Gloeothece sp.—Exploiting a New Source of Antioxidant, Anti-Inflammatory, and Antitumor Agents

Bioactive lipidic compounds of microalgae, such as polyunsaturated fatty acids (PUFA) and carotenoids, can avoid or treat oxidation-associated conditions and diseases like inflammation or cancer. This study aimed to assess the bioactive potential of lipidic extracts obtained from Gloeothece sp.–using Generally Recognized as Safe (GRAS) solvents like ethanol, acetone, hexane:isopropanol (3:2) (HI) and ethyl lactate. The bioactive potential of extracts was assessed in terms of antioxidant (ABTS•+, DPPH•, •NO and O2•assays), anti-inflammatory (HRBC membrane stabilization and Cox-2 screening assay), and antitumor capacity (death by TUNEL, and anti-proliferative by BrdU incorporation assay in AGS cancer cells); while its composition was characterized in terms of carotenoids and fatty acids, by HPLC-DAD and GC-FID methods, respectively. Results revealed a chemopreventive potential of the HI extract owing to its ability to: (I) scavenge -NO• radical (IC50, 1258 ± 0.353 µg·mL−1); (II) inhibit 50% of COX-2 expression at 130.2 ± 7.4 µg·mL−1; (III) protect 61.6 ± 9.2% of lysosomes from heat damage, and (IV) induce AGS cell death by 4.2-fold and avoid its proliferation up to 40% in a concentration of 23.2 ± 1.9 µg·mL−1. Hence, Gloeothece sp. extracts, namely HI, were revealed to have the potential to be used for nutraceutical purposes.


Introduction
The first reports on cyanobacteria date back to the time of Aztecs who used Spirulina (Arthrospira platensis, A. maxima) as food [1]. Nowadays the potential application of cyanobacteria in our daily lives has been well documented. Such microscopic organisms are indeed a universal source of a vast array of chemical products with applications in the feed, food, nutritional, cosmetic, and pharmaceutical industries [1][2][3]. The last decades have witnessed the massive development in the production of cyanobacteria through the improvement of processing methods, with particular emphasis on the extraction of high-value compounds to be used as nutraceuticals and pharmaceuticals [1,4].
Nevertheless, the exploitation of prokaryotic and eukaryotic microalgae is restricted to a few strains and most species remain largely unexplored. So far, till 2019, 260 families ) secrete or degrade inflammatory cytokines in regulating cytokines released by immune cells through a feedback mechanism. Phosphorylation of NF-κB activates several enzymes, e.g., cyclooxygenase(COX-2), oxidase, and iNOS, thus inducing the release of prostaglandins (PGE2), O2 •-and other molecules like anti-apoptotic factors, cell cycle regulators, adhesion molecules that are likely to be related to tumorogenesis, cancer cell growth and proliferation. The unbalanced increase of the former may lead to tumorogenesis and (among other events) cancer growth and proliferation.
Another common strategy followed in the formulation of anti-inflammatory agents is based on suppressing of production of inflammatory mediators, such as COX-2 inhibitors, that interfere with the initiation and progression of inflammation-associated diseases [18]. PGs were found in several kinds of tumors, like gastric cancer [19] or colon adenocarcinoma [20]; causing tumorigenic effects, such as stimulation of cell growth and angiogenesis, inhibition of apoptosis, and suppression of the immune system. Several studies also indicate that COX-2 inhibitors can reduce the risk of development of colon, lung, or skin cancer [21][22][23], and namely improve therapeutic effects on human cancers in combination with chemotherapeutic [24].
In practice, the synthetic drugs used to treat these disorders may bring about severe side effects; hence is important to find compounds from biological sources, such as cyanobacteria, lacking adverse effects [25]. Carotenoids and PUFA from microalgal sources have indeed been claimed to have anti-cancer and anti-inflammatory properties, having sometimes an antioxidant-based mechanism of action [26][27][28]. Some of them have even been proposed for the treatment and prevention of such chronic diseases [29,30]. Epidemiological studies suggest that carotenoids can prevent free radical-dependent oxidation of LDL, cholesterol, proteins or DNA, by capturing free radicals and thus reducing stress induced by ROS [31]. Furthermore, PUFA, namely n-3 PUFA, was described to hold antioxidant and anti-inflammatory effects [32][33][34].
In the particular case of cancer, some strategies of chemoprevention can be accom- Brief schematic representation of how oxidative stress, inflammation, and cancer development may be correlated. After the lipopolysaccharide (LPS) inflammatory activation pathway in a macrophage cell, secretory lysosomes ( ) secrete or degrade inflammatory cytokines in regulating cytokines released by immune cells through a feedback mechanism. Phosphorylation of NF-κB activates several enzymes, e.g., cyclooxygenase(COX-2), oxidase, and iNOS, thus inducing the release of prostaglandins (PGE2), O2 •-and other molecules like anti-apoptotic factors, cell cycle regulators, adhesion molecules that are likely to be related to tumorogenesis, cancer cell growth and proliferation. The unbalanced increase of the former may lead to tumorogenesis and (among other events) cancer growth and proliferation.
Another common strategy followed in the formulation of anti-inflammatory agents is based on suppressing of production of inflammatory mediators, such as COX-2 inhibitors, that interfere with the initiation and progression of inflammation-associated diseases [18]. PGs were found in several kinds of tumors, like gastric cancer [19] or colon adenocarcinoma [20]; causing tumorigenic effects, such as stimulation of cell growth and angiogenesis, inhibition of apoptosis, and suppression of the immune system. Several studies also indicate that COX-2 inhibitors can reduce the risk of development of colon, lung, or skin cancer [21][22][23], and namely improve therapeutic effects on human cancers in combination with chemotherapeutic [24].
In practice, the synthetic drugs used to treat these disorders may bring about severe side effects; hence is important to find compounds from biological sources, such as cyanobacteria, lacking adverse effects [25]. Carotenoids and PUFA from microalgal sources have indeed been claimed to have anti-cancer and anti-inflammatory properties, having sometimes an antioxidant-based mechanism of action [26][27][28]. Some of them have even been proposed for the treatment and prevention of such chronic diseases [29,30]. Epidemiological studies suggest that carotenoids can prevent free radical-dependent oxidation of LDL, cholesterol, proteins or DNA, by capturing free radicals and thus reducing stress induced by ROS [31]. Furthermore, PUFA, namely n-3 PUFA, was described to hold antioxidant and anti-inflammatory effects [32][33][34].
In the particular case of cancer, some strategies of chemoprevention can be accomplished by incorporating antioxidant compounds in the diet, which would block or delay ) secrete or degrade inflammatory cytokines in regulating cytokines released by immune cells through a feedback mechanism. Phosphorylation of NF-κB activates several enzymes, e.g., cyclooxygenase(COX-2), oxidase, and iNOS, thus inducing the release of prostaglandins (PGE2), O 2 •− and other molecules like anti-apoptotic factors, cell cycle regulators, adhesion molecules that are likely to be related to tumorogenesis, cancer cell growth and proliferation. The unbalanced increase of the former may lead to tumorogenesis and (among other events) cancer growth and proliferation.
In practice, the synthetic drugs used to treat these disorders may bring about severe side effects; hence is important to find compounds from biological sources, such as cyanobacteria, lacking adverse effects [25]. Carotenoids and PUFA from microalgal sources have indeed been claimed to have anti-cancer and anti-inflammatory properties, having sometimes an antioxidant-based mechanism of action [26][27][28]. Some of them have even been proposed for the treatment and prevention of such chronic diseases [29,30]. Epidemiological studies suggest that carotenoids can prevent free radical-dependent oxidation of LDL, cholesterol, proteins or DNA, by capturing free radicals and thus reducing stress induced by ROS [31]. Furthermore, PUFA, namely n-3 PUFA, was described to hold antioxidant and anti-inflammatory effects [32][33][34].
In the particular case of cancer, some strategies of chemoprevention can be accomplished by incorporating antioxidant compounds in the diet, which would block or delay cancer development, either in the initial phase of carcinogenesis or at the stage of progression of neoplastic cells to cancer [35]. A clear example is β-carotene, which protective effect against cancer was intimately associated with its antioxidant role [2] and COX-2 suppression abilities [36]. Moreover, the potential of microalgal lipidic components as Mar. Drugs 2021, 19, 623 4 of 18 chemopreventive agents was observed in colon, skin, and stomach cancer [2]. Also, other carotenoids such as violaxanthin, zeaxanthin, lutein, and fucoxanthin, or ethanol-based carotenoids-extracts, isolated from microalgae, exhibited antiproliferative activity against different cancer cells [27,35,[37][38][39][40].
For this study, a scarcely studied prokaryotic colonial microalga was selected, Gloeothece sp., with promising bioactive lipidic composition [41]. This study aimed to exploit the bioactive potential of its lipid extracts, as a new source of antioxidant, anti-inflammatory, and antitumor compounds-thus forecasting a possible application in the food and nutraceutical industry. Hence, GRAS (Generally Recognized as Safe) solvents-ethanol, acetone, ethyl lactate, and a mixture (3:2) of hexane/isopropanol, were selected to extract lipidic bioactive compounds from Gloeothece sp. [42,43].

Biochemical Composition of Extracts
Gloeothece sp. extracts may have the potential of application in the nutraceutical industry, due to their content in bioactive compounds as carotenoids, polyunsaturated fatty acids (PUFA), or phenolic compounds. First, a crude characterization of extracts composition in terms of each family of compounds (m C /m E , %) was done, as depicted in Figure 2.
For this study, a scarcely studied prokaryotic colonial microalga was selected, Gloeo thece sp., with promising bioactive lipidic composition [41]. This study aimed to exploi the bioactive potential of its lipid extracts, as a new source of antioxidant, anti-inflamma tory, and antitumor compounds-thus forecasting a possible application in the food and nutraceutical industry. Hence, GRAS (Generally Recognized as Safe) solvents-ethanol acetone, ethyl lactate, and a mixture (3:2) of hexane/isopropanol, were selected to extrac lipidic bioactive compounds from Gloeothece sp. [42,43].

Biochemical Composition of Extracts
Gloeothece sp. extracts may have the potential of application in the nutraceutical in dustry, due to their content in bioactive compounds as carotenoids, polyunsaturated fatty acids (PUFA), or phenolic compounds. First, a crude characterization of extracts compo sition in terms of each family of compounds (mC/mE, %) was done, as depicted in Figure  2.
It can be observed that A and E extracts are mainly composed of fatty acids, ca. 60 and 66%, respectively, most of them PUFA (more than 40%). Extract A also exhibited the highest percent composition in phenolic compounds (13%, mC/mE), followed by HI extrac (ca. 8%, mC/mE). The contents of carotenoids were ca. 4% in all extracts, except for E, which reaches 6.5%. A detailed fatty acids composition, available in Table 1, reveals different profiles in monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids, either in terms o concentration (µgFatty Acid·mgExtract −1 ) and content (%, mFatty Acid/mTotal Fatty Acid).
Concerning the MUFA C18:1 n9 c+t (oleic acid, OA), this is the one present in higher content and the 3rd in terms of all fatty acids. Its content in all extracts ranges between 14.4 (E) and 17.4% (EL), having a higher concentration in extract A, 53.796 ± 2.918 µgFatt Acid·mgExtract −1 -i.e., approximately half of concentration in E, and one quarter in HI and EL Figure 2. Gloeothece sp. extract's composition (m C /m E , %) in terms of against cancer was intimately associated with its antioxidant role [2] and COX-2 suppression abilities [36]. Moreover, the potential of microalgal lipidic components as chemopreventive agents was observed in colon, skin, and stomach cancer [2]. Also, other carotenoids such as violaxanthin, zeaxanthin, lutein, and fucoxanthin, or ethanol-based carotenoids-extracts, isolated from microalgae, exhibited antiproliferative activity against different cancer cells [27,35,[37][38][39][40].
For this study, a scarcely studied prokaryotic colonial microalga was selected, Gloeothece sp., with promising bioactive lipidic composition [41]. This study aimed to exploit the bioactive potential of its lipid extracts, as a new source of antioxidant, anti-inflammatory, and antitumor compounds-thus forecasting a possible application in the food and nutraceutical industry. Hence, GRAS (Generally Recognized as Safe) solvents-ethanol, acetone, ethyl lactate, and a mixture (3:2) of hexane/isopropanol, were selected to extract lipidic bioactive compounds from Gloeothece sp. [42,43].

Biochemical Composition of Extracts
Gloeothece sp. extracts may have the potential of application in the nutraceutical industry, due to their content in bioactive compounds as carotenoids, polyunsaturated fatty acids (PUFA), or phenolic compounds. First, a crude characterization of extracts composition in terms of each family of compounds (mC/mE, %) was done, as depicted in Figure  2.
It can be observed that A and E extracts are mainly composed of fatty acids, ca. 60 and 66%, respectively, most of them PUFA (more than 40%). Extract A also exhibited the highest percent composition in phenolic compounds (13%, mC/mE), followed by HI extract (ca. 8%, mC/mE). The contents of carotenoids were ca. 4% in all extracts, except for E, which reaches 6.5%. A detailed fatty acids composition, available in Table 1, reveals different profiles in monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids, either in terms of concentration (µgFatty Acid·mgExtract −1 ) and content (%, mFatty Acid/mTotal Fatty Acid).
Concerning the MUFA C18:1 n9 c+t (oleic acid, OA), this is the one present in higher content and the 3rd in terms of all fatty acids. Its content in all extracts ranges between 14.4 (E) and 17.4% (EL), having a higher concentration in extract A, 53.796 ± 2.918 µgFatty Acid·mgExtract −1 -i.e., approximately half of concentration in E, and one quarter in HI and EL.
For this study, a scarcely studied prokaryotic colonial microalga was selected, Gloeothece sp., with promising bioactive lipidic composition [41]. This study aimed to exploit the bioactive potential of its lipid extracts, as a new source of antioxidant, anti-inflammatory, and antitumor compounds-thus forecasting a possible application in the food and nutraceutical industry. Hence, GRAS (Generally Recognized as Safe) solvents-ethanol, acetone, ethyl lactate, and a mixture (3:2) of hexane/isopropanol, were selected to extract lipidic bioactive compounds from Gloeothece sp. [42,43].

Biochemical Composition of Extracts
Gloeothece sp. extracts may have the potential of application in the nutraceutical industry, due to their content in bioactive compounds as carotenoids, polyunsaturated fatty acids (PUFA), or phenolic compounds. First, a crude characterization of extracts composition in terms of each family of compounds (mC/mE, %) was done, as depicted in Figure  2.
It can be observed that A and E extracts are mainly composed of fatty acids, ca. 60 and 66%, respectively, most of them PUFA (more than 40%). Extract A also exhibited the highest percent composition in phenolic compounds (13%, mC/mE), followed by HI extract (ca. 8%, mC/mE). The contents of carotenoids were ca. 4% in all extracts, except for E, which reaches 6.5%. A detailed fatty acids composition, available in Table 1, reveals different profiles in monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids, either in terms of concentration (µgFatty Acid·mgExtract −1 ) and content (%, mFatty Acid/mTotal Fatty Acid).
Concerning the MUFA C18:1 n9 c+t (oleic acid, OA), this is the one present in higher content and the 3rd in terms of all fatty acids. Its content in all extracts ranges between 14.4 (E) and 17.4% (EL), having a higher concentration in extract A, 53.796 ± 2.918 µgFatty Acid·mgExtract −1 -i.e., approximately half of concentration in E, and one quarter in HI and EL.
PUFA, xample is β-carotene, which protective effect its antioxidant role [2] and COX-2 suppresicroalgal lipidic components as chemopreand stomach cancer [2]. Also, other carote-, and fucoxanthin, or ethanol-based caroteibited antiproliferative activity against difyotic colonial microalga was selected, Gloeomposition [41]. This study aimed to exploit a new source of antioxidant, anti-inflammasting a possible application in the food and ally Recognized as Safe) solvents-ethanol, exane/isopropanol, were selected to extract p. [42,43]. ential of application in the nutraceutical inounds as carotenoids, polyunsaturated fatty a crude characterization of extracts compo-(mC/mE, %) was done, as depicted in Figure   are mainly composed of fatty acids, ca. 60 ore than 40%). Extract A also exhibited the ounds (13%, mC/mE), followed by HI extract ere ca. 4% in all extracts, except for E, which E, %) in terms of MUFA, PUFA, nidentified compounds, obtained with acetone and ethyl lactate (EL). lable in Table 1, reveals different profiles in ated (PUFA) fatty acids, either in terms of t (%, mFatty Acid/mTotal Fatty Acid). c acid, OA), this is the one present in higher s. Its content in all extracts ranges between entration in extract A, 53.796 ± 2.918 µgFatty entration in E, and one quarter in HI and EL.

Biochemical Composition of Extracts
Gloeothece sp. extracts may have the potential of application in the nutraceutical industry, due to their content in bioactive compounds as carotenoids, polyunsaturated fatty acids (PUFA), or phenolic compounds. First, a crude characterization of extracts composition in terms of each family of compounds (mC/mE, %) was done, as depicted in Figure  2.
It can be observed that A and E extracts are mainly composed of fatty acids, ca. 60 and 66%, respectively, most of them PUFA (more than 40%). Extract A also exhibited the highest percent composition in phenolic compounds (13%, mC/mE), followed by HI extract (ca. 8%, mC/mE). The contents of carotenoids were ca. 4% in all extracts, except for E, which reaches 6.5%. A detailed fatty acids composition, available in Table 1, reveals different profiles in monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids, either in terms of concentration (µgFatty Acid·mgExtract −1 ) and content (%, mFatty Acid/mTotal Fatty Acid).
Concerning the MUFA C18:1 n9 c+t (oleic acid, OA), this is the one present in higher content and the 3rd in terms of all fatty acids. Its content in all extracts ranges between 14.4 (E) and 17.4% (EL), having a higher concentration in extract A, 53.796 ± 2.918 µgFatty Acid·mgExtract −1 -i.e., approximately half of concentration in E, and one quarter in HI and EL. It can be observed that A and E extracts are mainly composed of fatty acids, ca. 60 and 66%, respectively, most of them PUFA (more than 40%). Extract A also exhibited the highest percent composition in phenolic compounds (13%, m C /m E ), followed by HI extract (ca. 8%, m C /m E ). The contents of carotenoids were ca. 4% in all extracts, except for E, which reaches 6.5%.
A detailed fatty acids composition, available in Table 1, reveals different profiles in monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids, either in terms of concentration (µg Fatty Acid ·mg Extract −1 ) and content (%, m Fatty Acid /m Total Fatty Acid ). Concerning the MUFA C18:1 n9 c+t (oleic acid, OA), this is the one present in higher content and the 3rd in terms of all fatty acids. Its content in all extracts ranges between 14.4 (E) and 17.4% (EL), having a higher concentration in extract A, 53.796 ± 2.918 µg Fatty Acid ·mg Extract −1 -i.e., approximately half of concentration in E, and one quarter in HI and EL.

Antioxidant Capacity of Lipidic Extracts
The extracts were tested for their total antioxidant capacity (via ABTS •+ and DPPH • methods), and specific radical antioxidant capacity for radicals O2 •-and • NO.
As observed in Table 2, all extracts exhibited total antioxidant capacity-although in some assays the IC50 values could not be estimated within the range of concentrations tested, such as O2 •-assay.
No significant differences were found between E and A extracts (p < 0.05) in ABTS •+ assay, and A extract exhibited the lowest IC50 in DPPH • and • NO − assays. Although the IC50 for EL extract at • NO − assay could not be calculated in the range of concentrations tested, it was revealed to have antioxidant capacity.

Antitumoral Features of Lipidic Extracts
Among the available cancer adenocarcinoma cell lines, AGS highlights as being the gastric line most used in vitro study models [44]. Hence, antitumor capacities of all extracts were evaluated through different assays, using AGS cell line as a model. First, the cancer cell viability was evaluated by Sulforhodamine B assay, and IC50 was determined for each extract. The IC50 values of each extract were then used to determine whether the extracts were able to promote cell death via TUNEL assay; and whether the extracts were able to inhibit cancer cell proliferation, via cell proliferation BrdU assay.  oma cell lines, AGS highlights as being the [44]. Hence, antitumor capacities of all exs, using AGS cell line as a model. First, the odamine B assay, and IC50 was determined ct were then used to determine whether the UNEL assay; and whether the extracts were l proliferation BrdU assay. and HI, and 69% more than in EL, 3.19 ± 0.22 µgcarot·mgE −1 . Neoxanthin is the second most abundant xanthophyll, with 3.21 ± 0.23 µgcarot·mgE −1 in A, i.e., 1.5-fold that of E, 2.1-fold of HI, and 4.1-fold of EL. Moreover, A is the only extract than contains zeaxanthin 1.07 ± 0.12 µgcarot·mgE −1 , and the highest concentration of α-carotene, i.e., 0.53 ± 0.04 µgcarot·mgE −1 , and β-carotene, i.e., 1.60 ± 0.03 µgcarot·mgE −1 .

Antioxidant Capacity of Lipidic Extracts
The extracts were tested for their total antioxidant capacity (via ABTS •+ and DPPH • methods), and specific radical antioxidant capacity for radicals O2 •-and • NO.
As observed in Table 2, all extracts exhibited total antioxidant capacity-although in some assays the IC50 values could not be estimated within the range of concentrations tested, such as O2 •-assay.
No significant differences were found between E and A extracts (p < 0.05) in ABTS •+ assay, and A extract exhibited the lowest IC50 in DPPH • and • NO − assays. Although the IC50 for EL extract at • NO − assay could not be calculated in the range of concentrations tested, it was revealed to have antioxidant capacity.

Antitumoral Features of Lipidic Extracts
Among the available cancer adenocarcinoma cell lines, AGS highlights as being the gastric line most used in vitro study models [44]. Hence, antitumor capacities of all extracts were evaluated through different assays, using AGS cell line as a model. First, the cancer cell viability was evaluated by Sulforhodamine B assay, and IC50 was determined for each extract. The IC50 values of each extract were then used to determine whether the extracts were able to promote cell death via TUNEL assay; and whether the extracts were able to inhibit cancer cell proliferation, via cell proliferation BrdU assay. and HI, and 69% more than in EL, 3.19 ± 0.22 µgcarot·mgE −1 . Neoxanthin is the second most abundant xanthophyll, with 3.21 ± 0.23 µgcarot·mgE −1 in A, i.e., 1.5-fold that of E, 2.1-fold of HI, and 4.1-fold of EL. Moreover, A is the only extract than contains zeaxanthin 1.07 ± 0.12 µgcarot·mgE −1 , and the highest concentration of α-carotene, i.e., 0.53 ± 0.04 µgcarot·mgE −1 , and β-carotene, i.e., 1.60 ± 0.03 µgcarot·mgE −1 .

Antioxidant Capacity of Lipidic Extracts
The extracts were tested for their total antioxidant capacity (via ABTS •+ and DPPH • methods), and specific radical antioxidant capacity for radicals O2 •-and • NO.
As observed in Table 2, all extracts exhibited total antioxidant capacity-although in some assays the IC50 values could not be estimated within the range of concentrations tested, such as O2 •-assay.
No significant differences were found between E and A extracts (p < 0.05) in ABTS •+ assay, and A extract exhibited the lowest IC50 in DPPH • and • NO − assays. Although the IC50 for EL extract at • NO − assay could not be calculated in the range of concentrations tested, it was revealed to have antioxidant capacity.

Antitumoral Features of Lipidic Extracts
Among the available cancer adenocarcinoma cell lines, AGS highlights as being the gastric line most used in vitro study models [44]. Hence, antitumor capacities of all extracts were evaluated through different assays, using AGS cell line as a model. First, the cancer cell viability was evaluated by Sulforhodamine B assay, and IC50 was determined for each extract. The IC50 values of each extract were then used to determine whether the extracts were able to promote cell death via TUNEL assay; and whether the extracts were able to inhibit cancer cell proliferation, via cell proliferation BrdU assay. and HI, and 69% more than in EL, 3.19 ± 0.22 µgcarot·mgE −1 . Neoxanthin is the second most abundant xanthophyll, with 3.21 ± 0.23 µgcarot·mgE −1 in A, i.e., 1.5-fold that of E, 2.1-fold of HI, and 4.1-fold of EL. Moreover, A is the only extract than contains zeaxanthin 1.07 ± 0.12 µgcarot·mgE −1 , and the highest concentration of α-carotene, i.e., 0.53 ± 0.04 µgcarot·mgE −1 , and β-carotene, i.e., 1.60 ± 0.03 µgcarot·mgE −1 .

Antioxidant Capacity of Lipidic Extracts
The extracts were tested for their total antioxidant capacity (via ABTS •+ and DPPH • methods), and specific radical antioxidant capacity for radicals O2 •-and • NO.
As observed in Table 2, all extracts exhibited total antioxidant capacity-although in some assays the IC50 values could not be estimated within the range of concentrations tested, such as O2 •-assay.
No significant differences were found between E and A extracts (p < 0.05) in ABTS •+ assay, and A extract exhibited the lowest IC50 in DPPH • and • NO − assays. Although the IC50 for EL extract at • NO − assay could not be calculated in the range of concentrations tested, it was revealed to have antioxidant capacity.

Antitumoral Features of Lipidic Extracts
Among the available cancer adenocarcinoma cell lines, AGS highlights as being the gastric line most used in vitro study models [44]. Hence, antitumor capacities of all extracts were evaluated through different assays, using AGS cell line as a model. First, the cancer cell viability was evaluated by Sulforhodamine B assay, and IC50 was determined for each extract. The IC50 values of each extract were then used to determine whether the extracts were able to promote cell death via TUNEL assay; and whether the extracts were able to inhibit cancer cell proliferation, via cell proliferation BrdU assay.

Antioxidant Capacity of Lipidic Extracts
The extracts were tested for their total antioxidant capacity (via ABTS •+ and DPPH • methods), and specific radical antioxidant capacity for radicals O 2 •− and • NO. As observed in Table 2, all extracts exhibited total antioxidant capacity-although in some assays the IC 50 values could not be estimated within the range of concentrations tested, such as O 2 •− assay. No significant differences were found between E and A extracts (p < 0.05) in ABTS •+ assay, and A extract exhibited the lowest IC 50 in DPPH • and • NO − assays. Although the IC 50 for EL extract at • NO − assay could not be calculated in the range of concentrations tested, it was revealed to have antioxidant capacity.

Antitumoral Features of Lipidic Extracts
Among the available cancer adenocarcinoma cell lines, AGS highlights as being the gastric line most used in vitro study models [44]. Hence, antitumor capacities of all extracts were evaluated through different assays, using AGS cell line as a model. First, the cancer cell viability was evaluated by Sulforhodamine B assay, and IC 50 was determined for each extract. The IC 50 values of each extract were then used to determine whether the extracts were able to promote cell death via TUNEL assay; and whether the extracts were able to inhibit cancer cell proliferation, via cell proliferation BrdU assay.

Evaluation of Cancer Cell Viability by Sulforhodamine B Assay
Sulforhodamine B assay (SRB) uses the protein-binding dye SRB to indirectly assess cell growth [45,46]. Despite DMSO being widely described to be cytotoxic depending on its concentrationyet, it was used to suspend extracts at low and non-cytotoxic concentrations. DMSO was thus titrated in these cell lines and, it was found that a concentration of 0.25% (v/v) was innocuous to AGS cells (data not shown).
For each extract, a dose-response curve was established, allowing determination of the extract's concentration causing a cell growth inhibition of 50%, as shown in Table 2.
From the results calculated in Table 2, HI extract outstands for its lowest IC 50 values, reaching values 5-to 10-fold lower when compared to the other extracts. IC50 values determined for each extract were then used to perform the cancer cell death and proliferation assays.

Evaluation of Cancer Cell Death via TUNEL Assay
TUNEL is a common method for detecting DNA fragmentation that may result from cell death, either by apoptosis or necrosis [47]. Induction of DNA fragmentation in AGS cells, treated with the different extracts, at their IC 50 by 48 h of treatment, was examined using TUNEL. The results produced ( Figure 4) show that treatment with all four extracts results in a significantly increased cell death (p < 0.05), yet a stronger effect was observed for HI extract-which increased AGS cells death by c.a. of 4-fold. Sulforhodamine B assay (SRB) uses the protein-binding dye SRB to indirectly assess cell growth [45,46]. Despite DMSO being widely described to be cytotoxic depending on its concentration-yet, it was used to suspend extracts at low and non-cytotoxic concentrations. DMSO was thus titrated in these cell lines and, it was found that a concentration of 0.25% (v/v) was innocuous to AGS cells (data not shown).
For each extract, a dose-response curve was established, allowing determination of the extract's concentration causing a cell growth inhibition of 50%, as shown in Table 2.
From the results calculated in Table 2, HI extract outstands for its lowest IC50 values, reaching values 5-to 10-fold lower when compared to the other extracts. IC50 values determined for each extract were then used to perform the cancer cell death and proliferation assays.

Evaluation of Cancer Cell Death via TUNEL Assay
TUNEL is a common method for detecting DNA fragmentation that may result from cell death, either by apoptosis or necrosis [47]. Induction of DNA fragmentation in AGS cells, treated with the different extracts, at their IC50 by 48 h of treatment, was examined using TUNEL. The results produced ( Figure 4) show that treatment with all four extracts results in a significantly increased cell death (p ˂ 0.05), yet a stronger effect was observed for HI extract-which increased AGS cells death by c.a. of 4-fold.

Evaluation of Cancer Cell Proliferation
Assessment of cell proliferation by BrdU assay is based on the incorporation of BrdU into their replicating DNA, which can further be detected by immunofluorescence. For a quantitative approach, samples were analyzed by flow cytometry. Results revealed an anti-proliferative effect of the HI and EL extracts upon AGS, via 40% of inhibition of proliferation in ca., while cells treated with the E or A extracts behaved no differently from the negative control with DMSO (Figure 4), i.e., exhibited no antiproliferative effect.  Sulforhodamine B assay (SRB) uses the protein-binding dye SRB to indirectly assess cell growth [45,46]. Despite DMSO being widely described to be cytotoxic depending on its concentration-yet, it was used to suspend extracts at low and non-cytotoxic concentrations. DMSO was thus titrated in these cell lines and, it was found that a concentration of 0.25% (v/v) was innocuous to AGS cells (data not shown).
For each extract, a dose-response curve was established, allowing determination of the extract's concentration causing a cell growth inhibition of 50%, as shown in Table 2.
From the results calculated in Table 2, HI extract outstands for its lowest IC50 values, reaching values 5-to 10-fold lower when compared to the other extracts. IC50 values determined for each extract were then used to perform the cancer cell death and proliferation assays.

Evaluation of Cancer Cell Death via TUNEL Assay
TUNEL is a common method for detecting DNA fragmentation that may result from cell death, either by apoptosis or necrosis [47]. Induction of DNA fragmentation in AGS cells, treated with the different extracts, at their IC50 by 48 h of treatment, was examined using TUNEL. The results produced ( Figure 4) show that treatment with all four extracts results in a significantly increased cell death (p ˂ 0.05), yet a stronger effect was observed for HI extract-which increased AGS cells death by c.a. of 4-fold.

Evaluation of Cancer Cell Proliferation
Assessment of cell proliferation by BrdU assay is based on the incorporation of BrdU into their replicating DNA, which can further be detected by immunofluorescence. For a quantitative approach, samples were analyzed by flow cytometry. Results revealed an anti-proliferative effect of the HI and EL extracts upon AGS, via 40% of inhibition of proliferation in ca., while cells treated with the E or A extracts behaved no differently from the negative control with DMSO (Figure 4), i.e., exhibited no antiproliferative effect. Sulforhodamine B assay (SRB) uses the protein-binding dye SRB to indirectly assess cell growth [45,46]. Despite DMSO being widely described to be cytotoxic depending on its concentration-yet, it was used to suspend extracts at low and non-cytotoxic concentrations. DMSO was thus titrated in these cell lines and, it was found that a concentration of 0.25% (v/v) was innocuous to AGS cells (data not shown).

Acetone (A),
For each extract, a dose-response curve was established, allowing determination of the extract's concentration causing a cell growth inhibition of 50%, as shown in Table 2.
From the results calculated in Table 2, HI extract outstands for its lowest IC50 values, reaching values 5-to 10-fold lower when compared to the other extracts. IC50 values determined for each extract were then used to perform the cancer cell death and proliferation assays.

Evaluation of Cancer Cell Death via TUNEL Assay
TUNEL is a common method for detecting DNA fragmentation that may result from cell death, either by apoptosis or necrosis [47]. Induction of DNA fragmentation in AGS cells, treated with the different extracts, at their IC50 by 48 h of treatment, was examined using TUNEL. The results produced ( Figure 4) show that treatment with all four extracts results in a significantly increased cell death (p ˂ 0.05), yet a stronger effect was observed for HI extract-which increased AGS cells death by c.a. of 4-fold.

Evaluation of Cancer Cell Proliferation
Assessment of cell proliferation by BrdU assay is based on the incorporation of BrdU into their replicating DNA, which can further be detected by immunofluorescence. For a quantitative approach, samples were analyzed by flow cytometry. Results revealed an anti-proliferative effect of the HI and EL extracts upon AGS, via 40% of inhibition of proliferation in ca., while cells treated with the E or A extracts behaved no differently from the negative control with DMSO (Figure 4), i.e., exhibited no antiproliferative effect.

Antioxidant Capacity of Lipidic Extracts
The extracts were tested for their total antioxidant capacity (via methods), and specific radical antioxidant capacity for radicals O2 •-an As observed in Table 2, all extracts exhibited total antioxidant ca some assays the IC50 values could not be estimated within the rang tested, such as O2 •-assay. No significant differences were found between E and A extracts assay, and A extract exhibited the lowest IC50 in DPPH • and • NO − a IC50 for EL extract at • NO − assay could not be calculated in the rang tested, it was revealed to have antioxidant capacity.

Evaluation of Cancer Cell Viability by Sulforhodamine B Assay
Sulforhodamine B assay (SRB) uses the protein-binding dye SRB to indirectly assess cell growth [45,46]. Despite DMSO being widely described to be cytotoxic depending on its concentration-yet, it was used to suspend extracts at low and non-cytotoxic concentrations. DMSO was thus titrated in these cell lines and, it was found that a concentration of 0.25% (v/v) was innocuous to AGS cells (data not shown).
For each extract, a dose-response curve was established, allowing determination of the extract's concentration causing a cell growth inhibition of 50%, as shown in Table 2.
From the results calculated in Table 2, HI extract outstands for its lowest IC50 values, reaching values 5-to 10-fold lower when compared to the other extracts. IC50 values determined for each extract were then used to perform the cancer cell death and proliferation assays.

Evaluation of Cancer Cell Death via TUNEL Assay
TUNEL is a common method for detecting DNA fragmentation that may result from cell death, either by apoptosis or necrosis [47]. Induction of DNA fragmentation in AGS cells, treated with the different extracts, at their IC50 by 48 h of treatment, was examined using TUNEL. The results produced ( Figure 4) show that treatment with all four extracts results in a significantly increased cell death (p ˂ 0.05), yet a stronger effect was observed for HI extract-which increased AGS cells death by c.a. of 4-fold.

Evaluation of Cancer Cell Proliferation
Assessment of cell proliferation by BrdU assay is based on the incorporation of BrdU into their replicating DNA, which can further be detected by immunofluorescence. For a quantitative approach, samples were analyzed by flow cytometry. Results revealed an anti-proliferative effect of the HI and EL extracts upon AGS, via 40% of inhibition of proliferation in ca., while cells treated with the E or A extracts behaved no differently from the negative control with DMSO (Figure 4), i.e., exhibited no antiproliferative effect.

Evaluation of Cancer Cell Proliferation
Assessment of cell proliferation by BrdU assay is based on the incorporation of BrdU into their replicating DNA, which can further be detected by immunofluorescence. For a quantitative approach, samples were analyzed by flow cytometry. Results revealed an anti-proliferative effect of the HI and EL extracts upon AGS, via 40% of inhibition of proliferation in ca., while cells treated with the E or A extracts behaved no differently from the negative control with DMSO (Figure 4), i.e., exhibited no antiproliferative effect.

Anti-Inflammatory Potential of Lipidic Extracts
The mechanism of inflammation can be partially triggered via the release of ROS, from activated neutrophils and macrophages, thus leading to damage in macromolecules causing, namely, lipid peroxidation of membranes. ROS spread inflammation by stimulating the release of cytokines, regulated by lysosomes, which in turn stimulate the recruitment of additional neutrophils and macrophages. Lysosome structure conveys a physical and functional interface among cell organelles, as it plays a role in negative or positive modulation of the production of inflammatory cytokines [17,48]. Furthermore, free radicals are mediators that induce or sustain inflammatory processes; hence their neutralization by antioxidants and radical scavengers are fundamental to reducing inflammation [49]. In this context, extracts from Gloeothece sp. were screened for their potential anti-inflammatory features, by resorting to two different assays, one reflecting the stabilization of extracts on Human red blood cell (HRBC) membrane induced by heat, and another that ascertains the capacity of such extracts to inhibit the human enzyme COX-2.

Human Red Blood Cell (HRBC) Membrane Stabilization Assay
This assay allows the characterization of the capacity of Gloeothece sp. extracts to protect erythrocytes from hemolysis when heat is supplied. Since the erythrocyte membrane is quite similar to the lysosomal one, indirectly is possible to conclude if any Gloeothece sp. extract holds any capacity in the stabilization of lysosomal membranes [50], and so, if they have the potential to be used as a non-steroidal drug-the common anti-inflammatory drug that inhibits lysosomal enzymes or stabilizes their membrane.
Results show that the HI 3:2 (v/v) extract is the most promising as it exhibits a protection capacity of 61.6 ± 9.6%; nonetheless, EL extract also appears to hold some potential in protecting HRBC membranes. Conversely, the E and A extracts did not show significant protective capacity (see Table 3). Table 3. Anti-inflammatory potential of Gloeothece sp. lipidic extracts, upon the protection of HRBC membranes (average ± standard deviation) from heat, expressed in percentage of stabilization and IC50 (average ± standard deviation) values of extracts obtained at of COX-2 enzymatic activity inhibition.

Cox Human Inhibitory Assay
Cyclooxygenases (COXs) catalyze reactions that lead to the formation of pro-inflammatory prostaglandins (PG), thromboxanes, and prostacyclins. Hence, the ability of extracts to inhibit the conversion of AA to Prostaglandin H2 (PGH2) via inhibition of COX-2 was determined. All concentrations tested exhibit anti-inflammatory activity in vitro, by inhibiting PG production in a dose-dependent manner. However, the extracts exhibited different behaviors within the range of concentrations tested, data not shown.
While A and EL at lower extract concentration induces a higher inhibition, a linear percent of inhibition is of E concentration was observed, whereas a non-significantly percentage of inhibition variation was detected with HI concentration. In terms of total inhibition capacity of COX-2 enzymatic activity, one notices that A, E, and HI performed equally well beyond 50% with no significant differences between them (p < 0.05); however, the corresponding IC50 values (see Table 3) revealed that A and HI extracts attained the lowest values, without significant differences (p < 0.05).

Cytotoxicity
For a putative application of Gloeothece sp. extracts as a nutraceutical ingredient, it is mandatory that extracts do not exhibit any cytotoxicity to non-cancer cells. Therefore, cytotoxicity effects upon HCMEC cells were assessed after 24 h (see Figure 5A) and 48 h (see Figure 5B), using DMSO 1% as a negative control. Results show that A extract is cytotoxic, although its cytotoxicity decreases after 48 h. However, promising results were observed concerning the E extract, since there was no evidence of cytotoxicity at all concentrations tested. On the other hand, both HI and EL extracts were not lethal up to 100 µg·mL −1 ; the highest concentrations tested were toxic, although toxicity decreases with time.
totoxicity effects upon HCMEC cells were assessed after 24 h (see Figure 5A) and 48 h (see Figure 5B), using DMSO 1% as a negative control. Results show that A extract is cytotoxic, although its cytotoxicity decreases after 48 h. However, promising results were observed concerning the E extract, since there was no evidence of cytotoxicity at all concentrations tested. On the other hand, both HI and EL extracts were not lethal up to 100 µg·mL −1 ; the highest concentrations tested were toxic, although toxicity decreases with time.

Discussion
Drugs commonly used to treat inflammation and cancer raise severe side effects, such as toxicity and decreased life quality [51,52]. In this regard, this work aimed at making a preliminary test of Gloeothece sp. extracts to be eventually used as a natural source in nutraceuticals, and/or as a potential chemopreventive agent-based on the composition in carotenoids and PUFA, coupled with antioxidant, antitumoral, and anti-inflammatory features. Pearson correlations were calculated (data not shown) between composition (carotenoids and PUFA) and bioactive features, however possible synergetic effects among the molecules, that were not possible to measure, may contribute to its bioactive potential. Hence, these features will be discussed separately, and then in an integrated manner.

Antioxidant Capacity of Lipidic Crude Extracts
The antioxidant capacity of cyanobacterial carotenoids is well established-particularly concerning lutein and β-carotene [27,29,30,53], and long-chain fatty acids such ω3 PUFA [30,32]. Analyzing the extract contents in PUFA (see Table 1), carotenoids (see Figure 2) and, it results of total antioxidant capacity, it is possible to correlate extract concentration of carotenoids and PUFA with antioxidant bioactivity-at which A extract, stands out due to its lowest IC50 values at all antioxidant assays. As observed previously, lutein probably contributes the most to said bioactivity, owing to its higher concentration [54]. However, other carotenoids (e.g., β-carotene and neoxanthin) should not be overlooked owing to their concentrations, as well as such PUFA as 18:1 n9, 18:2 n6, and 18:3 n3 based on the IC50 values of Gloeothece sp. extracts (A > E > HI > EL). Particularly, a correlation was found with C18:2 n6 (r = 1, p < 0.083).
Concerning the specific radical's scavenger capacity, results reveal the same trend, particularly in NO • assay, in which the lowest IC50 was again observed in the A extract. The high concentration of total carotenoids and PUFA, namely lutein and C18:2 n6, may account for their important antioxidant role (r = 1, p < 0.083), as reported before [55][56][57].  ipidic Extracts ancer adenocarcinoma cell lines, AGS highlights as being the itro study models [44]. Hence, antitumor capacities of all exgh different assays, using AGS cell line as a model. First, the aluated by Sulforhodamine B assay, and IC50 was determined lues of each extract were then used to determine whether the te cell death via TUNEL assay; and whether the extracts were roliferation, via cell proliferation BrdU assay.

50,
ative application of Gloeothece sp. extracts as a nutraceutical ingredient, it is at extracts do not exhibit any cytotoxicity to non-cancer cells. Therefore, cycts upon HCMEC cells were assessed after 24 h (see Figure 5A) and 48 h (see ing DMSO 1% as a negative control. Results show that A extract is cytotoxic, ytotoxicity decreases after 48 h. However, promising results were observed e E extract, since there was no evidence of cytotoxicity at all concentrations other hand, both HI and EL extracts were not lethal up to 100 µg·mL −1 ; the ntrations tested were toxic, although toxicity decreases with time. mmonly used to treat inflammation and cancer raise severe side effects, such decreased life quality [51,52]. In this regard, this work aimed at making a est of Gloeothece sp. extracts to be eventually used as a natural source in , and/or as a potential chemopreventive agent-based on the composition and PUFA, coupled with antioxidant, antitumoral, and anti-inflammatory son correlations were calculated (data not shown) between composition (ca-PUFA) and bioactive features, however possible synergetic effects among , that were not possible to measure, may contribute to its bioactive potential. eatures will be discussed separately, and then in an integrated manner.

100,
ative application of Gloeothece sp. extracts as a nutraceutical ingredient, it is at extracts do not exhibit any cytotoxicity to non-cancer cells. Therefore, cycts upon HCMEC cells were assessed after 24 h (see Figure 5A) and 48 h (see ing DMSO 1% as a negative control. Results show that A extract is cytotoxic, ytotoxicity decreases after 48 h. However, promising results were observed e E extract, since there was no evidence of cytotoxicity at all concentrations other hand, both HI and EL extracts were not lethal up to 100 µg·mL −1 ; the ntrations tested were toxic, although toxicity decreases with time. mmonly used to treat inflammation and cancer raise severe side effects, such d decreased life quality [51,52]. In this regard, this work aimed at making a est of Gloeothece sp. extracts to be eventually used as a natural source in , and/or as a potential chemopreventive agent-based on the composition s and PUFA, coupled with antioxidant, antitumoral, and anti-inflammatory son correlations were calculated (data not shown) between composition (ca-PUFA) and bioactive features, however possible synergetic effects among , that were not possible to measure, may contribute to its bioactive potential. features will be discussed separately, and then in an integrated manner.

and
For a putative application of Gloeothece sp. extracts as a mandatory that extracts do not exhibit any cytotoxicity to no totoxicity effects upon HCMEC cells were assessed after 24 h Figure 5B), using DMSO 1% as a negative control. Results sh although its cytotoxicity decreases after 48 h. However, prom concerning the E extract, since there was no evidence of cyto tested. On the other hand, both HI and EL extracts were not highest concentrations tested were toxic, although toxicity d

Discussion
Drugs commonly used to treat inflammation and cancer as toxicity and decreased life quality [51,52]. In this regard, preliminary test of Gloeothece sp. extracts to be eventually nutraceuticals, and/or as a potential chemopreventive agen in carotenoids and PUFA, coupled with antioxidant, antitum features. Pearson correlations were calculated (data not show rotenoids and PUFA) and bioactive features, however poss the molecules, that were not possible to measure, may contri Hence, these features will be discussed separately, and then

Antioxidant Capacity of Lipidic Crude Extracts
The antioxidant capacity of cyanobacterial carotenoids larly concerning lutein and β-carotene [27,29,30,53], and lo PUFA [30,32]. Analyzing the extract contents in PUFA (see T ure 2) and, it results of total antioxidant capacity, it is possib tration of carotenoids and PUFA with antioxidant bioactivity out due to its lowest IC50 values at all antioxidant assays. A probably contributes the most to said bioactivity, owing to However, other carotenoids (e.g., β-carotene and neoxanthi owing to their concentrations, as well as such PUFA as 18:1 n on the IC50 values of Gloeothece sp. extracts (A > E > HI > E was found with C18:2 n6 (r = 1, p < 0.083).
Concerning the specific radical's scavenger capacity, r particularly in NO • assay, in which the lowest IC50 was aga The high concentration of total carotenoids and PUFA, nam account for their important antioxidant role (r = 1, p < 0.083), 300 µg·mL −1 , by 24 h (A) and 48 h (B). Bars marked with the same letter in the same superscript have no significant difference relative to the control (p < 0.05).

Discussion
Drugs commonly used to treat inflammation and cancer raise severe side effects, such as toxicity and decreased life quality [51,52]. In this regard, this work aimed at making a preliminary test of Gloeothece sp. extracts to be eventually used as a natural source in nutraceuticals, and/or as a potential chemopreventive agent-based on the composition in carotenoids and PUFA, coupled with antioxidant, antitumoral, and anti-inflammatory features. Pearson correlations were calculated (data not shown) between composition (carotenoids and PUFA) and bioactive features, however possible synergetic effects among the molecules, that were not possible to measure, may contribute to its bioactive potential. Hence, these features will be discussed separately, and then in an integrated manner.

Antioxidant Capacity of Lipidic Crude Extracts
The antioxidant capacity of cyanobacterial carotenoids is well established-particularly concerning lutein and β-carotene [27,29,30,53], and long-chain fatty acids such ω3 PUFA [30,32]. Analyzing the extract contents in PUFA (see Table 1), carotenoids (see Figure 2) and, it results of total antioxidant capacity, it is possible to correlate extract concentration of carotenoids and PUFA with antioxidant bioactivity-at which A extract, stands out due to its lowest IC 50 values at all antioxidant assays. As observed previously, lutein probably contributes the most to said bioactivity, owing to its higher concentration [54]. However, other carotenoids (e.g., β-carotene and neoxanthin) should not be overlooked owing to their concentrations, as well as such PUFA as 18:1 n9, 18:2 n6, and 18:3 n3 based on the IC 50 values of Gloeothece sp. extracts (A > E > HI > EL). Particularly, a correlation was found with C18:2 n6 (r = 1, p < 0.083).
Concerning the specific radical's scavenger capacity, results reveal the same trend, particularly in NO • assay, in which the lowest IC 50 was again observed in the A extract. The high concentration of total carotenoids and PUFA, namely lutein and C18:2 n6, may account for their important antioxidant role (r = 1, p < 0.083), as reported before [55][56][57].
Although the IC 50 values for the O 2 •− assay could not be found at the tested concentrations, some scavenging effects were detected at E and EL extracts-data not shown.
Hence, owing to the antioxidant scavenging capacity of A and E extracts against NO • and O 2 •− radicals in vitro, a similar capacity is expected in vivo-with a preventive role of chronic inflammatory diseases, cancer, or neurodegenerative disorders [58,59].

Antitumoral Features of Cyanobacterial Extracts
Unlike observed with antioxidant capacity, the most promising extracts, in terms of inducing AGS cell death and cell proliferation, are HI and EL extracts; where it cannot be established a clear correlation of antitumor capacity and high content in carotenoids and fatty acids.
Despite a possible interaction of all extracts' compounds, some evidence relate such bioactivities with some compounds identified in Gloeothece sp. extracts, such as phenolic compounds. Although these compounds have not been characterized, the content in aromatic compounds is described to exert effects in bioactivities, particularly in antitumor and anti-inflammatory agents [60].
From a nutraceutical point of view, dietary supplementation of β-carotene in animal models of colon carcinogenesis has revealed anticancer capacities for that compound [61], as well as growth-inhibitory and pro-apoptotic effects in human colon cancer cell lines [36]. It has also been demonstrated that such chemopreventive activity is dose-dependent, a high dose proving to be harmful and likely to have a proliferative effect upon some cancer cells lines [1]; this may explain why the HI and EL extracts, characterized by the lowest levels of β-carotene and lowest IC50 values, exhibited the best results upon cancer cell death and proliferation. Additionally, such xanthophylls, violaxanthin have been found to possess antiproliferative activity against different cancer cells [35], and in fact, HI extract exhibited the highest level of violaxanthin.
Some PUFA, particularly ω-3, have been reported to possess in vitro and in vivo anticancer effects, via modulation of tumor growth or increase of cell death rate [62,63], this is the particular case of EPA, able to inhibit some cancer cell lines proliferation in a dose-dependent and time-dependent manner [62]. However, particular attention should go to LA. Studies reveal that treatment of AGS and MKN cells with linoleic acid (C18:2n6), in which EL extract has the higher content, led to an increase in a proapoptotic protein expression and a decrease of an anti-apoptotic protein expression, as well as inhibits the production of PGE2 and activity of telomerase by suppressing COX-2 and hTERT expression, in a dose-dependent manner [64,65], which may be in line with our results in AGS cell death. Indeed, in our study, a correlation was found between cell death and C18:2n6 content (r = 1, p < 0.083).

Anti-Inflammatory Potential of Lipidic Crude Extracts
The anti-inflammatory potential of Gloeothece sp. extracts was assessed by two assays. In the HRBC assay, HI extracts stood out in terms of inhibition capacity of 61%; hence, this HI extract may potentially stabilize cell membrane and thus prevent stress-induced decay, as well as stabilize the lysosomal membrane. This feature is crucial in the prevention of an anti-inflammatory response induced by the release of lysosomal constituents, which cause further tissue inflammation and damage upon extracellular release [50].
As seen before, the ability to inactivate COX-2 is indicative of the potential of an extract to be used as an anti-inflammatory drug. All extracts of Gloeothece sp. exhibited that ability, some of them having a dose-dependent response, like E extracts. However, extract A exhibited the best performance at a concentration of 75 µg·mL −1 , inhibiting in ca. 57% of COX-2 enzymatic activity; however, the possible application of A extracts use must be discarded due to its cytotoxicity to HCMEC cells. Nonetheless, HI extract follows as most promising due to ca. 48% of inactivation capacity and with no cytotoxicity associated.
A number of anti-inflammatory molecules obtained from microalgae have been shown to display high antioxidant capacity, that is in the composition of A and HI, such as βcarotene, lutein, zeaxanthin, and ω3 PUFA [66]. Some of the anti-inflammatory ability could be attributed to violaxanthin. This xanthophyll isolated from C. ellipsoidea showed anti-inflammatory activity when it was tested on LPS-stimulated RAW 264.7 mouse macrophages, by inhibiting NF-κB activation and NO and prostaglandin E2 (PGE2) production [67].

Potential of Application of Gloeothece sp. Extracts
Chemoprevention consists of the use of pharmaceutical drugs, or nutritional supplements to reduce the risk of developing or having a recurrence of cancer. Several in vitro and animal studies showed the chemopreventive properties of a few metabolites from microalgae (e.g., carotenoids, fatty acids, polysaccharides, and proteins), namely against colon and skin cancer [2].
Performance recorded for Gloeothece sp. extracts, particularly the A and HI shows that they are a promising source in the eventual formulation of some nutraceutical products bearing antioxidant, anticancer, and anti-inflammatory capacities. But despite the notable antioxidant features of the A extract, particularly its ability to inhibit the radical NO • , its potential application as a nutraceutical is limited due to its cytotoxicity.
Experimental and epidemiological evidence reported before suggests that antiinflammatory drugs may also decrease the incidence of some types of cancer, as well as tumor burden and volume [68,69]. An attempt to provide a global overview of the potential of action of HI and A extracts is conveyed by Figure 6.
as β-carotene, lutein, zeaxanthin, and ω3 PUFA [66]. Some of the anti-inflammatory abil could be attributed to violaxanthin. This xanthophyll isolated from C. ellipsoidea show anti-inflammatory activity when it was tested on LPS-stimulated RAW 264.7 mouse ma rophages, by inhibiting NF-κB activation and NO and prostaglandin E2 (PGE2) produ tion [67].

Potential of Application of Gloeothece sp. Extracts
Chemoprevention consists of the use of pharmaceutical drugs, or nutritional supp ments to reduce the risk of developing or having a recurrence of cancer. Several in vit and animal studies showed the chemopreventive properties of a few metabolites fro microalgae (e.g., carotenoids, fatty acids, polysaccharides, and proteins), namely again colon and skin cancer [2].
Performance recorded for Gloeothece sp. extracts, particularly the A and HI show that they are a promising source in the eventual formulation of some nutraceutical pro ucts bearing antioxidant, anticancer, and anti-inflammatory capacities. But despite the n table antioxidant features of the A extract, particularly its ability to inhibit the radical NO its potential application as a nutraceutical is limited due to its cytotoxicity.
Experimental and epidemiological evidence reported before suggests that anti-i flammatory drugs may also decrease the incidence of some types of cancer, as well tumor burden and volume [68,69]. An attempt to provide a global overview of the pote tial of action of HI and A extracts is conveyed by Figure 6.  . Schematic representation of how the HI (red cross) and A (yellow cross) extracts may modulate oxidative stress, inflammation, and cancer development. The HI extract protects membranes of secretory lysosomes, thus avoiding the release of inflammatory cytokines and consequent feedback mechanism. The phosphorylation of NF-κB is activated. A is able to reduce the produced NO radicals. HI and A are able to suppress cyclooxygenase (COX-2), and subsequent release of prostaglandins (PGE2), as well as anti-apoptotic factors, cell cycle regulators, adhesion molecules related to tumorogenesis, cancer cell growth, and proliferation. HI extract is able to inhibit cancer-related events such as cancer growth and proliferation.
Hence, the HI extracts of Gloethece sp. appeared to be the most promising as a chemopreventive agent in the nutraceutical industry because of their features as (1) antioxidant namely high total antioxidant capacity and scavenging capacity against -NO • radical; (2) antitumor induction of cell death upon AGS cells, along with anti-proliferative effects; and (3) anti-inflammatory, namely inability to inhibit COX-2 expression while protecting lysosomes.

Microorganism Source and Biomass Production
Gloeothece sp. (ATCC 27152) was purchased from ATCC-American Type Culture Collection (USA), and kept at 25 • C, using Blue Green (BG11) as culture medium [70]. For biomass production, in 4 L batch culture, first, a pre-inoculum, with an initial optical density of 0.1 at 680 nm, was cultivated for 10 days in 800 mL of BG11 medium, buffered at pH 8 with Tri-(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl)-ensuring that the microorganism was at the exponential growth phase at the time of inoculation for biomass production. Hence, biomass production was started with an initial optical density of 0.1 in BG11 medium buffered at pH 8 and was produced for 14 days under a continuous illumination with fluorescent BlOLUX lamps, with an intensity of 150 µmol photon ·m −2 ·s −1 , and air bubbling at a flow rate of 0.5 L· min −1 . Biomass was then collected by centrifugation at 18× g for 10 min, the supernatant was rejected and pellet freeze-dried, and stored under gaseous nitrogen until analyses were performed.

Chemical Characterization of Extracts
Fatty acids and carotenoids are among the most widely known bioactive compounds found in microalgae, which possess a high interest in the nutraceutical and pharmaceutical markets; hence, solvent extracts were evaporated and residue composition was determined for each Gloeothece sp. extract, as detailed below.

Profile and Content of Polyunsaturated Fatty Acids
The weighted residue was submitted to direct transesterification to produce fatty acid methyl esters according to the acidic method described by Lepage and Roy [71], after modifications introduced by Cohen et al. [72] using acetyl chloride (Sigma-Aldrich, St. Louis, MO, USA) as catalyst. The internal standard used was heptadecanoic (C17:0, Sigma-Aldrich, St. Louis, MO, USA) acid and esters were analyzed in a Varian Chrompack CP-3800 gas chromatograph (GC), using a flame ionization detector, and quantified with the software Varian Star Chromatography Workstation (USA, Version 5.50). Helium was employed as the carrier gas in splitless mode and the silica CP-WAX 52 CB (Agilent) column was used. The injector and detector were maintained at 260 and 280 • C, respectively, and the oven heating program was the same as described before [42]. To identify PUFA, chromatographic grade standards of fatty acids were used in methyl ester form CRM47885 (Supelco, St. Louis, MO, USA). Concentrations of each polyunsaturated fatty acid (PUFAs) were determined and mean values were used as a datum point.

Antioxidant Effects of Lipidic Extracts
The antioxidant capacity of each extract was evaluated via four spectrophotometric assays: two assessed total antioxidant capacity (ABTS +• , DPPH • ); while the other two were more specific for two biological radicals, superoxide (O 2 •− ) and nitric oxide ( • NO − )-with the later be known to be correlated with inflammation processes.
A positive control, Trolox, was used to validate the antioxidant capacity of extracts and putatively establish a calibration curve but comparing the antioxidant capacity of the extracts, their IC 50 values were established. A dilution series was accordingly prepared for each extract, with concentrations ranging from 0.440 to 7 mg·mL −1 -for ethanol, acetone, and HI extracts, and from 1.5 to 24 mg·mL −1 for ethyl lactate extract, in Phosphate Buffered Saline (PBS) containing 5% of DMSO. Each antioxidant assay was performed in triplicate, as described in the following sub-sections.

DPPH • Scavenging Capacity
The antioxidant capacity was determined, in triplicate, by reacting each extract with 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) (Sigma-Aldrich (St. Louis, MO, USA), after an incubation period of 30 min at room temperature in dark. The scavenging reaction was monitored at 515 nm, as implemented before by Amaro et al. [41].

Superoxide Radical (O 2
•− ) Scavenging Capacity Superoxide radicals are generated by the NADH/PMS system. The extract antioxidant capacity was determined by monitoring the absorbance of the reaction mixture, at 560 nm and room temperature, for 2 min, as previously performed by Amaro et al. [41].

Nitric Oxide Radical ( • NO − ) Scavenging Capacity
Each extract was incubated with sodium nitroprusside, for 60 min at room temperature, in the light. Griess reagent was added afterward, and the chromophore reaction was carried out in the dark for 10 min; absorbance was read at 562 nm [41]. AGS cells in a concentration of 1 × 10 4 were seeded in 96-wells plates and treated for 48 h with different concentrations of microalgal extracts (0 to 550 µg·mL −1 whenever possible) or DMSO (AppliChem, Darmstadt, Germany) as negative treatment control (0.05% v/v). As a positive control, DMSO 100%, was used to validate the antitumoral capacity of extracts. Then cells were fixed by the addition of 50 µL of cold 50% trichloroacetic acid (Merck Millipore, Kenilworth, NJ, USA) to each well, and incubating the plates at 4 • C for 1 h. Next the fixation step, the plates were washed three times with deionized water and dried at room temperature. The cells were then stained with 50 µL of 4% sulforhodamine B (SRB) (Sigma-Aldrich, St. Louis, MO, USA) in 1% acetic acid (Mallinckrodt Baker, Deventer, The Netherlands) for 30 min and then washed three times with deionized water. After the plates were dry, the cells were solubilized with 100 µL of 10 mM unbuffered Tris Base (Sigma-Aldrich, St. Louis, MO, USA), and the optical density at 510 nm was measured using the fluorimeter SynergyTM 4 Multi-Mode Microplate Reader (Biotek, Winooski, VT, USA). Results were plotted as dose-response curves, and the IC 50 for each extract was found and expressed as µg E ·mL −1 .

Cancer Cell Death TUNEL Assay
AGS cells were cultured in 6-well plates in a concentration of 7.5 × 10 5 , and treated for 48 h with the microalgal extracts at the IC 50 found at the SRB assay, for 48 h. DMSO (AppliChem, Darmstadt, Germany) was used as a positive control treatment. Cells were washed and trypsinized and the pellet obtained was fixed in 3 mL of ice-cold methanol for 15 min. Then, cells were washed and resuspended in 500 µL of PBS. Incubation with TUNEL reaction mix (1:9:10 concerning the Dilution Buffer reagent, according to manufacture instructions-In Situ Cell Death Detection Kit Fluorescein, Roche, Mannheim, Germany) was done for 1 h, at 37 • C, in the dark. Then, data were acquired using a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

Cancer Proliferative Assay
AGS cells were cultured in 6-well plates containing a concentration of 7.5 × 10 5 and treated with the extracts at the IC 50 found at the SRB assay, for 48 h, using DMSO (AppliChem, Darmstadt, Germany) as positive control treatment. 5-Bromo-2 -deoxyuridine (BrdU) (BrdU labeling and detection kit 1, Roche, Mannheim, Germany) was incorporated in the cell culture medium at the ratio of 1:1000, and underwent incubation for 1 h, at 37 • C. Straightaway the following incubation, the cells were harvested, washed with PBS, fixed in 1 mL of ice-cold methanol for 30 min, washed again, and resuspended in 500 µL of PBS. This was followed by the incubation with 1 mL of HCl 4 M (Mallinckrodt Baker, Deventer, The Netherlands), for 20 min, two washing steps with PBS, a blocking step (PBS containing 0.5% Tween 20 and 0.05% BSA), and finally 1 h incubation at room temperature with the primary antibody against BrdU (1:20, Bu20a, Dako, Glostrup, Denmark). Next, the cells were further washed with PBS and incubated with the secondary antibody labeled with FITC (1:200, polyclonal rabbit anti-mouse, Dako, Glostrup, Denmark), for 30 min at room temperature washed two times and resuspended in 500 µL of PBS. Data acquisition was performed with a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

Anti-Inflammatory Effects of Extracts
To assess the anti-inflammatory potential of the lipidic extracts, two assays were performed. The Human red blood cell (HRBC) membrane stabilization assay, induced by heat, was used first; it allowed to observe if any extract holds the potential to stabilize lysosomal membranes. The second assay is specific to a prostaglandin-endoperoxide synthase, human COX-2 enzymatic activity inhibition-and helps conclusion on whether any extract has the potential to be used as a non-steroidal anti-inflammatory agent. The study was conducted according to the guidelines of the Declaration of Helsinki, and ap-proved by the Institutional Ethics Committee of CIIMAR (protocol code 001/2020 and date of approval 8 June 2020).

Human Red Blood Cell (HRBC) Membrane Stabilization Assay
Human fresh blood was collected intravenously to heparinized tubes, from a healthy volunteer that was not taking any non-steroidal anti-inflammatory drugs (NSAIDs) for 2 weeks before the experiment. Blood was centrifuged at 700× g for 10 min and supernatant (plasma) was removed. Hence human red blood cells (HRBC) were washed three times with an equal volume of isotonic PBS (10 mM sodium phosphate buffer(Alfa Aesar, Massachusetts, US) pH 7.4) and then reconstituted at 40% (v/v) suspension. Salicylic acid at 500 µg mL −1 was used for positive control and PBS with 20% of DMSO (AppliChem, Darmstadt, Germany) for negative control.
Each extract, prepared as explained in Section 2.2, at concentrations of 130, 150, 120, and 450 mg·mL −1 , for A, E, HI, and EL, respectively, were resuspended in PBS containing 20% of DMSO, and then mixed in 1:1 (v/v) with a solution of HRBC in 2% in PBS. Samples were incubated at 56 • C for 20 min, cooled in tap water, and centrifuged at 700× g for 5 min, and the supernatant was collected. The absorbance of the supernatant was measured spectrophotometrically at 560 nm using a microplate reader (Thermofisher GO, New Hampshire, EUA) [73]. The percentage of inhibition was calculated for each extract as: % inhibition = [(Abs E − Abs EB ) − Abs C ]/Abs C × 100 (1) where Abs E denotes supernatant absorbance after reaction with extract; Abs EB denotes extract absorbance at 560 nm; and Abs C denotes the control absorbance of PBS with 20% of DMSO.

Cox Human Inhibitory Screening Assay
The anti-inflammatory potential of the extracts was assessed via an enzyme inhibitory assay-inhibition of COX-2 enzymatic activity, using the COX-2 Enzyme Activity Assay Kit (Cayman Chemical, Michigan, MI, US), according to the manufacturer's instructions. Dried lipidic extracts were diluted in DMSO, and assayed at different concentrations-75, 125, and 250 µg·mL −1 .
In this assay, arachidonic acid (AA) served as a substrate for the human recombinant COX-2 enzyme, thus leading to the production of prostaglandin. The assay measures PGF2α produced by SnCl2 reduction of COX-derived PGH2. The PGF2α levels produced in the presence versus absence of test products were quantified through an enzyme immunoassay-using an antibody that binds to all major prostaglandin compounds, results are expressed in percent of inhibition, calculated according to kit instructions.

Cytotoxicity Evaluation
Cytotoxicity of the extracts was evaluated by measuring the viability of Human Cardiac Microvascular Endothelial Cells (HCMEC) obtained from the American Type Culture Collection (ATCC). Cells were seeded in a 96-well plate with a final concentration of 10 × 10 4 cells mL −1 with Dulbecco's Modified Eagle Medium (DMEM) (Sigma-Aldrich (St. Louis, MO, USA) for 24 h.
The cellular viability was assessed by the mitochondrial-dependent reduction of 3-(4,5dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich (St. Louis, MO, USA) to formazan, quantified by optical density measurement at 510 nm, as described by Lopes et al. [74]. Several concentrations of the extracts were tested: 50, 100, 200, and 300 µg·mL −1 -using DMSO 1% as negative control and DMSO 20% as the positive control. The assay was independently repeated four times, with duplicate extracts. Cytotoxicity was expressed as a percentage of cell viability, considering the values of the negative control as 100% viability. Funding: This research was supported by national funds through FCT-Foundation for Science and Technology within the scope of UIDB/04423/2020, granted to CIIMAR and UIDB/00511/2020 granted to LEPABE funded by national funds through FCT/MCTES (PIDDAC).

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of CIIMAR (protocol code 001/2020 and date of approval 8 June 2020).