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<document id="013EC0D249337CE8078045D82AA9D333" ID-CLB-Dataset="55389" ID-DOI="10.1016/j.phytochem.2021.112861" ID-GBIF-Dataset="6fa470c7-85a4-4f3f-a59d-83b9995cf1a4" ID-ISSN="1873-3700" ID-Zenodo-Dep="8258041" IM.bibliography_approvedBy="julia" IM.illustrations_approvedBy="julia" IM.materialsCitations_approvedBy="julia" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="julia" IM.taxonomicNames_approvedBy="julia" IM.treatments_approvedBy="julia" checkinTime="1692304778008" checkinUser="felipe" docAuthor="Kolodziejczyk-Czepas, Joanna, Kozachok, Solomiia, Pecio, Łukasz, Marchyshyn, Svitlana &amp; Oleszek, Wiesław" docDate="2021" docId="039EC91BFFE8D7492D4AFEB1FAA4F864" docLanguage="en" docName="Phytochemistry.190.112861.pdf" docOrigin="Phytochemistry (112861) 190" docSource="http://dx.doi.org/10.1016/j.phytochem.2021.112861" docStyle="DocumentStyle:F36D69FC8B198FBE91029DF9C24697D3.5:Phytochemistry.2020-.journal_article" docStyleId="F36D69FC8B198FBE91029DF9C24697D3" docStyleName="Phytochemistry.2020-.journal_article" docStyleVersion="5" docTitle="Herniaria fractions" docType="treatment" docVersion="2" lastPageNumber="18" masterDocId="FFA7B163FFF9D7582D2EFFAEFFEFFFDA" masterDocTitle="Determination of phenolic profiles of Herniaria polygama and Herniaria incana fractions and their in vitro antioxidant and anti-inflammatory effects" masterLastPageNumber="21" masterPageNumber="1" pageNumber="18" updateTime="1692791916840" updateUser="ExternalLinkService">
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<mods:title id="FBDB9C2F0C71219C2C9E6CD0929A7A4D">Determination of phenolic profiles of Herniaria polygama and Herniaria incana fractions and their in vitro antioxidant and anti-inflammatory effects</mods:title>
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<mods:namePart id="42BE549502F1155331B571C52ABC29B5">Kolodziejczyk-Czepas, Joanna</mods:namePart>
<mods:affiliation id="B432DDA308321583D11B1FC8DC4E75A3">* &amp; Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141 / 143, 90 - 236, Lodz, Poland</mods:affiliation>
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<mods:namePart id="DD78EC949BD4395A2F29C7712BB19F75">Kozachok, Solomiia</mods:namePart>
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<mods:namePart id="3B6744E5489593DD09642EC4C26C0697">Pecio, Łukasz</mods:namePart>
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<mods:namePart id="7DC3D2EAC67AA9054E8258C6CE20D3A3">Marchyshyn, Svitlana</mods:namePart>
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<mods:date id="DD5E7A35D823DE9F04D24D5C599CBC76">2021</mods:date>
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<treatment id="039EC91BFFE8D7492D4AFEB1FAA4F864" ID-DOI="http://doi.org/10.5281/zenodo.8276325" ID-Zenodo-Dep="8276325" LSID="urn:lsid:plazi:treatment:039EC91BFFE8D7492D4AFEB1FAA4F864" httpUri="http://treatment.plazi.org/id/039EC91BFFE8D7492D4AFEB1FAA4F864" lastPageNumber="18" pageId="17" pageNumber="18">
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<paragraph id="8B88780DFFE8D7492D4AFEB1FD1DFEE8" blockId="17.[100,754,287,306]" box="[100,754,287,306]" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492D4AFEB1FD1DFEE8" bold="true" box="[100,754,287,306]" centered="true" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">
<emphasis id="B943A41FFFE8D7492D4AFEB1FD1DFEE8" bold="true" box="[100,754,287,306]" italics="true" pageId="17" pageNumber="18">
4.7. Evaluation of antioxidant effects of the examined
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fractions
</emphasis>
</heading>
</paragraph>
</subSubSection>
<subSubSection id="C32D2B86FFE8D7492D4AFEF9FAA4F864" pageId="17" pageNumber="18" type="description">
<paragraph id="8B88780DFFE8D7492D4AFEF9FE1EFEB0" blockId="17.[100,497,343,362]" box="[100,497,343,362]" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492D4AFEF9FE1EFEB0" bold="true" box="[100,497,343,362]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">
<emphasis id="B943A41FFFE8D7492D4AFEF9FE1EFEB0" bold="true" box="[100,497,343,362]" italics="true" pageId="17" pageNumber="18">4.7.1. Preparation of blood plasma samples</emphasis>
</heading>
</paragraph>
<paragraph id="8B88780DFFE8D7492DAAFEDDFF2AFCD6" blockId="17.[100,771,371,780]" pageId="17" pageNumber="18">
Blood plasma was isolated from human buffy coat units, commercially available at the
<location id="8EE82ED6FFE8D7492C6FFE21FD78FE78" LSID="urn:lsid:plazi:treatment:039EC91BFFE8D7492D4AFEB1FAA4F864:8EE82ED6FFE8D7492C6FFE21FD78FE78" box="[321,663,399,418]" country="Poland" municipality="Ethics of Research" name="Regional Centre of Blood Donation" pageId="17" pageNumber="18" stateProvince="Lodzkie">Regional Centre of Blood Donation</location>
and Blood Treatment in
<collectingRegion id="49F3B6EFFFE8D7492DC4FE05FEF6FE64" box="[234,281,427,446]" country="Poland" name="Lodzkie" pageId="17" pageNumber="18">Lodz</collectingRegion>
,
<collectingCountry id="F320389DFFE8D7492C08FE05FE85FE64" box="[294,362,427,446]" name="Poland" pageId="17" pageNumber="18">Poland</collectingCountry>
. Experiments were approved by the committee on the
<collectingMunicipality id="6BECE277FFE8D7492DD8FE69FE45FE00" box="[246,426,455,474]" pageId="17" pageNumber="18">Ethics of Research</collectingMunicipality>
at the University of
<collectingRegion id="49F3B6EFFFE8D7492FADFE69FD5DFE00" box="[643,690,455,474]" country="Poland" name="Lodzkie" pageId="17" pageNumber="18">Lodz</collectingRegion>
,
<collectingCountry id="F320389DFFE8D7492FEFFE69FCEDFE00" box="[705,770,455,474]" name="Poland" pageId="17" pageNumber="18">Poland</collectingCountry>
(
<collectorName id="26C21DDBFFE8D7492D44FE4CFF08FE2C" box="[106,231,482,502]" pageId="17" pageNumber="18">Protocol No.</collectorName>
10/KBBN–UŁ/
<date id="FF895ECDFFE8D7492C59FE4DFE51FE2C" box="[375,446,483,502]" bridgedPair="/" pageId="17" pageNumber="18" value="2017-01">
<collectingDate id="EFCDA725FFE8D7492C59FE4DFE51FE2C" box="[375,446,483,502]" bridgedPair="/" pageId="17" pageNumber="18" value="2017-01">I/2017</collectingDate>
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). Stock solutions of the analysed fractions were dissolved in 30% DMSO (dimethyl sulfoxide) to the final concentration of DMSO of ≤0.03%, in the assayed samples. Plasma samples were pre-incubated for 15 min at 37
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C with the fractions (1–50 μg/ml) or a reference compound,
<emphasis id="B943A41FFFE8D7492C85FDFCFE50FDBF" bold="true" box="[427,447,594,613]" italics="true" pageId="17" pageNumber="18">i.e</emphasis>
. Trolox (5 μg/ml), and then they were exposed to ONOO (at final concentrations of 100 μM or 150 μM). Samples containing blood plasma treated with ONOO in the absence of the examined substances were also prepared. Control plasma was treated with neither the investigated plant fractions/reference antioxidant nor ONOO (but it contained 0.03% DMSO, as a vehicle for the fractions).
</paragraph>
<paragraph id="8B88780DFFE8D7492D4AFC9FFD24FC9E" blockId="17.[100,715,817,836]" box="[100,715,817,836]" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492D4AFC9FFD24FC9E" bold="true" box="[100,715,817,836]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">
<emphasis id="B943A41FFFE8D7492D4AFC9FFD24FC9E" bold="true" box="[100,715,817,836]" italics="true" pageId="17" pageNumber="18">4.7.2. Measurements of oxidative stress biomarkers in blood plasma</emphasis>
</heading>
</paragraph>
<paragraph id="8B88780DFFE8D7492DAAFCE3FEE1FB75" blockId="17.[100,771,845,1199]" pageId="17" pageNumber="18">
Oxidative stress was induced by the exposure of blood plasma to 100 μM ONOO. Lipid peroxidation was determined based on the level of thiobarbituric acid-reactive substances (TBARS) (
<bibRefCitation id="EFA605FCFFE8D7492F03FC2BFD3CFC42" author="Wachowicz, B." box="[557,723,901,920]" pageId="17" pageNumber="18" pagination="167 - 170" refId="ref28806" refString="Wachowicz, B., 1984. Adenine nucleotides in thrombocytes of birds. Cell Biochem. Funct. 2, 167 - 170. https: // doi. org / 10.1002 / cbf. 290020310." type="journal article" year="1984">Wachowicz, 1984</bibRefCitation>
) and lipid hydroperoxides generation (ferric-xylenol orange assay (
<bibRefCitation id="EFA605FCFFE8D7492F9DFC0FFF07FC0A" author="Gay, C. &amp; Gebicki, J. M." pageId="17" pageNumber="18" pagination="217 - 220" refId="ref23934" refString="Gay, C., Gebicki, J. M., 2000. A critical evaluation of the effect of sorbitol on the ferric - xylenol orange hydroperoxide assay. Anal. Biochem. 284, 217 - 220. https: // doi. org / 10.1006 / abio. 2000.4696." type="journal article" year="2000">Gay and Gebicki, 2000</bibRefCitation>
)). Modifications of blood plasma proteins were analysed using 3-nitrotyrosine and thiol groups as oxidative stress biomarkers. Presence of 3-nitrotyrosine in blood plasma proteins was determined with the use of a competitive enzyme-linked immunosorbent assay (ELISA) (
<bibRefCitation id="EFA605FCFFE8D7492D92FB82FEB3FBE5" author="Olas, B. &amp; Nowak, P. &amp; Kolodziejczyk, J. &amp; Wachowicz, B." box="[188,348,1068,1088]" pageId="17" pageNumber="18" pagination="399 - 406" refId="ref26436" refString="Olas, B., Nowak, P., Kolodziejczyk, J., Wachowicz, B., 2004. The effects of antioxidants on peroxynitrite-induced changes in platelet proteins. Thromb. Res. 113, 399 - 406. https: // doi. org / 10.1016 / j. thromres. 2004.04.002." type="journal article" year="2004">Olas et al., 2004</bibRefCitation>
). Results from this assay were expressed as equivalents of 3-nitrotyrosine-containing standard protein (
<emphasis id="B943A41FFFE8D7492FB8FBE6FD5EFB81" bold="true" box="[662,689,1096,1115]" italics="true" pageId="17" pageNumber="18">i.e.</emphasis>
nitrated fibrinogen, 3-NT-Fg, per mg of blood plasma protein). The level of protein thiol groups was determined spectrophotometrically (
<bibRefCitation id="EFA605FCFFE8D7492F91FB2EFEEEFB75" author="Rice-Evans, C. A. &amp; Diplock, A. T. &amp; Symons, M. C. R." pageId="17" pageNumber="18" pagination="207 - 235" refId="ref27263" refString="Rice-Evans, C. A., Diplock, A. T., Symons, M. C. R., 1991. Detection of protein structural modifications induced by free radicals. In: Rice-Evans, C. A., Diplock, A. T., Symons, M. C. R. (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology. Elsevier, Amsterdam, London, New York, Tokyo, pp. 207 - 235. https: // doi. org / 10.1016 / S 0075 - 7535 (08) 70047 - 0." type="book chapter" year="1991">Rice-Evans et al., 1991</bibRefCitation>
).
</paragraph>
<paragraph id="8B88780DFFE8D7492D4AFB7AFD09FB3C" blockId="17.[100,742,1235,1255]" box="[100,742,1235,1255]" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492D4AFB7AFD09FB3C" bold="true" box="[100,742,1235,1255]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">
<emphasis id="B943A41FFFE8D7492D4AFB7AFD09FB3C" bold="true" box="[100,742,1235,1255]" italics="true" pageId="17" pageNumber="18">4.7.3. Determination of the ferric reducing ability of blood plasma (the</emphasis>
</heading>
</paragraph>
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<heading id="D0C0CF61FFE8D7492D4AFB41FF38FAD8" box="[100,215,1263,1282]" fontSize="8" level="3" pageId="17" pageNumber="18" reason="8">
<emphasis id="B943A41FFFE8D7492D4AFB41FF38FAD8" bold="true" box="[100,215,1263,1282]" italics="true" pageId="17" pageNumber="18">FRAP assay)</emphasis>
</heading>
</paragraph>
<paragraph id="8B88780DFFE8D7492DAAFAA5FE05FA1C" blockId="17.[100,770,1263,1478]" pageId="17" pageNumber="18">
Blood plasma was exposed to 150 μM ONOO in the presence or absence of the examined substances. The influence of
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<emphasis id="B943A41FFFE8D7492F78FA89FD44FAE0" bold="true" box="[598,683,1319,1338]" italics="true" pageId="17" pageNumber="18">Herniaria</emphasis>
</taxonomicName>
fractions on the non-enzymatic antioxidant capacity (NEAC) of blood plasma was established colorimetrically by measurements of the ability of the assayed samples to reduce ferric ions (Fe
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) to ferrous ions (Fe
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). The FRAP assay was conducted according to the previously described protocol (
<bibRefCitation id="EFA605FCFFE8D7492D8FFA1DFE32FA1C" author="Kolodziejczyk-Czepas, J. &amp; Nowak, P. &amp; Kowalska, I. &amp; Stochmal, A." box="[161,477,1459,1478]" pageId="17" pageNumber="18" pagination="1308 - 1314" refId="ref24780" refString="Kolodziejczyk-Czepas, J., Nowak, P., Kowalska, I., Stochmal, A., 2014. Biological activity of clovers - free radical scavenging ability and antioxidant action of six Trifolium species. Pharm. Biol. 52, 1308 - 1314. https: // doi. org / 10.3109 / 13880209.2014. 891042." type="journal article" year="2014">Kolodziejczyk-Czepas et al., 2014</bibRefCitation>
).
</paragraph>
<paragraph id="8B88780DFFE8D7492D4AFA44FEFEF9C0" blockId="17.[100,731,1514,1533]" lastBlockId="17.[100,770,1542,1868]" pageId="17" pageNumber="18">
<emphasis id="B943A41FFFE8D7492D4AFA44FEFEF9C0" bold="true" italics="true" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492D4AFA44FD34FA27" bold="true" box="[100,731,1514,1533]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">4.7.4. Electrophoretic analysis of the peroxynitrite-induced changes in</heading>
<heading id="D0C0CF61FFE8D7492D4AF9A8FEFEF9C0" box="[100,273,1542,1562]" fontSize="8" level="3" pageId="17" pageNumber="18" reason="8">fibrinogen structure</heading>
</emphasis>
</paragraph>
<paragraph id="8B88780DFFE8D7492DAAF98CFF2AF896" blockId="17.[100,770,1542,1868]" pageId="17" pageNumber="18">
Fibrinogen was isolated from human blood plasma based on a technique of precipitation with cold ethanol (
<bibRefCitation id="EFA605FCFFE8D7492F09F990FD1BF98B" author="Doolittle, R. F. &amp; Schubert, D. &amp; Schwartz, S. A." box="[551,756,1598,1618]" pageId="17" pageNumber="18" pagination="456 - 467" refId="ref23397" refString="Doolittle, R. F., Schubert, D., Schwartz, S. A., 1967. Amino acid sequence studies on artiodactyl fibrinopeptides. I. Dromedary camel, mule deer, and cape buffalo. Arch. Biochem. Biophys. 118, 456 - 467. https: // doi. org / 10.1016 / 0003 - 9861 (67) 90374 - 8." type="journal article" year="1967">Doolittle et al., 1967</bibRefCitation>
). Isolated protein (
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of fibrinogen/ml, in 0.01 M Tris/HCl buffer, pH 7.4) was pre-incubated (15 min, at 37
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C) with the examined
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<emphasis id="B943A41FFFE8D7492F83F9D8FCEDF953" bold="true" box="[685,770,1654,1673]" italics="true" pageId="17" pageNumber="18">Herniaria</emphasis>
</taxonomicName>
fractions (1–50 μg/ml) or the reference antioxidant, and then, exposed to peroxynitrite (100 μM). Protein samples were separated in 4–20% SDS-PAGE gradient gels (
<emphasis id="B943A41FFFE8D7492C79F964FE9DF907" bold="true" box="[343,370,1738,1757]" italics="true" pageId="17" pageNumber="18">i.e.</emphasis>
Mini-Protean TGX™ precast gels; 8 μg of protein per each lane), under reducing conditions. Electrophoresis was conducted using BioRad Mini Protean Tetra Cell equipment and BioRad reagents. Protein bands were visualized with the use of Coomassie Blue R250 dye.
</paragraph>
<paragraph id="8B88780DFFE8D7492E1CFF3AFBC5FF19" blockId="17.[818,1412,147,167]" lastBlockId="17.[818,1066,175,195]" pageId="17" pageNumber="18">
<emphasis id="B943A41FFFE8D7492E1CFF3AFBC5FF19" bold="true" italics="true" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492E1CFF3AFA6BFF7C" bold="true" box="[818,1412,147,167]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">4.8. COX-inhibitor screening and experiments on peripheral blood</heading>
<heading id="D0C0CF61FFE8D7492E1CFF01FBC5FF19" box="[818,1066,175,195]" fontSize="8" level="3" pageId="17" pageNumber="18" reason="8">mononuclear cells (PBMCs)</heading>
</emphasis>
</paragraph>
<paragraph id="8B88780DFFE8D7492E7FFF49FADBFEB0" blockId="17.[818,1488,231,362]" pageId="17" pageNumber="18">
Cells were isolated from human buffy coat units using density centrifugation in BioWestLymphosep medium (diatrizoic acid dihydrate –
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/l, EDTA tetrasodium salt dihydrate –
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/l, sodium hydroxide pellets –
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/l, polysaccharose 400–
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/l, water), according to the protocol provided by the manufacturer.
</paragraph>
<paragraph id="8B88780DFFE8D7492E1CFE21FB38FE7B" blockId="17.[818,1239,398,418]" box="[818,1239,398,418]" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492E1CFE21FB38FE7B" bold="true" box="[818,1239,398,418]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">
<emphasis id="B943A41FFFE8D7492E1CFE21FB38FE7B" bold="true" box="[818,1239,398,418]" italics="true" pageId="17" pageNumber="18">4.8.1. Determination of COX-inhibitory effects</emphasis>
</heading>
</paragraph>
<paragraph id="8B88780DFFE8D7492E7FFE05FC64FDBF" blockId="17.[818,1487,426,613]" pageId="17" pageNumber="18">
Inhibitory effects of the examined
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<emphasis id="B943A41FFFE8D74929B5FE05FB1FFE64" bold="true" box="[1179,1264,427,446]" italics="true" pageId="17" pageNumber="18">Herniaria</emphasis>
</taxonomicName>
fractions (1–50 μg/ml) on COX-1 and -2 activities were evaluated using a commercial colorimetric kit, measuring the enzymatic activity of the peroxidase component of COXs (responsible for the oxidation of the
<emphasis id="B943A41FFFE8D74929D5FE51FAE5FDC8" bold="true" box="[1275,1290,511,530]" italics="true" pageId="17" pageNumber="18">N</emphasis>
,
<emphasis id="B943A41FFFE8D749283EFE51FAF0FDC8" bold="true" box="[1296,1311,511,530]" italics="true" pageId="17" pageNumber="18">N</emphasis>
,
<emphasis id="B943A41FFFE8D749280BFE51FADBFDC8" bold="true" box="[1317,1332,511,530]" italics="true" pageId="17" pageNumber="18">N</emphasis>
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,
<emphasis id="B943A41FFFE8D749286EFE51FAA0FDC8" bold="true" box="[1344,1359,511,530]" italics="true" pageId="17" pageNumber="18">N</emphasis>
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-
<emphasis id="B943A41FFFE8D7492874FE50FCD3FDF7" bold="true" italics="true" pageId="17" pageNumber="18">tetramethylp</emphasis>
-phenylenediamine (TMPD) chromogenic substrate). Assays were carried out in triplicate, using indomethacin (1–50 μg/ml) as a reference inhibitor.
</paragraph>
<paragraph id="8B88780DFFE8D7492E1CFD24FABDFD47" blockId="17.[818,1362,649,669]" box="[818,1362,649,669]" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492E1CFD24FABDFD47" bold="true" box="[818,1362,649,669]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">
<emphasis id="B943A41FFFE8D7492E1CFD24FABDFD47" bold="true" box="[818,1362,649,669]" italics="true" pageId="17" pageNumber="18">4.8.2. Determination of anti-inflammatory effects in PBMCs</emphasis>
</heading>
</paragraph>
<paragraph id="8B88780DFFE8D7492E7FFD08FB7EFBDD" blockId="17.[818,1488,678,1031]" pageId="17" pageNumber="18">
Isolated PBMCs were suspended in RPMI 1640 medium (supplemented with 10% fetal calf serum and 0.1% of penicillin-streptomycin), at the density of 1.5 × 10
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cells/ml and pre-incubated for 60 min with the examined fractions, in a laboratory CO
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incubator. Then, PBMCs were stimulated with concanavalin A (Con A; at the final conc. of 10 μg/ ml) and cultured for 24 h (in 96-well microplates, at 37
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C, with 5% of CO
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concentration and 95% humidity). The next day, microplates were centrifuged to obtain supernatants (cell culture medium) for further analyses. Anti-inflammatory effect of the examined
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<emphasis id="B943A41FFFE8D749280FFC2BFA99FC42" bold="true" box="[1313,1398,901,920]" italics="true" pageId="17" pageNumber="18">Herniaria</emphasis>
</taxonomicName>
fractions was evaluated based on measurements of interleukin-2 and TNF-α secretion from PBMCs. Concentrations of these cytokines in supernatants were determined with commercial ELISA kits, according to protocols provided by the manufacturer.
</paragraph>
<paragraph id="8B88780DFFE8D7492E1CFB82FBF1FBE5" blockId="17.[818,1054,1068,1087]" box="[818,1054,1068,1087]" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492E1CFB82FBF1FBE5" bold="true" box="[818,1054,1068,1087]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">
<emphasis id="B943A41FFFE8D7492E1CFB82FBF1FBE5" bold="true" box="[818,1054,1068,1087]" italics="true" pageId="17" pageNumber="18">4.8.3. Cytotoxicity assays</emphasis>
</heading>
</paragraph>
<paragraph id="8B88780DFFE8D7492E7FFBE7FC02FA8C" blockId="17.[818,1488,1096,1645]" pageId="17" pageNumber="18">
For preliminary evaluation of cellular safety of the examined fractions, a short-term incubation cytotoxicity test was applied (
<bibRefCitation id="EFA605FCFFE8D74928BFFBCAFBA9FB49" author="Kolodziejczyk-Czepas, J. &amp; Sieradzka, M. &amp; Nowak, P. &amp; Stochmal, A." pageId="17" pageNumber="18" pagination="54 - 63" refId="ref24842" refString="Kolodziejczyk-Czepas, J., Krzyzanowska-Kowalczyk ˙, J., Sieradzka, M., Nowak, P., Stochmal, A., 2017. Clovamide and clovamide-rich extracts of three Trifolium species as antioxidants and moderate antiplatelet agents in vitro. Phytochemistry 143, 54 - 63. https: // doi. org / 10.1016 / j. phytochem. 2017.07.011." type="journal article" year="2017">Kolodziejczyk-Czepas et al., 2017</bibRefCitation>
;
<bibRefCitation id="EFA605FCFFE8D7492979FB2EFAD4FB49" author="Marchelak, A. &amp; Owczarek, A. &amp; Matczak, M. &amp; Pawlak, A. &amp; Kolodziejczyk-Czepas, J. &amp; Nowak, P. &amp; Olszewska, M. A." box="[1111,1339,1152,1171]" pageId="17" pageNumber="18" pagination="680" refId="ref26013" refString="Marchelak, A., Owczarek, A., Matczak, M., Pawlak, A., Kolodziejczyk-Czepas, J., Nowak, P., Olszewska, M. A., 2017. Bioactivity potential of Prunus spinosa L. Flower extracts: phytochemical profiling, cellular safety, pro-inflammatory enzymes inhibition and protective effects against oxidative stress in vitro. Front. Pharmacol. 8, 680. https: // doi. org / 10.3389 / fphar. 2017.00680." type="journal article" year="2017">Marchelak et al., 2017</bibRefCitation>
). PBMCs were suspended in 0.02 PBS only (1 ×
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PBMCs/ml) and incubated with the analysed
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<emphasis id="B943A41FFFE8D7492EA2FB16FC0EFB11" bold="true" box="[908,993,1208,1227]" italics="true" pageId="17" pageNumber="18">Herniaria</emphasis>
</taxonomicName>
fractions (5, 25 and 50 μg/ml) to assess the possibility of direct damage to the cell membrane. This cytotoxicity assay was based on a dye excluding test, employing 0.4% Trypan blue (
<bibRefCitation id="EFA605FCFFE8D749284DFB41FC5BFAC4" author="Strober, W. &amp; Strober, Warren" pageId="17" pageNumber="18" refId="ref27870" refString="Strober, W., Strober, Warren, 2001. Trypan blue exclusion test of cell viability. In: Current Protocols in Immunology. John Wiley &amp; Sons, Inc., Hoboken, NJ, USA https: // doi. org / 10.1002 / 0471142735. ima 03 bs 21. A. 3 B. 1 - A. 3 B. 2." type="book" year="2001">Strober and Strober, 2001</bibRefCitation>
). Cell viability was measured in a micro-chamber automated cell counter, after 6 h of incubation (at 37
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C, with gentle mixing on a rotary shaker).
</paragraph>
<paragraph id="8B88780DFFE8D7492E7FFAF1FBA9F9B7" blockId="17.[818,1488,1096,1645]" pageId="17" pageNumber="18">
Analyses based onTrypan blue exclusion were complemented with the resazurin-based cell viability assay. PBMCs (1.5 ×
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PBMCs/ml) were suspended in the RPMI-1640 medium (supplemented with 10% fetal calf serum and 0.1% of penicillin-streptomycin) and incubated with the fractions (5, 25 and 50 μg/ml) for 24 h (in 96-well microplates, at 37
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C, with 5% CO
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and 95% humidity). Then, resazurin solution was added to the final concentration of 10%. Measurements of cell viability were executed 4 h after resazurin application, using a BMG Labtech SectroStarNano microplate spectrophotometer, at λ =
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(
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was a reference wavelength).
</paragraph>
<paragraph id="8B88780DFFE8D7492E1CF901FBE8F918" blockId="17.[818,1031,1711,1730]" box="[818,1031,1711,1730]" pageId="17" pageNumber="18">
<heading id="D0C0CF61FFE8D7492E1CF901FBE8F918" bold="true" box="[818,1031,1711,1730]" fontSize="36" level="1" pageId="17" pageNumber="18" reason="1">
<emphasis id="B943A41FFFE8D7492E1CF901FBE8F918" bold="true" box="[818,1031,1711,1730]" italics="true" pageId="17" pageNumber="18">4.9. Statistical analysis</emphasis>
</heading>
</paragraph>
<paragraph id="8B88780DFFE8D7492E7FF946FAA4F864" blockId="17.[818,1487,1767,1982]" pageId="17" pageNumber="18">
Data are presented as mean values ± SD;
<emphasis id="B943A41FFFE8D74929CEF946FAEEF921" box="[1248,1281,1768,1787]" italics="true" pageId="17" pageNumber="18">
<emphasis id="B943A41FFFE8D74929CEF946FB05F921" bold="true" box="[1248,1258,1768,1787]" italics="true" pageId="17" pageNumber="18">p</emphasis>
&lt;
</emphasis>
0.05 was assumed as statistically significant, and “n” corresponded to the number of independent experiments/blood donors. Uncertain data were excluded by the Q-Dixon test. In experiments employing blood plasma, isolated fibrinogen and PBMCs, at least two independent pre-incubations of examined substances with biological material from each donor were performed. Statistical significance of obtained results was established by the Student’ s t-test or ANOVA and Tukey’ s post-hoc test.
</paragraph>
</subSubSection>
</treatment>
</document>
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