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<mods:title id="2E4AC1F10B2D0C2B21839E9BEFD75A9D">Effects of Cultivation Stress on the Glaucophyte Cyanophora paradoxa and Bioactive Potential in Human Cancer Cell Lines</mods:title>
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<mods:namePart id="2316B02B9D2594E309FBB8B750F034CE">Barone, Maria Elena</mods:namePart>
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The growth of
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<emphasis id="D7CEEA95DB0AA634FC14458FD08CFD0E" box="[1002,1244,648,672]" italics="true" pageId="4" pageNumber="281">Cyanophora paradoxa</emphasis>
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<paragraph id="E5053687DB0AA635FB19458FD6EFFF48" blockId="4.[829,1501,648,865]" lastBlockId="5.[125,797,142,230]" lastPageId="5" lastPageNumber="282" pageId="4" pageNumber="281">
under various cultivation conditions including control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM sodium chloride (NaCl) was monitored (
<figureCitation id="7D812A02DB0AA634FC0F45EFD07FFCAF" box="[1009,1071,744,769]" captionStart="Figs 1–4" captionStartId="4.[125,160,1880,1900]" captionTargetBox="[133,1497,927,1846]" captionTargetId="figure-428@4.[125,1501,923,1847]" captionTargetPageId="4" captionText="Figs 1–4. Growth (cells ml−1) and pigment production of Cyanophora paradoxa in various cultivation conditions: control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM of NaCl. Fig. 1. Growth (cells ml−1) of Cyanophora paradoxa for ~27 days. All data are mean ± SD (n = 3). Fig. 2. HPLC-UV chromatogram representation of identified pigments. Fig. 3. Pigment content (mg g−1) of Cyanophora paradoxa. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12). Fig. 4. Phycocyanin, allophycocyanin and phycoerythrin content (mg g−1) of Cyanophora paradoxa. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12)." figureDoi="http://doi.org/10.5281/zenodo.15520497" httpUri="https://zenodo.org/record/15520497/files/figure.png" pageId="4" pageNumber="281">Fig. 1</figureCitation>
). The BLU (0.03 d
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± 0.03) and NaCl (0.08 d
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± 0.03) treatments returned significantly lower specific growth rates as compared to the CONT (0.16 d
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± 0.01) and N- (0.14 d
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± 0.02) treatments (ANOVA,
<emphasis id="D7CEEA95DB0AA634FABD444ED100FCCE" box="[1347,1360,841,864]" italics="true" pageId="4" pageNumber="281">p</emphasis>
&lt;0.001, n = 12) (
<tableCitation id="A838033CDB0BA635FF4E4789D541FF08" box="[176,273,142,166]" captionStart="Table 1" captionStartId="5.[330,377,1718,1738]" captionTargetPageId="5" captionText="Table 1. Pigment and phycobiliprotein content (mg g−1) in the biomass of Cyanophora paradoxa strain CCAP 981/1" pageId="5" pageNumber="282">Table S1</tableCitation>
). Varying amounts of dried biomass were then recovered, following centrifugation, from the cultures (CONT
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, BLU
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, N-
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and NaCl
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).
</paragraph>
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<emphasis id="D7CEEA95DB0AA634FF83405FD49AF8C2" bold="true" box="[125,202,1880,1900]" pageId="4" pageNumber="281">Figs 1–4.</emphasis>
Growth (cells ml
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) and pigment production of
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<emphasis id="D7CEEA95DB0AA634FD9E405FD744F8C2" box="[608,788,1880,1900]" italics="true" pageId="4" pageNumber="281">Cyanophora paradoxa</emphasis>
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in various cultivation conditions: control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM of NaCl.
<emphasis id="D7CEEA95DB0AA634FF69408FD49AF832" bold="true" box="[151,202,1928,1948]" pageId="4" pageNumber="281">Fig. 1</emphasis>
. Growth (cells ml
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) of
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<emphasis id="D7CEEA95DB0AA634FE6B408FD61AF832" box="[405,586,1928,1948]" italics="true" pageId="4" pageNumber="281">Cyanophora paradoxa</emphasis>
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for ~27 days. All data are mean ± SD (n = 3).
<emphasis id="D7CEEA95DB0AA634FF6940A7D49AF81A" bold="true" box="[151,202,1952,1972]" pageId="4" pageNumber="281">Fig. 2</emphasis>
. HPLC-UV chromatogram representation of identified pigments.
<emphasis id="D7CEEA95DB0AA634FF6940BFD499F862" bold="true" box="[151,201,1976,1996]" pageId="4" pageNumber="281">Fig. 3</emphasis>
. Pigment content (mg g
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) of
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<emphasis id="D7CEEA95DB0AA634FE3240BFD6D0F862" box="[460,640,1976,1996]" italics="true" pageId="4" pageNumber="281">Cyanophora paradoxa</emphasis>
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. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA,
<emphasis id="D7CEEA95DB0AA634FAC140BFD119F862" box="[1343,1353,1976,1996]" italics="true" pageId="4" pageNumber="281">p</emphasis>
&lt;0.001, n = 12).
<emphasis id="D7CEEA95DB0AA634FF6940D7D49DF84A" bold="true" box="[151,205,2000,2020]" pageId="4" pageNumber="281">Fig. 4</emphasis>
. Phycocyanin, allophycocyanin and phycoerythrin content (mg g
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) of
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<emphasis id="D7CEEA95DB0AA634FCBB40D7D7ADF84A" box="[837,1021,2000,2020]" italics="true" pageId="4" pageNumber="281">Cyanophora paradoxa</emphasis>
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. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA,
<emphasis id="D7CEEA95DB0AA634FE6040EFD5F8F852" box="[414,424,2024,2044]" italics="true" pageId="4" pageNumber="281">p</emphasis>
&lt;0.001, n = 12).
</paragraph>
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<emphasis id="D7CEEA95DB0BA635FF83462ED5B5FECC" bold="true" pageId="5" pageNumber="282">Effect of Cultivation Conditions on Pigment and Phycobiliprotein Composition</emphasis>
</paragraph>
<paragraph id="E5053687DB0BA635FF83467ED6BFFC9F" blockId="5.[124,797,377,1009]" pageId="5" pageNumber="282">
Alterations to the culture medium of
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<emphasis id="D7CEEA95DB0BA635FDBB467ED681FE3F" box="[581,721,377,401]" italics="true" pageId="5" pageNumber="282">C. paradoxa</emphasis>
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influenced pigment abundance as shown in
<tableCitation id="A838033CDB0BA635FDC9469ED6DAFE1F" box="[567,650,409,433]" captionStart="Table 1" captionStartId="5.[330,377,1718,1738]" captionTargetPageId="5" captionText="Table 1. Pigment and phycobiliprotein content (mg g−1) in the biomass of Cyanophora paradoxa strain CCAP 981/1" pageId="5" pageNumber="282">Table 1</tableCitation>
. Chlorophyll
<emphasis id="D7CEEA95DB0BA635FF8346BED4DAFE7E" box="[125,138,441,464]" italics="true" pageId="5" pageNumber="282">a</emphasis>
, β-carotene and zeaxanthin were detected in all extracts (
<figureCitation id="7D812A02DB0BA635FF7846DED4A1FE5F" box="[134,241,473,497]" captionStart="Figs 1–4" captionStartId="4.[125,160,1880,1900]" captionTargetBox="[133,1497,927,1846]" captionTargetId="figure-428@4.[125,1501,923,1847]" captionTargetPageId="4" captionText="Figs 1–4. Growth (cells ml−1) and pigment production of Cyanophora paradoxa in various cultivation conditions: control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM of NaCl. Fig. 1. Growth (cells ml−1) of Cyanophora paradoxa for ~27 days. All data are mean ± SD (n = 3). Fig. 2. HPLC-UV chromatogram representation of identified pigments. Fig. 3. Pigment content (mg g−1) of Cyanophora paradoxa. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12). Fig. 4. Phycocyanin, allophycocyanin and phycoerythrin content (mg g−1) of Cyanophora paradoxa. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12)." figureDoi="http://doi.org/10.5281/zenodo.15520497" httpUri="https://zenodo.org/record/15520497/files/figure.png" pageId="5" pageNumber="282">Figs 2, 3</figureCitation>
). A significant 3-fold decrease in the yield of zeaxanthin was observed following BLU treatment (
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g
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) compared to the CONT treatment (
<quantity id="22429B62DB0BA635FD7D451ED68CFD9F" box="[643,732,537,561]" metricMagnitude="-6" metricUnit="kg" metricValue="6.8" pageId="5" pageNumber="282" unit="mg" value="6.8">6.8 mg</quantity>
g
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) (ANOVA,
<emphasis id="D7CEEA95DB0BA635FF0D453ED550FDFE" box="[243,256,569,592]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12). This pattern was also observed for β-carotene, with BLU treatment having significantly less pigment (
<quantity id="22429B62DB0BA635FF1A457ED57FFD3F" box="[228,303,633,657]" metricMagnitude="-6" metricUnit="kg" metricValue="2.1" pageId="5" pageNumber="282" unit="mg" value="2.1">2.1 mg</quantity>
g
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) as compared to the CONT (
<quantity id="22429B62DB0BA635FD67457ED6B4FD3F" box="[665,740,633,657]" metricMagnitude="-6" metricUnit="kg" metricValue="7.5" pageId="5" pageNumber="282" unit="mg" value="7.5">7.5 mg</quantity>
g
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), N- (
<quantity id="22429B62DB0BA635FF54459ED4A6FD1F" box="[170,246,665,689]" metricMagnitude="-6" metricUnit="kg" metricValue="4.6" pageId="5" pageNumber="282" unit="mg" value="4.6">4.6 mg</quantity>
g
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) and NaCl (
<quantity id="22429B62DB0BA635FE52459ED5A9FD1F" box="[428,505,665,689]" metricMagnitude="-6" metricUnit="kg" metricValue="8.7" pageId="5" pageNumber="282" unit="mg" value="8.7">8.7 mg</quantity>
g
<superScript id="12CF9BCFDB0BA635FDF04594D673FD0D" attach="left" box="[526,547,659,675]" fontSize="7" pageId="5" pageNumber="282">−1</superScript>
) treatments (ANOVA,
<emphasis id="D7CEEA95DB0BA635FF8245BED4D9FD7E" box="[124,137,697,720]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12). There were also significant differences in chlorophyll
<emphasis id="D7CEEA95DB0BA635FEFB45DED542FD5E" box="[261,274,729,752]" italics="true" pageId="5" pageNumber="282">a</emphasis>
yields, with BLU and N- treatments having significantly less (~
<quantity id="22429B62DB0BA635FEAC45FED5F4FCBF" box="[338,420,761,785]" metricMagnitude="-6" metricUnit="kg" metricValue="7.5" pageId="5" pageNumber="282" unit="mg" value="7.5">7.5 mg</quantity>
g
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) than the CONT and NaCl treatments (~19.0 mg g
<superScript id="12CF9BCFDB0BA635FE804414D5C2FC8D" attach="right" box="[382,402,787,803]" fontSize="7" pageId="5" pageNumber="282">−1</superScript>
) (ANOVA,
<emphasis id="D7CEEA95DB0BA635FDE3441ED67AFC9E" box="[541,554,793,816]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12).
</paragraph>
<paragraph id="E5053687DB0BA635FF62443ED6A2FC5F" blockId="5.[124,797,377,1009]" pageId="5" pageNumber="282">
Both the phycocyanin and allophycocyanin contents were significantly higher in the CONT as compared to the three treatments (ANOVA,
<emphasis id="D7CEEA95DB0BA635FF11447ED544FC3E" box="[239,276,889,912]" italics="true" pageId="5" pageNumber="282">p &lt;</emphasis>
0.001, n = 12) (
<figureCitation id="7D812A02DB0BA635FE3F447ED5ACFC3F" box="[449,508,889,913]" captionStart="Figs 1–4" captionStartId="4.[125,160,1880,1900]" captionTargetBox="[133,1497,927,1846]" captionTargetId="figure-428@4.[125,1501,923,1847]" captionTargetPageId="4" captionText="Figs 1–4. Growth (cells ml−1) and pigment production of Cyanophora paradoxa in various cultivation conditions: control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM of NaCl. Fig. 1. Growth (cells ml−1) of Cyanophora paradoxa for ~27 days. All data are mean ± SD (n = 3). Fig. 2. HPLC-UV chromatogram representation of identified pigments. Fig. 3. Pigment content (mg g−1) of Cyanophora paradoxa. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12). Fig. 4. Phycocyanin, allophycocyanin and phycoerythrin content (mg g−1) of Cyanophora paradoxa. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12)." figureDoi="http://doi.org/10.5281/zenodo.15520497" httpUri="https://zenodo.org/record/15520497/files/figure.png" pageId="5" pageNumber="282">Fig. 4</figureCitation>
). The phycocyanin content ranged from
<quantity id="22429B62DB0BA635FEFD449ED532FC1F" box="[259,354,921,945]" metricMagnitude="-4" metricUnit="kg" metricValue="1.139" pageId="5" pageNumber="282" unit="mg" value="113.9">113.9 mg</quantity>
g
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−
<quantity id="22429B62DB0BA635FE7F4494D5D8FC0D" box="[385,392,915,931]" metricMagnitude="-2" metricUnit="m" metricValue="2.54" pageId="5" pageNumber="282" unit="in" value="1.0">1</quantity>
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in the CONT treatment to
<quantity id="22429B62DB0BA635FD5D449ED6A6FC1F" box="[675,758,921,945]" metricMagnitude="-5" metricUnit="kg" metricValue="1.94" pageId="5" pageNumber="282" unit="mg" value="19.4">19.4 mg</quantity>
g
<superScript id="12CF9BCFDB0BA635FCF64494D74CFC0D" attach="left" box="[776,796,915,931]" fontSize="7" pageId="5" pageNumber="282">
−
<quantity id="22429B62DB0BA635FCEB4494D74CFC0D" box="[789,796,915,931]" metricMagnitude="-2" metricUnit="m" metricValue="2.54" pageId="5" pageNumber="282" unit="in" value="1.0">1</quantity>
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in the BLU treatment. The allophycocyanin yields were 71.1 and
<quantity id="22429B62DB0BA635FF8244DED49FFC5F" box="[124,207,985,1009]" metricMagnitude="-5" metricUnit="kg" metricValue="1.26" pageId="5" pageNumber="282" unit="mg" value="12.6">12.6 mg</quantity>
g
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for the CONT and BLU treatments, respectively.
</paragraph>
<paragraph id="E5053687DB0BA635FF834333D5BCFBC3" blockId="5.[125,678,1076,1133]" pageId="5" pageNumber="282">
<emphasis id="D7CEEA95DB0BA635FF834333D5BCFBC3" bold="true" pageId="5" pageNumber="282">Effect of Cultivation Conditions on Fatty Acid Composition and EPA Content</emphasis>
</paragraph>
<paragraph id="E5053687DB0BA635FF834383D0BBFEA8" blockId="5.[124,797,1156,1660]" lastBlockId="5.[828,1500,142,262]" pageId="5" pageNumber="282">
Significant changes in the relative proportions of some FAMEs were observed in response to stress conditions (
<figureCitation id="7D812A02DB0BA635FD5643A3D75EFB12" box="[680,782,1187,1212]" captionStart="Figs 5–7" captionStartId="6.[125,160,1144,1164]" captionTargetBox="[140,1483,145,1107]" captionTargetId="figure-283@6.[137,1487,142,1110]" captionTargetPageId="6" captionText="Figs 5–7. Fatty acid analysis of Cyanophora paradoxa cultivated under various conditions: control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM NaCl. Fig. 5. GC-MS chromatogram representation of identified FAMEs. Fig. 6. FAME proportions (%). Fig. 7. EPA (mg g−1) content. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12)." figureDoi="http://doi.org/10.5281/zenodo.15520499" httpUri="https://zenodo.org/record/15520499/files/figure.png" pageId="5" pageNumber="282">Figs 5, 6</figureCitation>
). There was a significant decrease in the proportions of C20:4 following the BLU treatment (7.1%) compared to the CONT (22.7%), N- (24.5%) and NaCl (22.3%) treatments (ANOVA,
<emphasis id="D7CEEA95DB0BA635FF824223D4D9FA95" box="[124,137,1316,1339]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12) (
<tableCitation id="A838033CDB0BA635FE994223D59CFA92" box="[359,460,1316,1340]" captionStart="Table 2" captionStartId="6.[283,330,1313,1333]" captionTargetPageId="6" captionText="Table 2. Fatty acids proportions (%) in the biomass of Cyanophora paradoxa strain CCAP 981/1 under various conditions:" pageId="5" pageNumber="282">Table S2</tableCitation>
). There was also a significant increase in C16:1 following the BLU treatment (11.2%) as compared to the CONT (1.3%), N- (1.4%) and NaCl (1.8%) treatments (ANOVA,
<emphasis id="D7CEEA95DB0BA635FE944283D527FA35" box="[362,375,1412,1435]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12). In addition, the BLU treatment enhanced the accumulation of unsaturated forms of C18 (34.3%) compared to the CONT (6.5%), N- (6.6%) and NaCl (19.4%) treatments (ANOVA,
<emphasis id="D7CEEA95DB0BA635FDEB42E3D672FA55" box="[533,546,1508,1531]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12). As shown in
<tableCitation id="A838033CDB0BA635FF144103D56BF9B2" box="[234,315,1540,1564]" captionStart="Table 2" captionStartId="6.[283,330,1313,1333]" captionTargetPageId="6" captionText="Table 2. Fatty acids proportions (%) in the biomass of Cyanophora paradoxa strain CCAP 981/1 under various conditions:" pageId="5" pageNumber="282">Table 2</tableCitation>
, the BLU treatment also led to a significant reduction in polyunsaturated fatty acids (PUFA) proportions, while increasing those of monounsaturated fatty acids (MUFA) (ANOVA,
<emphasis id="D7CEEA95DB0BA635FF0F4163D4AEF9D5" box="[241,254,1636,1659]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12). The EPA content was negatively affected by all the treatments compared to the CONT (ANOVA,
<emphasis id="D7CEEA95DB0BA635FC4D47A9D790FF6B" box="[947,960,174,197]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12) (
<figureCitation id="7D812A02DB0BA635FB6F47A9D09FFF6B" box="[1169,1231,174,198]" captionStart="Figs 5–7" captionStartId="6.[125,160,1144,1164]" captionTargetBox="[140,1483,145,1107]" captionTargetId="figure-283@6.[137,1487,142,1110]" captionTargetPageId="6" captionText="Figs 5–7. Fatty acid analysis of Cyanophora paradoxa cultivated under various conditions: control (CONT), blue light (BLU), nitrogen limitation (N-) and 18 mM NaCl. Fig. 5. GC-MS chromatogram representation of identified FAMEs. Fig. 6. FAME proportions (%). Fig. 7. EPA (mg g−1) content. All data are mean ± SD (n = 3). Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12)." figureDoi="http://doi.org/10.5281/zenodo.15520499" httpUri="https://zenodo.org/record/15520499/files/figure.png" pageId="5" pageNumber="282">Fig. 7</figureCitation>
). The highest EPA yield obtained for the CONT (
<quantity id="22429B62DB0BA635FBB347C9D0C8FF48" box="[1101,1176,206,230]" metricMagnitude="-6" metricUnit="kg" metricValue="1.1" pageId="5" pageNumber="282" unit="mg" value="1.1">1.1 mg</quantity>
g
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) was ~4-fold greater than that of the BLU treatment (
<quantity id="22429B62DB0BA635FB9847E9D0E2FEA8" box="[1126,1202,238,262]" metricMagnitude="-7" metricUnit="kg" metricValue="3.0" pageId="5" pageNumber="282" unit="mg" value="0.3">0.3 mg</quantity>
g
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).
</paragraph>
<paragraph id="E5053687DB0BA635FCC24642D046FED0" blockId="5.[828,1501,325,382]" pageId="5" pageNumber="282">
<emphasis id="D7CEEA95DB0BA635FCC24642D046FED0" bold="true" pageId="5" pageNumber="282">Antiproliferative Effect of Crude Extracts on Cancer Cell Lines - MTS Assay</emphasis>
</paragraph>
<paragraph id="E5053687DB0BA635FCC34692D1DCFD03" blockId="5.[828,1501,400,688]" pageId="5" pageNumber="282">
Extracts of Et
<subScript id="793E34C2DB0BA635FC3046A7D787FE1E" attach="both" box="[974,983,416,432]" fontSize="7" pageId="5" pageNumber="282">2</subScript>
O, EA and MeOH (100 µg ml
<superScript id="12CF9BCFDB0BA635FAE34697D161FE0E" attach="left" box="[1309,1329,400,416]" fontSize="7" pageId="5" pageNumber="282">−1</superScript>
) obtained from each treatment were used to monitor their effect on the cell viability of human lung (A549) and breast (MCF-7) cancer cell lines. There was no significant difference in A549 cell viability with any extract (
<figureCitation id="7D812A02DB0BA635FC014512D06CFD83" box="[1023,1084,533,557]" captionStart="Figs 8, 9" captionStartId="7.[125,160,609,629]" captionTargetBox="[132,1493,145,561]" captionTargetId="figure-386@7.[129,1497,142,576]" captionTargetPageId="7" captionText="Figs 8, 9. Relative cell viability of human cancer cell lines exposed to diethyl ether (Et2O), ethyl acetate (EA) and methanol (MeOH) extracts derived from Cyanophora paradoxa at a concentration of 100 µg ml−1 over 72 h. Values are expressed as mean ± SD of three separate experiments, each assay was performed in triplicate. Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12). Fig. 8. A549 human cancer cell line. Fig. 9. MCF-7 human cancer cell line." figureDoi="http://doi.org/10.5281/zenodo.15520501" httpUri="https://zenodo.org/record/15520501/files/figure.png" pageId="5" pageNumber="282">Fig. 8</figureCitation>
). The MCF-7 cell line appeared more sensitive to extracts, in particular to the EA extract obtained following the N- treatment, for which a significant reduction in viability was observed (~70%) (ANOVA,
<emphasis id="D7CEEA95DB0BA635FB054571D158FD23" box="[1275,1288,630,653]" italics="true" pageId="5" pageNumber="282">p</emphasis>
&lt;0.001, n = 12) as compared to the Et
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O extracts (CONT and N-) (
<figureCitation id="7D812A02DB0BA635FABC4592D12DFD03" box="[1346,1405,661,685]" captionStart="Figs 8, 9" captionStartId="7.[125,160,609,629]" captionTargetBox="[132,1493,145,561]" captionTargetId="figure-386@7.[129,1497,142,576]" captionTargetPageId="7" captionText="Figs 8, 9. Relative cell viability of human cancer cell lines exposed to diethyl ether (Et2O), ethyl acetate (EA) and methanol (MeOH) extracts derived from Cyanophora paradoxa at a concentration of 100 µg ml−1 over 72 h. Values are expressed as mean ± SD of three separate experiments, each assay was performed in triplicate. Lettering indicates homogenous subsets (ANOVA, p &lt;0.001, n = 12). Fig. 8. A549 human cancer cell line. Fig. 9. MCF-7 human cancer cell line." figureDoi="http://doi.org/10.5281/zenodo.15520501" httpUri="https://zenodo.org/record/15520501/files/figure.png" pageId="5" pageNumber="282">Fig. 9</figureCitation>
).
</paragraph>
<paragraph id="E5053687DB0BA635FCC345EAD13FFCA8" blockId="5.[829,1391,749,774]" box="[829,1391,749,774]" pageId="5" pageNumber="282">
<emphasis id="D7CEEA95DB0BA635FCC345EAD13FFCA8" bold="true" box="[829,1391,749,774]" pageId="5" pageNumber="282">Genotoxicity Assay - Acridine Orange Staining</emphasis>
</paragraph>
<paragraph id="E5053687DB0BA635FCC3441BD0B4FBFA" blockId="5.[829,1501,796,1108]" pageId="5" pageNumber="282">
A549 and MCF-7 human cancer exposed to
<taxonomicName id="22BA4D04DB0BA635FAAC441AD18DFC9A" box="[1362,1501,796,820]" class="Glaucophyceae" family="Glaucocystaceae" genus="Cyanophora" kingdom="Plantae" order="Glaucocystales" pageId="5" pageNumber="282" phylum="Glaucophyta" rank="species" species="paradoxa">
<emphasis id="D7CEEA95DB0BA635FAAC441AD18DFC9A" box="[1362,1501,796,820]" italics="true" pageId="5" pageNumber="282">C. paradoxa</emphasis>
</taxonomicName>
extracts were stained with acridine orange and examined microscopically to assess the presence of genotoxic effects after 72 h. The nuclei of treated cells appeared uniformly bright green, indicating that DNA within the nucleus remained undamaged, with a cellular structure comparable to that of the untreated control cells (
<figureCitation id="7D812A02DB0BA635FB1C44DBD17DFC5A" box="[1250,1325,988,1012]" captionStart="Fig" captionStartId="7.[125,153,1323,1343]" captionTargetBox="[128,1498,795,1286]" captionTargetId="figure-474@7.[125,1501,792,1289]" captionTargetPageId="7" captionText="Fig. 10. Epi-fluorescence microscopy images of acridine orange stained A549 (A-E) and MCF-7 (F-J) cells following treatment (72 h) with 100 µg ml−1 of Cyanophora paradoxa derived extracts. Untreated cells (A and F) are also included. Scale bar = 100 µm." figureDoi="http://doi.org/10.5281/zenodo.15520503" httpUri="https://zenodo.org/record/15520503/files/figure.png" pageId="5" pageNumber="282">Fig. 10</figureCitation>
). Only a minor population of MCF-7 cells (exposed to NaCl derived extract) exhibited red and orange fluorescence outside the nucleus, presumably associated with lysosomes.
</paragraph>
<paragraph id="E5053687DB0BA635FCC34393D0A2FB03" blockId="5.[829,1266,1172,1197]" box="[829,1266,1172,1197]" pageId="5" pageNumber="282">
<emphasis id="D7CEEA95DB0BA635FCC34393D0A2FB03" bold="true" box="[829,1266,1172,1197]" pageId="5" pageNumber="282">Principal Component Analysis (PCA)</emphasis>
</paragraph>
<paragraph id="E5053687DB0BA635FCC343C3D009F9B2" blockId="5.[829,1501,1220,1660]" pageId="5" pageNumber="282">
A PCA examined patterns among the treatments and parameters measured. The PCA revealed two components, accounting for 88% of the variance (
<figureCitation id="7D812A02DB0BA635FB134203D16DFAB2" box="[1261,1341,1284,1308]" captionStart="Fig" captionStartId="8.[125,153,686,706]" captionTargetBox="[131,789,155,649]" captionTargetId="figure-938@8.[128,793,152,652]" captionTargetPageId="8" captionText="Fig. 11. Principal Component Analysis (PCA) projections indicating the relationships between the treatment conditions applied to C. paradoxa and the variables measured (compounds including carotenoids, fatty acids and phycobiliproteins , bioactivity, biomass production)." figureDoi="http://doi.org/10.5281/zenodo.15520505" httpUri="https://zenodo.org/record/15520505/files/figure.png" pageId="5" pageNumber="282">Fig. 11</figureCitation>
). Component 1 (CP1) was positively related to variables such as phycobiliproteins and Et
<subScript id="793E34C2DB0BA635FBEF4248D04BFAF1" attach="both" box="[1041,1051,1359,1375]" fontSize="7" pageId="5" pageNumber="282">2</subScript>
O extract bioactivity on A549 cells and negatively associated with the Et
<subScript id="793E34C2DB0BA635FB4B4268D0EFFAD1" attach="both" box="[1205,1215,1391,1407]" fontSize="7" pageId="5" pageNumber="282">2</subScript>
O and EA extract bioactivities on A549 and MCF-7 cells. Component 2 (CP2) was more closely aligned with the fatty acid content. In addition, both the zeaxanthin content and MeOH extract bioactivity on A549 cells were strongly associated with the positive domain on CP2.
</paragraph>
<paragraph id="E5053687DB0BA636FCA34123D6ACF9CA" blockId="5.[829,1501,1220,1660]" lastBlockId="6.[125,797,1580,1636]" lastPageId="6" lastPageNumber="283" pageId="5" pageNumber="282">The four treatments applied in this study were projected in different locations of the PCA plane. In particular, the CONT and N- treatments were firmly associated to the positive axes of CP1 and CP2, respectively, while the BLU treatment was instead located along the negative domain of CP2.</paragraph>
</subSubSection>
</treatment>
</document>