposure likely boosting β-carotene production in mixotrophic cultures due to its role in photoprotection and light harvesting.\u003c/p\u003e\u003cp\u003eThe methanolic extract of \u003cem\u003eMicractinium\u003c/em\u003e sp., which demonstrated the highest antioxidant activity under mixotrophic cultivation, underwent further evaluation for its potential to mitigate intracellular oxidative stress and apoptosis induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in MCF-7 cells. Utilizing the DCFH-DA assay, a well-established method for measuring intracellular ROS levels, our study revealed a significant reduction in ROS in a concentration-dependent manner following pre-incubation with the microalgal extract (Oparka et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This substantial decrease in ROS levels is particularly noteworthy as it highlights the potent antioxidant capacity of extracts in protecting cells from oxidative damage induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, a stable ROS generator known to cause significant cellular damage at elevated concentrations (Zhuang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The concentration-dependent response observed in this study aligns with the notion that higher concentrations of antioxidants can more effectively neutralize ROS, thereby providing greater protection against oxidative stress.\u003c/p\u003e\u003cp\u003eApoptosis, or programmed cell death, represents a fundamental cellular process crucial for maintaining tissue homeostasis and eliminating damaged or aberrant cells. Dysregulation of apoptotic pathways is closely associated with various pathological conditions, including cancer (Vitale et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In our study, we explored the potential of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts in modulating apoptotic responses induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, a potent oxidizing agent known to trigger apoptotic cascades in cancer cells (Zhuang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our findings reveal a significant reduction in apoptotic rates in MCF-7 breast adenocarcinoma cells pre-treated with \u003cem\u003eMicractinium\u003c/em\u003e sp. methanolic extracts, suggesting a cytoprotective effect against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis. This finding suggests that the methanolic extract of \u003cem\u003eMicractinium\u003c/em\u003e sp. not only scavenges ROS effectively but also enhances cell survival under oxidative stress conditions. The significant improvement in cell viability and prevention of necrotic and apoptotic cell death pathways underscore the therapeutic potential of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts in combating oxidative stress-related cellular damage.\u003c/p\u003e\u003cp\u003eThese findings align with prior research indicating that microalgal extracts possess robust antioxidant properties and effectively alleviate oxidative stress in diverse cell lines (Sansone et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Vahdati et al. 2020; Bulut et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In a previous study, Bechelli et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) investigated the cytotoxic effects of algae, including \u003cem\u003eDunaliella salina\u003c/em\u003e extracts, on normal hematopoietic and leukemia cells by Annexin staining, demonstrating a significant reduction in cell viability induced by \u003cem\u003eD. salina\u003c/em\u003e ethanolic extracts. Similarly, Karakaş et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) demonstrated that the cytotoxic effects of extracts from \u003cem\u003eChlorella protothecoides\u003c/em\u003e and \u003cem\u003eNannochloropsis oculate\u003c/em\u003e on human brain glioblastoma and colon colorectal carcinoma cell lines. To the best of our knowledge, the current study marks the first demonstration of in vitro cytoprotective activity in cell extracts from a \u003cem\u003eMicractinium\u003c/em\u003e species. Furthermore, while other studies have explored the cytotoxic effects of various algae extracts on different cell lines, this study uniquely demonstrates the in vitro cytoprotective activity of \u003cem\u003eMicractinium\u003c/em\u003e species, opening avenues for further investigations into specific bioactive compounds.\u003c/p\u003e\u003cp\u003eThe ability of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts to modulate cell death pathways and enhance cellular viability in the face of oxidative stress holds significant implications for biomedical applications. While our study provides valuable insights into the cytoprotective effects of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress in breast adenocarcinoma cells, several avenues for future research warrant exploration. Further elucidation of the underlying molecular mechanisms governing the cytoprotective activity of \u003cem\u003eMicractinium\u003c/em\u003e sp. extracts, including their impact on apoptotic signaling pathways and cellular redox balance, is essential for fully harnessing their therapeutic potential.\u003c/p\u003e\u003cp\u003eIn addition to whole cell extracts, specific bioactive compounds derived from microalgae have been examined for their antioxidant activity on cell lines. For instance, β-carotene extracted from \u003cem\u003eD. salina\u003c/em\u003e strongly reduced cell viability of prostate cancer cells (Jayappriyan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Another carotenoid, violaxanthin isolated from \u003cem\u003eD. tertiolecta\u003c/em\u003e showed anti-cancer activity on MCF-7 cells (Pasquet et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Polyunsaturated fatty acids extracted from \u003cem\u003eNannochloropsis salina\u003c/em\u003e also exhibited in vitro anti-proliferative effect on MCF-7 cells (Sayegh et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). While these studies highlight the potential of individual compounds, the use of crude extracts is also important. Crude extracts contain a complex mixture of various bioactive compounds that can work synergistically, potentially enhancing their overall antioxidant and cytotoxic effects. This synergism can lead to a more effective mitigation of oxidative stress and inhibition of cancer cell proliferation compared to isolated compounds. Therefore, exploring the bioactivity of crude extracts provides a holistic understanding of their therapeutic potential and can uncover interactions that may be missed when studying single compounds. \u003cem\u003eMicractinium\u003c/em\u003e sp. contains a rich profile of fatty acids, which may collectively contribute to its antioxidant activity.\u003c/p\u003e\u003cp\u003eThe versatile characteristics of \u003cem\u003eMicractinium\u003c/em\u003e sp., including its adaptability to both mixotrophic and heterotrophic conditions, wide temperature range (16°C-50°C), and diverse biochemical composition, position it as an ideal candidate for mass cultivation with promising applications in the nutraceutical and food industries. Our study represents the first attempt to quantify specific phenolic compounds in a \u003cem\u003eMicractinium\u003c/em\u003e species and compare their concentrations under different cultivation methods. Significantly, the antioxidant-rich extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp. exhibited a notable inhibitory effect on ROS production and apoptosis induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in MCF-7 cells. This discovery provides valuable insights into the relatively unexplored field of in vitro antioxidant activity of green microalgae for potential biotechnological applications. Future investigations focusing on the identification and characterization of specific bioactive compounds derived from \u003cem\u003eMicractinium\u003c/em\u003e sp. can further enhance our understanding of its antioxidant activity, both in vitro and in vivo, thus contributing to the advancement of microalgal biotechnology.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the evaluation of antioxidant activity in mixotrophically and heterotrophically grown \u003cem\u003eMicractinium\u003c/em\u003e sp. cells using six different solvents for extraction has yielded significant insights. Among these solvents, methanol emerged as particularly effective, with \u003cem\u003eMicractinium\u003c/em\u003e sp. methanolic extracts demonstrating the highest antioxidant activity. The notable reduction in oxidative stress and the observed cytoprotective effects on MCF-7 cells underscore the therapeutic potential of \u003cem\u003eMicractinium\u003c/em\u003e sp., particularly in addressing oxidative stress-related disorders.\u003c/p\u003e \u003cp\u003eA comprehensive comparative analysis revealed intriguing distinctions between mixotrophically and heterotrophically grown microalgal extracts. Overall, mixotrophic samples exhibited a superior antioxidant capacity, accompanied by higher levels of total phenolics, flavonoids, and carotenoids. This suggests that the cultivation method has a significant impact on the biochemical composition of \u003cem\u003eMicractinium\u003c/em\u003e sp., influencing its potential health-promoting attributes.\u003c/p\u003e \u003cp\u003eSpecifically, mixotrophic samples displayed elevated concentrations of gallic acid and rutin, compounds associated with various health benefits. In contrast, heterotrophic samples showcased substantial accumulations of 4-hydroxy benzoic acid and cinnamic acid, indicating a distinct biochemical profile under these growth conditions.\u003c/p\u003e \u003cp\u003eThis study breaks new ground by quantifying the amounts of these phenolic compounds in a \u003cem\u003eMicractinium\u003c/em\u003e species for the first time. Moreover, it pioneers the documentation of the antioxidant and cytoprotective activities of \u003cem\u003eMicractinium\u003c/em\u003e sp., expanding the understanding of its potential applications in microalgal biotechnology.\u003c/p\u003e \u003cp\u003eFuture investigations could focus on the targeted extraction of specific bioactive compounds from \u003cem\u003eMicractinium\u003c/em\u003e sp. This approach would allow for a more detailed exploration of the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e antioxidant activities, both in isolation and in conjunction with whole cell extracts. Such focused studies will undoubtedly contribute to unraveling the therapeutic potential and specific health benefits associated with \u003cem\u003eMicractinium\u003c/em\u003e sp. ME05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Konya Food and Agriculture University (KFAU) Strategic Products Research and Development Center (SARGEM) for technical support with the HPLC analysis and KFAU Research and Development Center for Diagnostic Kits (KIT-ARGEM) for the use of the facilities. The MCF-7 cells are a kind gift of Hasan Huseyin Kazan. We especially appreciate Assoc. Prof. Dr. Okan Bulut from the University of Alberta for his help and guidance with statistical analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by the Konya Food and Agriculture University Research Project BAP-2019/0040.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Onur Bulut and Işkın Engin. The first draft of the manuscript was written by Çağla Sönmez and Onur Bulut, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbbaszadeh H, Keikhaei B, Mottaghi S (2019) A review of molecular mechanisms involved in anticancer and antiangiogenic effects of natural polyphenolic compounds. Phytother Res 33:2002-2014. https://doi.org/10.1002/ptr.6403\u003c/li\u003e\n \u003cli\u003eAbd El-Hack ME, Abdelnour S, Alagawany M, Abdo M, Sakr MA, Khafaga AF, Mahgoub SA, Elnesr SS, Gebriel MG (2019) Microalgae in modern cancer therapy: Current knowledge. Biomed Pharmacother 111:42-50. https://doi.org/10.1016/j.biopha.2018.12.069\u003c/li\u003e\n \u003cli\u003eAbou-Shanab RA, El-Dalatony MM, El-Sheekh MM, Ji MK, Salama ES, Kabra AN, Jeon BH (2014) Cultivation of a new microalga, \u003cem\u003eMicractinium reisseri\u003c/em\u003e, in municipal wastewater for nutrient removal, biomass, lipid, and fatty acid production. Biotechnol Bioprocess Eng 19:510-518. https://doi.org/10.1007/s12257-013-0485-z\u003c/li\u003e\n \u003cli\u003eAbreu AP, Morais RC, Teixeira JA, Nunes J (2022) A comparison between microalgal autotrophic growth and metabolite accumulation with heterotrophic, mixotrophic and photoheterotrophic cultivation modes. Renew Sustain Energy Rev 159:112247. https://doi.org/10.1016/j.rser.2022.112247\u003c/li\u003e\n \u003cli\u003eAlmendinger M, Saalfrank F, Rohn S, Kurth E, Springer M, Pleissner D (2021) Characterization of selected microalgae and cyanobacteria as sources of compounds with antioxidant capacity. Algal Res 53:102168. https://doi.org/10.1016/j.algal.2020.102168\u003c/li\u003e\n \u003cli\u003eAmbriz-P\u0026eacute;rez DL, Leyva-L\u0026oacute;pez N, Gutierrez-Grijalva EP, Heredia, JB (2016) Phenolic compounds: Natural alternative in inflammation treatment. A Review. Cogent Food \u0026amp; Agriculture, 2(1), 1131412. https://doi.org/10.1080/23311932.2015.1131412\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBechelli J, Coppage M, Rosell K, Liesveld J (2011) Cytotoxicity of algae extracts on normal and malignant cells. Leuk Res Treat 2011:1-6. https://doi.org/10.4061/2011/373519\u003c/li\u003e\n \u003cli\u003eBrewer MS (2011) Natural antioxidants: sources, compounds, mechanisms of action, and potential applications. Compr Rev Food Sci Food Saf 10:221-247. https://doi.org/10.1111/j.1541-4337.2011.00156.x\u003c/li\u003e\n \u003cli\u003eBulut O, Akın D, S\u0026ouml;nmez \u0026Ccedil;, \u0026Ouml;ktem A, Y\u0026uuml;cel M, \u0026Ouml;ktem HA (2019) Phenolic compounds, carotenoids, and antioxidant capacities of a thermo-tolerant Scenedesmus sp. (Chlorophyta) extracted with different solvents. J Appl Phycol 31:1675-1683. https://doi.org/10.1007/s10811-018-1726-5\u003c/li\u003e\n \u003cli\u003eBulut O, S\u0026ouml;nmez \u0026Ccedil;, \u0026Ouml;ktem HA (2023) \u003cem\u003eHindakia tetrachotoma\u003c/em\u003e ME03 (Chlorophyta) has high phenolic content, antioxidant capacity, and attenuates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress and apoptosis in human cells. Phycologia 62(4), 293-302. https://doi.org/10.1080/00318884.2023.2202520\u003c/li\u003e\n \u003cli\u003eCheng Z, Moore J, Yu L (2006) High-throughput relative DPPH radical scavenging capacity assay. J Agric Food Chem 54:7429-7436. https://doi.org/10.1021/jf0611668\u003c/li\u003e\n \u003cli\u003eCoulombier N, Jauffrais T, Lebouvier N (2021) Antioxidant compounds from microalgae: A review. Mar Drugs 19:549. https://doi.org/10.3390/md19100549\u003c/li\u003e\n \u003cli\u003eDai J, Mumper RJ (2010) Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 15:7313-7352. https://doi.org/10.3390/molecules15107313\u003c/li\u003e\n \u003cli\u003eEl-Baz FK, Hussein RA, Mahmoud K, Abdo SM (2018) Cytotoxic activity of carotenoid-rich fractions from \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e and \u003cem\u003eDunaliella salina\u003c/em\u003e microalgae and the identification of the phytoconstituents using LC-DAD/ESI-MS. Phytother Res 32:298-304. https://doi.org/10.1002/ptr.5976\u003c/li\u003e\n \u003cli\u003eEngin IK, Cekmecelioglu D, Y\u0026uuml;cel AM, Oktem HA (2018a) Heterotrophic growth and oil production from Micractinium sp. ME05 using molasses. J Appl Phycol 30:3483-3492. https://doi.org/10.1007/s10811-018-1486-2\u003c/li\u003e\n \u003cli\u003eEngin IK, Cekmecelioglu D, Y\u0026uuml;cel AM, Oktem HA (2018b) Evaluation of heterotrophic and mixotrophic cultivation of novel Micractinium sp. ME05 on vinasse and its scale up for biodiesel production. Bioresour Technol 251:128-134. https://doi.org/10.1016/j.biortech.2017.12.023\u003c/li\u003e\n \u003cli\u003eEngin IK, Cekmecelioglu D, Y\u0026uuml;cel AM, Oktem HA (2018c) Enhancement of heterotrophic biomass production by Micractinium sp. ME05. Waste Biomass Valorization 9:811-820. https://doi.org/10.1007/s12649-017-9846-8\u003c/li\u003e\n \u003cli\u003eFarvin KS, Jacobsen C (2013) Phenolic compounds and antioxidant activities of selected species of seaweeds from Danish coast. Food Chem 138:1670-1681. https://doi.org/10.1016/j.foodchem.2012.10.078\u003c/li\u003e\n \u003cli\u003eFerdous UT, Yusof ZNB (2021) Medicinal prospects of antioxidants from algal sources in cancer therapy. Front Pharmacol 12:593116. https://doi.org/10.3389/fphar.2021.593116\u003c/li\u003e\n \u003cli\u003eFiruzi O, Lacanna A, Petrucci R, Marrosu G, Saso L (2005) Evaluation of the antioxidant activity of flavonoids by \u0026ldquo;ferric reducing antioxidant power\u0026rdquo; assay and cyclic voltammetry. Biochim Biophys Acta Gen Subj 1721:174-184. https://doi.org/10.1016/j.bbagen.2004.11.001\u003c/li\u003e\n \u003cli\u003eForman HJ, Zhang H (2021) Targeting oxidative stress in disease: Promise and limitations of antioxidant therapy. Nat Rev Drug Discov 20:689-709. https://doi.org/10.1038/s41573-021-00233-1\u003c/li\u003e\n \u003cli\u003eFranco R, Navarro G, Mart\u0026iacute;nez-Pinilla E (2019) Antioxidants versus food antioxidant additives and food preservatives. Antioxidants 8:542. https://doi.org/10.3390/antiox8110542\u003c/li\u003e\n \u003cli\u003eGauthier MR, Senhorinho GNA, Scott JA (2020) Microalgae under environmental stress as a source of antioxidants. Algal Res 52:102104. https://doi.org/10.1016/j.algal.2020.102104\u003c/li\u003e\n \u003cli\u003eGoiris K, Muylaert K, Fraeye I, Foubert I, De Brabanter J, De Cooman L (2012) Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. J Appl Phycol 24:1477-1486. https://doi.org/10.1007/s10811-012-9804-6\u003c/li\u003e\n \u003cli\u003eGoiris K, Muylaert K, Voorspoels S, Noten B, De Paepe D, Baart GJ, De Cooman L (2014) Detection of flavonoids in microalgae from different evolutionary lineages. J Phycol 50:483-492. https://doi.org/10.1111/jpy.12180\u003c/li\u003e\n \u003cli\u003eHerald TJ, Gadgil P, Tilley M (2012) High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour. J Sci Food Agric 92:2326-2331. https://doi.org/10.1002/jsfa.5633\u003c/li\u003e\n \u003cli\u003eJayappriyan\u0026nbsp;KR, Rajkumar R, Venkatakrishnan V, Nagaraj S, Rengasamy R (2013) In vitro anticancer activity of natural \u0026beta;-carotene from \u003cem\u003eDunaliella salina\u003c/em\u003e EU5891199 in PC-3 cells. Biomed Prev Nutr 3:99-105. https://doi.org/10.1016/j.bionut.2012.08.003\u003c/li\u003e\n \u003cli\u003eJerez-Martel I, Garc\u0026iacute;a-Poza S, Rodr\u0026iacute;guez-Martel G, Rico M, Afonso-Olivares C, G\u0026oacute;mez-Pinchetti JL (2017) Phenolic profile and antioxidant activity of crude extracts from microalgae and cyanobacteria strains. J Food Qual 2017:1-10. https://doi.org/10.1155/2017/2924508\u003c/li\u003e\n \u003cli\u003eKarakaş CY, Tekarslan Şahin H, Inan B, \u0026Ouml;z\u0026ccedil;imen D, Erginer Y\u0026Ouml; (2019) In vitro cytotoxic activity of microalgal extracts loaded nano-micro particles produced via electrospraying and microemulsion methods. Biotechnol Prog 35. https://doi.org/10.1002/btpr.2876\u003c/li\u003e\n \u003cli\u003eKozłowska A, Szostak-Węgierek D (2019) Flavonoids \u0026ndash; Food sources, health benefits, and mechanisms involved. In: M\u0026eacute;rillon JM, Ramawat K (eds) Bioactive molecules in food. Reference Series in Phytochemistry. Springer, Cham, pp 1-20. https://doi.org/10.1007/978-3-319-54528-8_54-1\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKrivina E, Sinetova M, Savchenko T, Degtyaryov E, Tebina E, Temraleeva A (2023) \u003cem\u003eMicractinium lacustre\u003c/em\u003e and \u003cem\u003eM. thermotolerans\u003c/em\u003e spp. nov. (Trebouxiophyceae, Chlorophyta): Taxonomy, temperature-dependent growth, photosynthetic characteristics and fatty acid composition. Algal Res 71:103042. https://doi.org/10.1016/j.algal.2023.103042\u003c/li\u003e\n \u003cli\u003eLi HB, Cheng KW, Wong CC, Fan KW, Chen F, Jiang Y (2007) Evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae. Food Chem 102:771-776. https://doi.org/10.1016/j.foodchem.2006.06.022\u003c/li\u003e\n \u003cli\u003eLichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: Measurement and characterization by UV-VIS spectroscopy. Curr Protoc Food Anal Chem, 1(1), F4-3. https://doi.org/10.1002/0471142913.faf0403s01\u003c/li\u003e\n \u003cli\u003eLobiuc A, Pavăl NE, Mangalagiu II, Gheorghiță R, Teliban GC, Amăriucăi-Mantu D, Stoleru V (2023) Future antimicrobials: Natural and functionalized phenolics. Molecules 28:1114. https://doi.org/10.3390/molecules28031114\u003c/li\u003e\n \u003cli\u003eMachu L, Misurcova L, Vavra Ambrozova J, Orsavova J, Mlcek J, Sochor J, Jurikova T (2015) Phenolic content and antioxidant capacity in algal food products. Molecules 20:1118-1133. https://doi.org/10.3390/molecules20011118\u003c/li\u003e\n \u003cli\u003eMalavasi V, Soru S, Cao G (2020) Extremophile microalgae: the potential for biotechnological application. J Phycol 56:559-573. https://doi.org/10.1111/jpy.12965\u003c/li\u003e\n \u003cli\u003eMaoka T (2020) Carotenoids as natural functional pigments. J Nat Med 74:1-16. https://doi.org/10.1007/s11418-019-01364-x\u003c/li\u003e\n \u003cli\u003eMonteiro M, Santos RA, Iglesias P, Couto A, Serra CR, Gouvinhas I, Barros A, Olivia-Teles A, Enes P, D\u0026iacute;az-Rosales P (2020) Effect of extraction method and solvent system on the phenolic content and antioxidant activity of selected macro- and microalgae extracts. J Appl Phycol 32:349-362. https://doi.org/10.1007/s10811-019-01927-1\u003c/li\u003e\n \u003cli\u003eMunteanu IG, Apetrei C (2021) Analytical methods used in determining antioxidant activity: A review. Int J Mol Sci 22:3380. https://doi.org/10.3390/ijms22073380\u003c/li\u003e\n \u003cli\u003eOnay M, Sonmez C, Oktem HA, Yucel AM (2014) Thermo-resistant green microalgae for effective biodiesel production: isolation and characterization of unialgal species from geothermal flora of Central Anatolia. Bioresour Technol 169:62-71. https://doi.org/10.1016/j.biortech.2014.06.078\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOparka M, Walczak J, Malinska D, van Oppen LM, Szczepanowska J, Koopman WJ, Wieckowski MR (2016) Quantifying ROS levels using CM-H\u003csub\u003e2\u003c/sub\u003eDCFDA and HyPer. Methods 109:3-11. https://doi.org/10.1016/j.ymeth.2016.06.008\u003c/li\u003e\n \u003cli\u003ePaliwal C, Ghosh T, George B, Pancha I, Maurya R, Chokshi K, Ghosh A, Mishra S (2016) Microalgal carotenoids: Potential nutraceutical compounds with chemotaxonomic importance. Algal Res 15:24-31. https://doi.org/10.1016/j.algal.2016.01.017\u003c/li\u003e\n \u003cli\u003eParra-Riofrio G, Moreno P, Garc\u0026iacute;a-Rosado E, Alonso MC, Uribe-Tapia E, Abdala-Diaz RT, Bejar J (2023) \u003cem\u003eTetraselmis suecica\u003c/em\u003e and \u003cem\u003ePorphyridium cruentum\u003c/em\u003e exopolysaccharides show anti-VHSV activity on RTG-2 cells. Aquac Int 31(6):3145-3157. https://doi.org/10.1007/s10499-023-01202-8\u003c/li\u003e\n \u003cli\u003ePasquet V, Morisset P, Ihammouine S, Chepied A, Aumailley L, Berard JB, Serive B, Kaas R, Lanneluc I, Thiery V, Lafferriere M, Picot JM, Patrice T, Cadoret JP, Picot L (2011) Antiproliferative activity of violaxanthin isolated from bioguided fractionation of \u003cem\u003eDunaliella tertiolecta\u003c/em\u003e extracts. Mar Drugs 9:819-831. https://doi.org/10.3390/md9050819\u003c/li\u003e\n \u003cli\u003ePoljsak B, Kovač V, Milisav I (2021) Antioxidants, food processing and health. Antioxidants 10:433. https://doi.org/10.3390/antiox10030433\u003c/li\u003e\n \u003cli\u003eSafafar H, Van Wagenen J, M\u0026oslash;ller P, Jacobsen C (2015) Carotenoids, phenolic compounds and tocopherols contribute to the antioxidative properties of some microalgae species grown on industrial wastewater. Mar Drugs 13:7339-7356. https://doi.org/10.3390/md13127069\u003c/li\u003e\n \u003cli\u003eSanjeewa KKA, Fernando IPS, Samarakoon KW, Lakmal HH, Kim EA, Kwon ON, Dilshara MG, Lee JB, Jeon YJ (2016) Anti-inflammatory and anti-cancer activities of sterol-rich fraction of cultured marine microalga \u003cem\u003eNannochloropsis oculata\u003c/em\u003e. Algae 31:277-287. https://doi.org/10.4490/algae.2016.31.6.29\u003c/li\u003e\n \u003cli\u003eSansone C, Galasso C, Orefice I, Nuzzo G, Luongo E, Cutignano A, Romano G, Brunet C, Fontana A, Esposito F, Ianora A (2017) The green microalga \u003cem\u003eTetraselmis suecica\u003c/em\u003e reduces oxidative stress and induces repairing mechanisms in human cells. Sci Rep 7:1-12. https://doi.org/10.1038/srep41215\u003c/li\u003e\n \u003cli\u003eSathasivam R, Ki JS (2018) A review of the biological activities of microalgal carotenoids and their potential use in healthcare and cosmetic industries. Mar Drugs 16(1):26. https://doi.org/10.3390/md16010026\u003c/li\u003e\n \u003cli\u003eSayegh F, Elazzazy A, Bellou S, Moustogianni A, Elkady AI, Baeshen MN, Aggelis G (2016) Production of polyunsaturated single cell oils possessing antimicrobial and anticancer properties. Ann Microbiol 66:937-948. https://doi.org/10.1007/s13213-015-1176-0\u003c/li\u003e\n \u003cli\u003eShetty V, Sibi G (2015) Relationship between total phenolics content and antioxidant activities of microalgae under autotrophic, heterotrophic and mixotrophic growth. J Food Res Sci 4:1-9. https://doi.org/10.3923/jfrs.2015.1.9\u003c/li\u003e\n \u003cli\u003eS\u0026ouml;nmez \u0026Ccedil;, El\u0026ccedil;in E, Akın D, \u0026Ouml;ktem HA, Y\u0026uuml;cel M (2016) Evaluation of novel thermo-resistant Micractinium and Scenedesmus sp. for efficient biomass and lipid production under different temperature and nutrient regimes. Bioresour Technol 211:422-428. https://doi.org/10.1016/j.biortech.2016.03.125\u003c/li\u003e\n \u003cli\u003eVacca RA, Valenti D, Caccamese S, Daglia M, Braidy N, Nabavi SM (2016) Plant polyphenols as natural drugs for the management of Down syndrome and related disorders. Neurosci Biobehav Rev 71:865-877. https://doi.org/10.1016/j.neubiorev.2016.10.023\u003c/li\u003e\n \u003cli\u003eVahdati SN, Lashkari A, Navasatli SA, Ardestani SK, Safavi M (2022) Butylated hydroxyl-toluene, 2,4-Di-tert-butylphenol, and phytol of Chlorella sp. protect the PC12 cell line against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced neurotoxicity. Biomed Pharmacother 145:112415. https://doi.org/10.1016/j.biopha.2021.112415\u003c/li\u003e\n \u003cli\u003eVitale I, Pietrocola F, Guilbaud E, Aaronson SA, Abrams JM, Adam D, ... Pasparakis M (2023) Apoptotic cell death in disease\u0026mdash;Current understanding of the NCCD 2023. Cell Death Differ 30:1097-1154. https://doi.org/10.1038/s41418-023-01153-w\u003c/li\u003e\n \u003cli\u003eWang T, Jonsdottir R, \u0026Oacute;lafsd\u0026oacute;ttir G (2009) Total phenolic compounds, radical scavenging and metal chelation of extracts from Icelandic seaweeds. Food Chem 116:240-248. https://doi.org/10.1016/j.foodchem.2009.02.041\u003c/li\u003e\n \u003cli\u003eYuan M, Zhang G, Bai W, Han X, Li C, Bian S (2022) The role of bioactive compounds in natural products extracted from plants in cancer treatment and their mechanisms related to anticancer effects. Oxid Med Cell Longev 2022. https://doi.org/10.1155/2022/1429869\u003c/li\u003e\n \u003cli\u003eZhang H, Tsao R (2016) Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr Opin Food Sci 8:33-42. https://doi.org/10.1016/j.cofs.2016.02.002\u003c/li\u003e\n \u003cli\u003eZheng X, Shao C, Zhu J, Zhang L, Huang Q (2023) Study of the antioxidant capacity of astaxanthin in cells against radiation-induced strong oxidative stress. Aquac Int 31(5):2705-2725. https://doi.org/10.1007/s10499-023-01105-8\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhuang Y, Ma Q, Guo Y, Sun L (2017) Protective effects of rambutan (Nephelium lappaceum) peel phenolics on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative damages in HepG2 cells and d-galactose-induced aging mice. Food Chem Toxicol 108:554-562. https://doi.org/10.1016/j.fct.2017.01.022\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microalgae, antioxidants, gallic acid, 4-hydroxy benzoic acid, oxidative stress, cytoprotective effect","lastPublishedDoi":"10.21203/rs.3.rs-4690459/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4690459/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn response to the growing demand for high-value bioactive compounds, microalgae cultivation has gained a significant acceleration in recent years. Among these compounds, antioxidants have emerged as essential constituents in the food, pharmaceutical, and cosmetics industries. This study focuses on \u003cem\u003eMicractinium\u003c/em\u003e sp. ME05, a green microalgal strain previously isolated from hot springs flora in our laboratory. \u003cem\u003eMicractinium\u003c/em\u003e sp. cells were extracted using six different solvents, and their antioxidant capacity, as well as total phenolic, flavonoid, and carotenoid contents, were evaluated. The methanolic extracts demonstrated the highest antioxidant capacity, measuring 7.72 and 93.80 µmol trolox equivalents.g\u003csup\u003e-1\u003c/sup\u003e dry weight (DW) according to the DPPH and FRAP assays, respectively. To further characterize the biochemical profile, reverse phase high-performance chromatography (RP-HPLC) was employed to quantify twelve different phenolics, including rutin, gallic acid, benzoic acid, cinnamic acid, and β-carotene, in the microalgal extracts. Notably, the acetone extracts of \u003cem\u003eMicractinium\u003c/em\u003e sp. grown mixotrophically contained a high amount of gallic acid (469.21 ± 159.74 µg.g\u003csup\u003e-1\u003c/sup\u003e DW), while 4-hydroxy benzoic acid (403.93 ± 20.98 µg.g\u003csup\u003e-1\u003c/sup\u003e DW) was the main phenolic compound in the methanolic extracts under heterotrophic cultivation. Moreover, extracts from \u003cem\u003eMicractinium\u003c/em\u003e sp. exhibited remarkable cytoprotective activity by effectively inhibiting hydrogen peroxide-induced oxidative stress and cell death in human breast adenocarcinoma (MCF-7) cells. In conclusion, with its diverse biochemical composition and adaptability to different growth regimens, \u003cem\u003eMicractinium\u003c/em\u003e sp. emerges as a robust candidate for mass cultivation in nutraceutical and food applications.\u003c/p\u003e","manuscriptTitle":"Antioxidant activity of Micractinium sp. (Chlorophyta) extracts against H2O2 induced oxidative stress in human breast adenocarcinoma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-24 05:20:47","doi":"10.21203/rs.3.rs-4690459/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc69a9fc-7349-411e-8f38-63d8778cae6a","owner":[],"postedDate":"July 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-24T05:20:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-24 05:20:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4690459","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4690459","identity":"rs-4690459","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}