1298 lines
158 KiB
XML
1298 lines
158 KiB
XML
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<mods:title id="8E292A5D5B544B3B22556438ABF1FB28">Effects of N source concentration and NH / NO ratio on phenylethanoid glycoside pattern in tissue cultures of Plantago lanceolata L.: A metabolomics driven full-factorial experiment with LC-ESI-MS</mods:title>
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<mods:namePart id="DA5B3BC98D7A4A83647F7540F9B20CC8">Gonda, Sándor</mods:namePart>
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<mods:affiliation id="EA559888F123A8FF45BCA523213C8833">University of Debrecen, Department of Botany, Division of Pharmacognosy, Egyetem tér 1, H- 4010 Debrecen, Hungary</mods:affiliation>
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<mods:namePart id="3E1B9187411068FA526B28807ED8A8A0">Kiss-Szikszai, Attila</mods:namePart>
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<mods:namePart id="C286903E8089747F3A6FFCF4EE3CB5CC">Szucs, Zsolt</mods:namePart>
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<mods:namePart id="BB50ED492BBB8F161B242D61850377CE">Máthé, Csaba</mods:namePart>
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<mods:namePart id="1FEEC2463C3626BF0A92D30A74D0CB5B">Vasas, Gábor</mods:namePart>
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<mods:title id="28B8C0B5B32D4AA40D33388FBF2A1199">Phytochemistry</mods:title>
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<mods:date id="940FADAD3BBB3E8A0505748435D61987">2014</mods:date>
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<treatment id="03AE2D4F0B50615EFCD4F9D6FBF47632" ID-DOI="http://doi.org/10.5281/zenodo.10561675" ID-GBIF-Taxon="220883739" ID-Zenodo-Dep="10561675" LSID="urn:lsid:plazi:treatment:03AE2D4F0B50615EFCD4F9D6FBF47632" httpUri="http://treatment.plazi.org/id/03AE2D4F0B50615EFCD4F9D6FBF47632" lastPageId="7" lastPageNumber="51" pageId="1" pageNumber="45">
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<subSubSection id="C31DCFD20B506158FCD4F9D6FAC2767A" box="[831,1358,1632,1652]" pageId="1" pageNumber="45" type="nomenclature">
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<paragraph id="8BB89C590B506158FCD4F9D6FAC2767A" blockId="1.[831,1358,1632,1652]" box="[831,1358,1632,1652]" pageId="1" pageNumber="45">
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<heading id="D0F02B350B506158FCD4F9D6FAC2767A" box="[831,1358,1632,1652]" fontSize="36" level="2" pageId="1" pageNumber="45" reason="3">
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<emphasis id="B973404B0B506158FCD4F9D6FAC2767A" box="[831,1358,1632,1652]" italics="true" pageId="1" pageNumber="45">
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2.1. Effects of N source on growth of
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||
<taxonomicName id="4C07E7DA0B506158FB4EF9D6FAAC767A" ID-CoL="4JLMM" authorityName="L." box="[1189,1312,1632,1652]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="1" pageNumber="45" phylum="Tracheophyta" rank="species" species="lanceolata">P. lanceolata</taxonomicName>
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calli
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</emphasis>
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</heading>
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</paragraph>
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<paragraph id="8BB89C590B506158FCB5F92FFA03770E" blockId="1.[831,1501,1688,2017]" pageId="1" pageNumber="45">
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||
The calli of
|
||
<taxonomicName id="4C07E7DA0B506158FC3CF92EFBDE76A2" box="[983,1106,1688,1708]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="1" pageNumber="45" phylum="Tracheophyta" rank="species" species="lanceolata">
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||
<emphasis id="B973404B0B506158FC3CF92EFBDE76A2" box="[983,1106,1688,1708]" italics="true" pageId="1" pageNumber="45">P. lanceolata</emphasis>
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</taxonomicName>
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||
were cultured on modified Murashige Skoog media with different NH
|
||
<subScript id="17839E1C0B506158FB90F908FB0876C4" attach="left" box="[1147,1156,1726,1738]" fontSize="5" pageId="1" pageNumber="45">4</subScript>
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<superScript id="7C7231110B506158FB90F907FB0876B3" attach="left" box="[1147,1156,1713,1725]" fontSize="5" pageId="1" pageNumber="45">+</superScript>
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/NO
|
||
<subScript id="17839E1C0B506158FB47F908FB3976C4" attach="left" box="[1196,1205,1726,1738]" fontSize="5" pageId="1" pageNumber="45">3</subScript>
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<emphasis id="B973404B0B506158FB47F907FB3476B3" box="[1196,1208,1713,1725]" italics="true" pageId="1" pageNumber="45">
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<superScript id="7C7231110B506158FB47F907FB3476B3" attach="left" box="[1196,1208,1713,1725]" fontSize="5" pageId="1" pageNumber="45">–</superScript>
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</emphasis>
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ratios and N source concentrations in a full factorial experiment. For experimental design, nomenclature of media and details, please see Section 4.4.
|
||
</paragraph>
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||
<paragraph id="8BB89C590B50615BFCB5F8BEFDE171EE" blockId="1.[831,1501,1688,2017]" lastBlockId="2.[87,757,178,481]" lastPageId="2" lastPageNumber="46" pageId="1" pageNumber="45">
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Growth indices on different media ranged from 4.52 ± 0.10 (40(0.33)) to 9.87 ± 3.31 (20(0)). Unsurprisingly, the concentration and composition of the N source influenced the growth of the
|
||
<taxonomicName id="4C07E7DA0B506158FCD4F8EDFC3B7761" authority="TC. A" authorityName="TC. A" box="[831,951,1883,1903]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="1" pageNumber="45" phylum="Tracheophyta" rank="species" species="lanceolata">
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<emphasis id="B973404B0B506158FCD4F8EDFC3B7761" box="[831,951,1883,1903]" italics="true" pageId="1" pageNumber="45">P. lanceolata</emphasis>
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</taxonomicName>
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||
TC. A more detailed insight was obtained by subjecting all known medium parameters to ANOVA models, with the growth index being the response variable. It was shown, that N source concentration (ranging from 10 to 60 mM) did not significantly alter growth of the calli, while NH
|
||
<subScript id="17839E1C0B506158FB80F863FBF877EF" attach="left" box="[1131,1140,2005,2017]" fontSize="5" pageId="1" pageNumber="45">4</subScript>
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<superScript id="7C7231110B506158FB80F87EFBF877DA" attach="left" box="[1131,1140,1992,2004]" fontSize="5" pageId="1" pageNumber="45">+</superScript>
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/NO
|
||
<subScript id="17839E1C0B506158FB70F863FB2877EF" attach="left" box="[1179,1188,2005,2017]" fontSize="5" pageId="1" pageNumber="45">3</subScript>
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<emphasis id="B973404B0B506158FB70F87EFB2B77DA" box="[1179,1191,1992,2004]" italics="true" pageId="1" pageNumber="45">
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<superScript id="7C7231110B506158FB70F87EFB2B77DA" attach="left" box="[1179,1191,1992,2004]" fontSize="5" pageId="1" pageNumber="45">–</superScript>
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</emphasis>
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||
ratio had a more pronounced effect. Addition of different amounts of NH
|
||
<subScript id="17839E1C0B53615BFDC8FF09FDA070C5" attach="left" box="[547,556,191,203]" fontSize="5" pageId="2" pageNumber="46">4</subScript>
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<superScript id="7C7231110B53615BFDC8FF04FDA070B0" attach="left" box="[547,556,178,190]" fontSize="5" pageId="2" pageNumber="46">+</superScript>
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and NO
|
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<subScript id="17839E1C0B53615BFD63FF09FD1D70C5" attach="left" box="[648,657,191,203]" fontSize="5" pageId="2" pageNumber="46">3</subScript>
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<emphasis id="B973404B0B53615BFD63FF05FD1870B1" box="[648,660,179,191]" italics="true" pageId="2" pageNumber="46">
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<superScript id="7C7231110B53615BFD63FF05FD1870B1" attach="left" box="[648,660,179,191]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
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</emphasis>
|
||
required counter-ions, which were Na
|
||
<superScript id="7C7231110B53615BFE68FF78FE0070D4" attach="left" box="[387,396,206,218]" fontSize="5" pageId="2" pageNumber="46">+</superScript>
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and Cl
|
||
<emphasis id="B973404B0B53615BFE33FF79FE6870D5" box="[472,484,207,219]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFE33FF79FE6870D5" attach="left" box="[472,484,207,219]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
, added in the range 0–41 and 0–16 mM, respectively, to the different media. The amount of these ions was shown to have only insignificant effects on growth of the TC. Higher concentrations of NH
|
||
<superScript id="7C7231110B53615BFDB4FE94FDE47120" attach="left" box="[607,616,290,302]" fontSize="5" pageId="2" pageNumber="46">+</superScript>
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<subScript id="17839E1C0B53615BFDB4FE99FDE47135" attach="left" box="[607,616,303,315]" fontSize="5" pageId="2" pageNumber="46">4</subScript>
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have led to decreased growth of the tissue cultures. This is in accordance with that described by
|
||
<bibRefCitation id="EF96E1A80B53615BFEE7FEE8FD91717F" author="Budzianowska, A. & Skrzypczak, L. & Budzianowski, J." box="[268,541,350,369]" pageId="2" pageNumber="46" pagination="834 - 840" refId="ref11162" refString="Budzianowska, A., Skrzypczak, L., Budzianowski, J., 2004. Phenylethanoid glucosides from in vitro propagated plants and callus cultures of Plantago lanceolata. Planta Med. 70, 834 - 840. http: // dx. doi. org / 10.1055 / s- 2004 - 827232." type="journal article" year="2004">Budzianowska et al. (2004)</bibRefCitation>
|
||
and is usually attributed to the direct toxicity of excess NH
|
||
<superScript id="7C7231110B53615BFE00FEC0FE78718C" attach="left" box="[491,500,374,386]" fontSize="5" pageId="2" pageNumber="46">+</superScript>
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<subScript id="17839E1C0B53615BFE00FE35FE787181" attach="left" box="[491,500,387,399]" fontSize="5" pageId="2" pageNumber="46">4</subScript>
|
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at higher concentrations (
|
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<bibRefCitation id="EF96E1A80B53615BFFB4FE20FE9B71A7" author="George, E. F. & Hall, M. A. & Klerk, G. - J. D." box="[95,279,405,425]" pageId="2" pageNumber="46" pagination="186" refId="ref11390" refString="George, E. F., Hall, M. A., Klerk, G. - J. D., 2007. Plant Propagation by Tissue Culture: Volume 1. The Background, first ed. Springer, pp. 71, 73, 186." type="journal article" year="2007">George et al., 2007</bibRefCitation>
|
||
). Examination of the effects on growth was secondary, our main focus was to detect the changes in the metabolome, which will be detailed in the following sections.
|
||
</paragraph>
|
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<paragraph id="8BB89C590B53615BFFBCFDB1FDC97215" blockId="2.[87,581,519,539]" box="[87,581,519,539]" pageId="2" pageNumber="46">
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<heading id="D0F02B350B53615BFFBCFDB1FDC97215" box="[87,581,519,539]" fontSize="36" level="2" pageId="2" pageNumber="46" reason="3">
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<emphasis id="B973404B0B53615BFFBCFDB1FDC97215" box="[87,581,519,539]" italics="true" pageId="2" pageNumber="46">
|
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2.2. Phenylethanoid glycosides in
|
||
<taxonomicName id="4C07E7DA0B53615BFE76FDB1FD947215" authorityName="L." box="[413,536,519,539]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="2" pageNumber="46" phylum="Tracheophyta" rank="subSpecies" species="lanceolata">P. lanceolata</taxonomicName>
|
||
calli
|
||
</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B53615BFF9DFD89FD9E738B" blockId="2.[87,757,575,1292]" pageId="2" pageNumber="46">
|
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After a few preliminary tests, calli from media with 10 mM N were selected for the qualitative study. Ten microliters of the concentrated MeOH extract were injected into a semi-preparative HPLC system (Supelco C
|
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<subScript id="17839E1C0B53615BFEFFFD2AFEAB72A6" attach="left" box="[276,295,668,680]" fontSize="5" pageId="2" pageNumber="46">18</subScript>
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column,
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<quantity id="4CFF31BC0B53615BFE61FD25FE5172A9" box="[394,477,659,679]" metricMagnitude="-1" metricUnit="m" metricValue="2.5" pageId="2" pageNumber="46" unit="mm" value="250.0">250 mm</quantity>
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<emphasis id="B973404B0B53615BFE08FD22FE7872A8" box="[483,500,660,678]" italics="true" pageId="2" pageNumber="46">×</emphasis>
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<emphasis id="B973404B0B53615BFDAEFD22FDDA72A8" box="[581,598,660,678]" italics="true" pageId="2" pageNumber="46">×</emphasis>
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5 µm; gradient was adapted from the LC–MS method described in Section 4.5) for HPLC-UV–Vis measurement. Most of the NPs in the polarity range examined later were found to have the UV–Vis spectrum characteristic to phenylethanoid glycosides, i.e. maxima around 248, 290 and
|
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<quantity id="4CFF31BC0B53615BFFBCFCA9FF2F733C" box="[87,163,799,818]" metricMagnitude="-7" metricUnit="m" metricValue="3.3" pageId="2" pageNumber="46" unit="nm" value="330.0">330 nm</quantity>
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were found (
|
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<bibRefCitation id="EF96E1A80B53615BFEC7FCA8FE4C733C" author="Shi, Y. & Wu, C. & Chen, Y. & Liu, W. & Feng, F. & Xie, N." box="[300,448,798,818]" pageId="2" pageNumber="46" pagination="239 - 247" refId="ref12642" refString="Shi, Y., Wu, C., Chen, Y., Liu, W., Feng, F., Xie, N., 2013. Comparative analysis of three Callicarpa herbs using high performance liquid chromatography with diode array detector and electrospray ionization - trap mass spectrometry method. J. Pharm. Biomed. Anal. 75, 239 - 247. http: // dx. doi. org / 10.1016 / j. jpba. 2012.11.038." type="journal article" year="2013">Shi et al., 2013</bibRefCitation>
|
||
). These wavelengths are unaffected by methylation of the aglyca (
|
||
<bibRefCitation id="EF96E1A80B53615BFE2FFC8CFDD97340" author="Shi, Y. & Wu, C. & Chen, Y. & Liu, W. & Feng, F. & Xie, N." box="[452,597,826,846]" pageId="2" pageNumber="46" pagination="239 - 247" refId="ref12642" refString="Shi, Y., Wu, C., Chen, Y., Liu, W., Feng, F., Xie, N., 2013. Comparative analysis of three Callicarpa herbs using high performance liquid chromatography with diode array detector and electrospray ionization - trap mass spectrometry method. J. Pharm. Biomed. Anal. 75, 239 - 247. http: // dx. doi. org / 10.1016 / j. jpba. 2012.11.038." type="journal article" year="2013">Shi et al., 2013</bibRefCitation>
|
||
). The same pattern was found by
|
||
<bibRefCitation id="EF96E1A80B53615BFEFBFCE0FD917367" author="Budzianowska, A. & Skrzypczak, L. & Budzianowski, J." box="[272,541,854,874]" pageId="2" pageNumber="46" pagination="834 - 840" refId="ref11162" refString="Budzianowska, A., Skrzypczak, L., Budzianowski, J., 2004. Phenylethanoid glucosides from in vitro propagated plants and callus cultures of Plantago lanceolata. Planta Med. 70, 834 - 840. http: // dx. doi. org / 10.1055 / s- 2004 - 827232." type="journal article" year="2004">Budzianowska et al. (2004)</bibRefCitation>
|
||
, who reported lack of other phenolic NP groups in
|
||
<taxonomicName id="4C07E7DA0B53615BFE9FFCC7FE61738B" authority="TC." authorityName="TC." box="[372,493,881,901]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="2" pageNumber="46" phylum="Tracheophyta" rank="species" species="lanceolata">
|
||
<emphasis id="B973404B0B53615BFE9FFCC7FE61738B" box="[372,493,881,901]" italics="true" pageId="2" pageNumber="46">P. lanceolata</emphasis>
|
||
</taxonomicName>
|
||
TC.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B53615BFF9DFC38FAA970E8" blockId="2.[87,757,575,1292]" lastBlockId="2.[805,1475,182,1290]" pageId="2" pageNumber="46">
|
||
Negative mode LC–ESI–MS
|
||
<superScript id="7C7231110B53615BFE61FC3CFE1F7398" attach="left" box="[394,403,906,918]" fontSize="5" pageId="2" pageNumber="46">3</superScript>
|
||
was shown to be a very suitable method for characterization of the NPs from the MeOH extracts of
|
||
<taxonomicName id="4C07E7DA0B53615BFF92FC73FF7673D7" box="[121,250,965,985]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="2" pageNumber="46" phylum="Tracheophyta" rank="species" species="lanceolat">
|
||
<emphasis id="B973404B0B53615BFF92FC73FF6373D7" box="[121,239,965,985]" italics="true" pageId="2" pageNumber="46">P. lanceolat</emphasis>
|
||
a
|
||
</taxonomicName>
|
||
calli. A high number of [M
|
||
<emphasis id="B973404B0B53615BFDD3FC70FDC573D6" box="[568,585,966,984]" italics="true" pageId="2" pageNumber="46">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B53615BFD8AFC74FDE173C0" box="[609,621,962,974]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFD8AFC74FDE173C0" attach="left" box="[609,621,962,974]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
peaks were detected in the total ion chromatogram (
|
||
<figureCitation id="133C80DC0B53615BFE05FC54FDAF73FB" box="[494,547,994,1013]" captionStart="Fig" captionStartId="2.[87,113,1930,1944]" captionTargetBox="[209,1337,1371,1901]" captionTargetId="figure-896@2.[208,1344,1370,1901]" captionTargetPageId="2" captionText="Fig. 2. LC–ESI–MS chromatogram of the MeOH extract of a P.lanceolata callus grown on medium 10(0.33) (25 mg dry wt./ml, total ion chromatogram in negative ion mode). Note that in this injection, the area under curve of plantamajoside was beyond the linear range of determination, and thus, major NPs were quantified in the 100-fold dilution, and not from the chromatogram presented here.Main peaks are marked with the metabolite numbers used in text, see Table 1 for putative identification.NPs compared with standards are marked with an asterisk. For m/z values, and putative identification, see Table 1. Abbreviations: AC, acteoside; PM, plantamajoside." figureDoi="http://doi.org/10.5281/zenodo.10491348" httpUri="https://zenodo.org/record/10491348/files/figure.png" pageId="2" pageNumber="46">Fig. 2</figureCitation>
|
||
). Putative identification was carried out using detailed study of the fragmentation patterns of the two authentic standards plantamajoside and acteoside, application to the similar PGs, and consulting the scientific literature. Fragmentation patterns of most PGs also present in these samples are described in
|
||
<bibRefCitation id="EF96E1A80B53615BFEB1FBDBFE63748E" author="Li, L. & Liu, C. & Liu, Z. & Tsao, R. & Liu, S." box="[346,495,1133,1153]" pageId="2" pageNumber="46" pagination="541 - 545" refId="ref12101" refString="Li, L., Liu, C., Liu, Z., Tsao, R., Liu, S., 2009. Identification of phenylethanoid glycosides in plant extract of Plantago asiatica L. by liquid chromatography - electrospray ionization mass spectrometry. Chin. J. Chem. 27, 541 - 545. http: // dx. doi. org / 10.1002 / cjoc. 200990088." type="journal article" year="2009">Li et al. (2009)</bibRefCitation>
|
||
,
|
||
<bibRefCitation id="EF96E1A80B53615BFE11FBDBFD5F748E" author="Petreska, J. & Stefkov, G. & Kulevanova, S. & Alipieva, K. & Bankova, V. & Stefova, M." box="[506,723,1133,1153]" pageId="2" pageNumber="46" pagination="21 - 30" refId="ref12270" refString="Petreska, J., Stefkov, G., Kulevanova, S., Alipieva, K., Bankova, V., Stefova, M., 2011. Phenolic compounds of mountain tea from the Balkans: LC / DAD / ESI / MS n profile and content. Nat. Prod. Commun. 6, 21 - 30." type="journal article" year="2011">Petreska et al. (2011)</bibRefCitation>
|
||
,
|
||
<bibRefCitation id="EF96E1A80B53615BFD35FBDBFF437492" author="Qi, M. & Xiong, A. & Geng, F. & Yang, L. & Wang, Z." pageId="2" pageNumber="46" pagination="1470 - 1478" refId="ref12400" refString="Qi, M., Xiong, A., Geng, F., Yang, L., Wang, Z., 2012. A novel strategy for target profiling analysis of bioactive phenylethanoid glycosides in Plantago medicinal plants using ultra-performance liquid chromatography coupled with tandem quadrupole mass spectrometry. J. Sep. Sci. 35, 1470 - 1478. http: // dx. doi. org / 10.1002 / jssc. 201200010." type="journal article" year="2012">Qi et al. (2012)</bibRefCitation>
|
||
and
|
||
<bibRefCitation id="EF96E1A80B53615BFEEBFB3FFE107492" author="Shi, Y. & Wu, C. & Chen, Y. & Liu, W. & Feng, F. & Xie, N." box="[256,412,1161,1181]" pageId="2" pageNumber="46" pagination="239 - 247" refId="ref12642" refString="Shi, Y., Wu, C., Chen, Y., Liu, W., Feng, F., Xie, N., 2013. Comparative analysis of three Callicarpa herbs using high performance liquid chromatography with diode array detector and electrospray ionization - trap mass spectrometry method. J. Pharm. Biomed. Anal. 75, 239 - 247. http: // dx. doi. org / 10.1016 / j. jpba. 2012.11.038." type="journal article" year="2013">Shi et al. (2013)</bibRefCitation>
|
||
. For practical reasons and to show the ‘‘taxonomy’’ of the detected molecules and fragments, the abbreviations of the PG subunits (glucose (Glc), caffeic acid (CA), hydroxytyrosol (HT), etc.) will be used to indicate what parts make up a particular fragment: e.g. [HT-Glc(Glc)-CA – H]
|
||
<emphasis id="B973404B0B53615BFDBEFB43FDED750F" box="[597,609,1269,1281]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFDBEFB43FDED750F" attach="left" box="[597,609,1269,1281]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
is the
|
||
<emphasis id="B973404B0B53615BFD4FFB4EFD3B7502" box="[676,695,1272,1292]" italics="true" pageId="2" pageNumber="46">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B53615BFD54FB4EFD457502" box="[703,713,1272,1292]" italics="true" pageId="2" pageNumber="46">z</emphasis>
|
||
639 ion shown in
|
||
<figureCitation id="133C80DC0B53615BFC53FF00FC7D70C7" box="[952,1009,182,201]" captionStart="Fig" captionStartId="3.[113,139,1320,1334]" captionTargetBox="[348,1255,182,1289]" captionTargetId="figure-1@3.[348,1258,181,1290]" captionTargetPageId="3" captionText="Fig. 3. Proposed fragmentation pattern of plantamajoside, the main phenylethanoid glycoside of the P. lanceolata callus extract in negative mode LC–ESI–MS3. The main fragmentation route is highlighted with bold arrows and bold font." figureDoi="http://doi.org/10.5281/zenodo.10491350" httpUri="https://zenodo.org/record/10491350/files/figure.png" pageId="2" pageNumber="46">Fig. 3</figureCitation>
|
||
. The NPs that could be putatively identified from the MS
|
||
<superScript id="7C7231110B53615BFC4FFF78FC2170D4" attach="left" box="[932,941,206,218]" fontSize="5" pageId="2" pageNumber="46">3</superScript>
|
||
fragmentation are shown in
|
||
<tableCitation id="C685A9E20B53615BFB3DFF64FAAD70EB" box="[1238,1313,210,229]" captionStart="Table 1" captionStartId="3.[114,158,1416,1430]" captionTargetPageId="3" captionText="Table 1 Fragmentation pattern of putatively identified major peaks obtained by LC–ESI–MS3 analysis of defatted Plantago lanceolata calli MeOH extracts in negative ion mode. MS2 ions are CID products of the [M–H] –, while MS3 ions are CID products of the most abundant MS2 ion (MS2a)." httpUri="http://table.plazi.org/id/DF78CCD10B52615AFF99FA3EFC3D75CD" pageId="2" pageNumber="46" tableUuid="DF78CCD10B52615AFF99FA3EFC3D75CD">Table 1</tableCitation>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B53615BFCAFFF58FA1C723A" blockId="2.[805,1475,182,1290]" pageId="2" pageNumber="46">
|
||
Major PGs were shown to be plantamajoside (
|
||
<emphasis id="B973404B0B53615BFAD5FF58FADB710F" bold="true" box="[1342,1367,238,257]" pageId="2" pageNumber="46">18</emphasis>
|
||
, [M
|
||
<emphasis id="B973404B0B53615BFA6EFF59FA1A710F" box="[1413,1430,239,257]" italics="true" pageId="2" pageNumber="46">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B53615BFA45FF5CFA3670F8" box="[1454,1466,234,246]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFA45FF5CFA3670F8" attach="none" box="[1454,1466,234,246]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
: [HT-Glc(Glc)-CA – H]
|
||
<emphasis id="B973404B0B53615BFC17FEB0FB84711C" box="[1020,1032,262,274]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFC17FEB0FB84711C" attach="none" box="[1020,1032,262,274]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
) and acteoside (verbascoside,
|
||
<emphasis id="B973404B0B53615BFAA9FEBCFAD77113" bold="true" box="[1346,1371,266,285]" pageId="2" pageNumber="46">27</emphasis>
|
||
, [M
|
||
<emphasis id="B973404B0B53615BFA6EFEBDFA1A7113" box="[1413,1430,267,285]" italics="true" pageId="2" pageNumber="46">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B53615BFA45FEB0FA36711C" box="[1454,1466,262,274]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFA45FEB0FA36711C" attach="none" box="[1454,1466,262,274]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
: [HT-Glc(Rha)-CA – H]
|
||
<emphasis id="B973404B0B53615BFBEDFE94FB9E7120" box="[1030,1042,290,302]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFBEDFE94FB9E7120" attach="none" box="[1030,1042,290,302]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
), their retention times and mass spectra were identical with our authentic standards (
|
||
<figureCitation id="133C80DC0B53615BFB04FEF4FAA9715B" box="[1263,1317,322,341]" captionStart="Fig" captionStartId="2.[87,113,1930,1944]" captionTargetBox="[209,1337,1371,1901]" captionTargetId="figure-896@2.[208,1344,1370,1901]" captionTargetPageId="2" captionText="Fig. 2. LC–ESI–MS chromatogram of the MeOH extract of a P.lanceolata callus grown on medium 10(0.33) (25 mg dry wt./ml, total ion chromatogram in negative ion mode). Note that in this injection, the area under curve of plantamajoside was beyond the linear range of determination, and thus, major NPs were quantified in the 100-fold dilution, and not from the chromatogram presented here.Main peaks are marked with the metabolite numbers used in text, see Table 1 for putative identification.NPs compared with standards are marked with an asterisk. For m/z values, and putative identification, see Table 1. Abbreviations: AC, acteoside; PM, plantamajoside." figureDoi="http://doi.org/10.5281/zenodo.10491348" httpUri="https://zenodo.org/record/10491348/files/figure.png" pageId="2" pageNumber="46">Fig. 2</figureCitation>
|
||
). Their isomers
|
||
<emphasis id="B973404B0B53615BFCCEFEEBFCCA717E" bold="true" box="[805,838,349,368]" pageId="2" pageNumber="46">(43</emphasis>
|
||
for
|
||
<emphasis id="B973404B0B53615BFC9BFEE8FC05717F" bold="true" box="[880,905,350,369]" pageId="2" pageNumber="46">27</emphasis>
|
||
and
|
||
<emphasis id="B973404B0B53615BFC57FEE8FC59717F" bold="true" box="[956,981,350,369]" pageId="2" pageNumber="46">37</emphasis>
|
||
,
|
||
<emphasis id="B973404B0B53615BFC09FEE8FC77717F" bold="true" box="[994,1019,350,369]" pageId="2" pageNumber="46">49</emphasis>
|
||
for
|
||
<emphasis id="B973404B0B53615BFBCEFEE8FBB2717F" bold="true" box="[1061,1086,350,369]" pageId="2" pageNumber="46">18</emphasis>
|
||
) were also abundant, these can either be isoacteoside and isoplantamajoside, or cis-acteoside and cis-plantamajoside (
|
||
<bibRefCitation id="EF96E1A80B53615BFC19FE23FBF071A7" author="Li, L. & Liu, C. & Liu, Z. & Tsao, R. & Liu, S." box="[1010,1148,405,425]" pageId="2" pageNumber="46" pagination="541 - 545" refId="ref12101" refString="Li, L., Liu, C., Liu, Z., Tsao, R., Liu, S., 2009. Identification of phenylethanoid glycosides in plant extract of Plantago asiatica L. by liquid chromatography - electrospray ionization mass spectrometry. Chin. J. Chem. 27, 541 - 545. http: // dx. doi. org / 10.1002 / cjoc. 200990088." type="journal article" year="2009">Li et al., 2009</bibRefCitation>
|
||
) – cis/trans pairs can appear as separate peaks in HPLC according to
|
||
<bibRefCitation id="EF96E1A80B53615BFB4EFE07FA3171CA" author="Budzianowska, A. & Skrzypczak, L. & Budzianowski, J." box="[1189,1469,433,453]" pageId="2" pageNumber="46" pagination="834 - 840" refId="ref11162" refString="Budzianowska, A., Skrzypczak, L., Budzianowski, J., 2004. Phenylethanoid glucosides from in vitro propagated plants and callus cultures of Plantago lanceolata. Planta Med. 70, 834 - 840. http: // dx. doi. org / 10.1055 / s- 2004 - 827232." type="journal article" year="2004">Budzianowska et al. (2004)</bibRefCitation>
|
||
, even from solution of purified standards. The fragmentation routes of
|
||
<emphasis id="B973404B0B53615BFCD5FE5FFCDB71F2" bold="true" box="[830,855,489,508]" pageId="2" pageNumber="46">18</emphasis>
|
||
and
|
||
<emphasis id="B973404B0B53615BFC6CFE5FFC2C71F2" bold="true" box="[903,928,489,508]" pageId="2" pageNumber="46">27</emphasis>
|
||
were identical to that of acteoside (
|
||
<bibRefCitation id="EF96E1A80B53615BFAECFE5FFA0671F2" author="Li, L. & Liu, C. & Liu, Z. & Tsao, R. & Liu, S." box="[1287,1418,489,508]" pageId="2" pageNumber="46" pagination="541 - 545" refId="ref12101" refString="Li, L., Liu, C., Liu, Z., Tsao, R., Liu, S., 2009. Identification of phenylethanoid glycosides in plant extract of Plantago asiatica L. by liquid chromatography - electrospray ionization mass spectrometry. Chin. J. Chem. 27, 541 - 545. http: // dx. doi. org / 10.1002 / cjoc. 200990088." type="journal article" year="2009">Li et al., 2009</bibRefCitation>
|
||
). The proposed fragmentation pattern of plantamajoside is presented in
|
||
<figureCitation id="133C80DC0B53615BFCCEFD97FCD7723A" box="[805,859,545,564]" captionStart="Fig" captionStartId="3.[113,139,1320,1334]" captionTargetBox="[348,1255,182,1289]" captionTargetId="figure-1@3.[348,1258,181,1290]" captionTargetPageId="3" captionText="Fig. 3. Proposed fragmentation pattern of plantamajoside, the main phenylethanoid glycoside of the P. lanceolata callus extract in negative mode LC–ESI–MS3. The main fragmentation route is highlighted with bold arrows and bold font." figureDoi="http://doi.org/10.5281/zenodo.10491350" httpUri="https://zenodo.org/record/10491350/files/figure.png" pageId="2" pageNumber="46">Fig. 3</figureCitation>
|
||
, that of acteoside is presented in Supplementary
|
||
<figureCitation id="133C80DC0B53615BFABFFD97FA07723A" box="[1364,1419,545,564]" captionStart="Fig" captionStartId="1.[113,139,470,484]" captionTargetBox="[259,635,182,440]" captionTargetId="figure-1094@1.[258,638,181,441]" captionTargetPageId="1" captionText="Fig. 1. Selected phenylethanoid glycosides detected from the MeOH extracts of P. lanceolata calli by LC–ESI–MS3. Plantamajoside, R 1 = Glc, R 2 = H; Acteoside, R 1 = Rha, R2 = H; Desrhamnosyl-acteoside, R1 = R2 = H; Lavandulifolioside, R1 = Ara(1->2)Rha, R2 = H; Leucosceptoside A, R1 = Rha, R2 = Me. For details of identification, see text and Table 1. Abbreviations: Ara, arabinose; Glc, glucose; Rha, rhamnose." figureDoi="http://doi.org/10.5281/zenodo.10491346" httpUri="https://zenodo.org/record/10491346/files/figure.png" pageId="2" pageNumber="46">Fig. 1</figureCitation>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B53615BFCAFFD8BFC03738D" blockId="2.[805,1475,182,1290]" pageId="2" pageNumber="46">
|
||
More polar PGs were detected at lower retention times.
|
||
<emphasis id="B973404B0B53615BFA6DFD8BFA1F725E" bold="true" box="[1414,1427,573,592]" pageId="2" pageNumber="46">8</emphasis>
|
||
was putatively identified as a PG bearing an additional glucose (hexose) on plantamajoside, presenting the [HT-Glc(Glc,Glc)-CA – H]
|
||
<emphasis id="B973404B0B53615BFA5DFDC7FA4E7273" box="[1462,1474,625,637]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFA5DFDC7FA4E7273" attach="none" box="[1462,1474,625,637]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
deprotonated molecular ion (
|
||
<emphasis id="B973404B0B53615BFB99FD39FB0972AD" box="[1138,1157,655,675]" italics="true" pageId="2" pageNumber="46">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B53615BFB67FD39FB1A72AD" box="[1164,1174,655,675]" italics="true" pageId="2" pageNumber="46">z</emphasis>
|
||
801). In MS
|
||
<superScript id="7C7231110B53615BFADEFD3BFAB27297" attach="left" box="[1333,1342,653,665]" fontSize="5" pageId="2" pageNumber="46">2</superScript>
|
||
, it yielded [HT-Glc(Glc)-CA – H]
|
||
<emphasis id="B973404B0B53615BFC13FD1FFB8872BB" box="[1016,1028,681,693]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFC13FD1FFB8872BB" attach="left" box="[1016,1028,681,693]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
([M
|
||
<emphasis id="B973404B0B53615BFBC5FD1BFBB372B1" box="[1070,1087,685,703]" italics="true" pageId="2" pageNumber="46">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B53615BFBBCFD1FFBEF72BB" box="[1111,1123,681,693]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFBBCFD1FFBEF72BB" attach="left" box="[1111,1123,681,693]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
of
|
||
<emphasis id="B973404B0B53615BFB68FD1AFB1072B1" bold="true" box="[1155,1180,684,703]" pageId="2" pageNumber="46">18</emphasis>
|
||
), which could be fragmented further, and yielded the same fragments, as
|
||
<emphasis id="B973404B0B53615BFB07FD7EFA8972D5" bold="true" box="[1260,1285,712,731]" pageId="2" pageNumber="46">18</emphasis>
|
||
(
|
||
<emphasis id="B973404B0B53615BFAFDFD71FAA572D5" box="[1302,1321,711,731]" italics="true" pageId="2" pageNumber="46">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B53615BFADAFD71FAB772D5" box="[1329,1339,711,731]" italics="true" pageId="2" pageNumber="46">z</emphasis>
|
||
477 and
|
||
<emphasis id="B973404B0B53615BFA75FD71FA3D72D5" box="[1438,1457,711,731]" italics="true" pageId="2" pageNumber="46">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B53615BFA53FD71FA4E72D5" box="[1464,1474,711,731]" italics="true" pageId="2" pageNumber="46">z</emphasis>
|
||
315).
|
||
<emphasis id="B973404B0B53615BFC8AFD52FCF672F9" bold="true" box="[865,890,740,759]" pageId="2" pageNumber="46">19</emphasis>
|
||
was putatively identified as lavandulifolioside, showing fragmentation described in
|
||
<bibRefCitation id="EF96E1A80B53615BFBA9FCB6FA90731D" author="Schmidt, S. & Jakab, M. & Jav, S. & Streif, D. & Pitschmann, A. & Zehl, M. & Purevsuren, S. & Glasl, S. & Ritter, M." box="[1090,1308,768,788]" pageId="2" pageNumber="46" pagination="85 - 94" refId="ref12566" refString="Schmidt, S., Jakab, M., Jav, S., Streif, D., Pitschmann, A., Zehl, M., Purevsuren, S., Glasl, S., Ritter, M., 2013. Extracts from Leonurus sibiricus L. increase insulin secretion and proliferation of rat INS- 1 E insulinoma cells. J. Ethnopharmacol. 150, 85 - 94." type="journal article" year="2013">Schmidt et al. (2013)</bibRefCitation>
|
||
. The compound was described in
|
||
<taxonomicName id="4C07E7DA0B53615BFC3EFCADFBDC7321" box="[981,1104,795,815]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="2" pageNumber="46" phylum="Tracheophyta" rank="species" species="lanceolata">
|
||
<emphasis id="B973404B0B53615BFC3EFCADFBDC7321" box="[981,1104,795,815]" italics="true" pageId="2" pageNumber="46">P. lanceolata</emphasis>
|
||
</taxonomicName>
|
||
leaves, and micropropagated plants, but could not be detected from calli so far (
|
||
<bibRefCitation id="EF96E1A80B53615BFB1BFC8EFCDB7369" author="Budzianowska, A. & Skrzypczak, L. & Budzianowski, J." pageId="2" pageNumber="46" pagination="834 - 840" refId="ref11162" refString="Budzianowska, A., Skrzypczak, L., Budzianowski, J., 2004. Phenylethanoid glucosides from in vitro propagated plants and callus cultures of Plantago lanceolata. Planta Med. 70, 834 - 840. http: // dx. doi. org / 10.1055 / s- 2004 - 827232." type="journal article" year="2004">Budzianowska et al., 2004</bibRefCitation>
|
||
). Proposed fragmentation scheme is available in Supplementary
|
||
<figureCitation id="133C80DC0B53615BFCBFFCC6FC07738D" box="[852,907,880,899]" captionStart="Fig" captionStartId="2.[87,113,1930,1944]" captionTargetBox="[209,1337,1371,1901]" captionTargetId="figure-896@2.[208,1344,1370,1901]" captionTargetPageId="2" captionText="Fig. 2. LC–ESI–MS chromatogram of the MeOH extract of a P.lanceolata callus grown on medium 10(0.33) (25 mg dry wt./ml, total ion chromatogram in negative ion mode). Note that in this injection, the area under curve of plantamajoside was beyond the linear range of determination, and thus, major NPs were quantified in the 100-fold dilution, and not from the chromatogram presented here.Main peaks are marked with the metabolite numbers used in text, see Table 1 for putative identification.NPs compared with standards are marked with an asterisk. For m/z values, and putative identification, see Table 1. Abbreviations: AC, acteoside; PM, plantamajoside." figureDoi="http://doi.org/10.5281/zenodo.10491348" httpUri="https://zenodo.org/record/10491348/files/figure.png" pageId="2" pageNumber="46">Fig. 2</figureCitation>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B53615BFCAFFC3DFB2F7470" blockId="2.[805,1475,182,1290]" pageId="2" pageNumber="46">
|
||
Simpler PGs were also found in the calli, as also presented in
|
||
<bibRefCitation id="EF96E1A80B53615BFCCEFC11FBB773B5" author="Budzianowska, A. & Skrzypczak, L. & Budzianowski, J." box="[805,1083,935,955]" pageId="2" pageNumber="46" pagination="834 - 840" refId="ref11162" refString="Budzianowska, A., Skrzypczak, L., Budzianowski, J., 2004. Phenylethanoid glucosides from in vitro propagated plants and callus cultures of Plantago lanceolata. Planta Med. 70, 834 - 840. http: // dx. doi. org / 10.1055 / s- 2004 - 827232." type="journal article" year="2004">Budzianowska et al. (2004)</bibRefCitation>
|
||
.
|
||
<emphasis id="B973404B0B53615BFBA3FC11FBED73B4" bold="true" box="[1096,1121,935,954]" pageId="2" pageNumber="46">22</emphasis>
|
||
and
|
||
<emphasis id="B973404B0B53615BFB7DFC11FB2373B4" bold="true" box="[1174,1199,935,954]" pageId="2" pageNumber="46">30</emphasis>
|
||
(
|
||
<emphasis id="B973404B0B53615BFB2BFC10FB5F73B4" box="[1216,1235,934,954]" italics="true" pageId="2" pageNumber="46">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B53615BFB31FC10FB6873B4" box="[1242,1252,934,954]" italics="true" pageId="2" pageNumber="46">z</emphasis>
|
||
477) were putatively identified as desrhamnosyl-acteoside (=calceorioside B) and its isomer. Distinguishment of the suggested deprotonated molecular ion [HT-Glc-CA – H]
|
||
<emphasis id="B973404B0B53615BFC2CFC4EFC5F740A" box="[967,979,1016,1028]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFC2CFC4EFC5F740A" attach="left" box="[967,979,1016,1028]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
from [HT-Glc-Glc – H]
|
||
<emphasis id="B973404B0B53615BFB5CFC4EFB4F740A" box="[1207,1219,1016,1028]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFB5CFC4EFB4F740A" attach="left" box="[1207,1219,1016,1028]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
(both
|
||
<emphasis id="B973404B0B53615BFAEEFC4CFA947400" box="[1285,1304,1018,1038]" italics="true" pageId="2" pageNumber="46">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B53615BFACBFC4CFAA67400" box="[1312,1322,1018,1038]" italics="true" pageId="2" pageNumber="46">z</emphasis>
|
||
477) was done on basis of retention time (polarity in RP-HPLC), and the presence of fragments, that can be linked to CA, and cannot be explained by fragmentation of glucose. The proposed fragmentation scheme of
|
||
<emphasis id="B973404B0B53615BFCCEFBDDFCB27470" bold="true" box="[805,830,1131,1150]" pageId="2" pageNumber="46">22</emphasis>
|
||
is plotted in Supplementary
|
||
<figureCitation id="133C80DC0B53615BFB8CFBDDFB127470" box="[1127,1182,1131,1150]" captionStart="Fig" captionStartId="3.[113,139,1320,1334]" captionTargetBox="[348,1255,182,1289]" captionTargetId="figure-1@3.[348,1258,181,1290]" captionTargetPageId="3" captionText="Fig. 3. Proposed fragmentation pattern of plantamajoside, the main phenylethanoid glycoside of the P. lanceolata callus extract in negative mode LC–ESI–MS3. The main fragmentation route is highlighted with bold arrows and bold font." figureDoi="http://doi.org/10.5281/zenodo.10491350" httpUri="https://zenodo.org/record/10491350/files/figure.png" pageId="2" pageNumber="46">Fig. 3</figureCitation>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B53615DFCAFFB31FFF571EE" blockId="2.[805,1475,182,1290]" lastBlockId="4.[87,757,179,1205]" lastPageId="4" lastPageNumber="48" pageId="2" pageNumber="46">
|
||
In the higher retention time range, several less polar PGs were detected. Some of them could also be putatively identified, some were found to be methylated PGs. Of these, many had methyl-caffeic acid (MeCA) as a phenylpropanoid aglycon, and this was found to be the case with
|
||
<emphasis id="B973404B0B53615BFC06FB40FB8A7507" bold="true" box="[1005,1030,1270,1289]" pageId="2" pageNumber="46">59</emphasis>
|
||
(leucosceptoside A, [M
|
||
<emphasis id="B973404B0B53615BFB19FB41FA8F7507" box="[1266,1283,1271,1289]" italics="true" pageId="2" pageNumber="46">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B53615BFAF0FB45FAAB74F1" box="[1307,1319,1267,1279]" italics="true" pageId="2" pageNumber="46">
|
||
<superScript id="7C7231110B53615BFAF0FB45FAAB74F1" attach="none" box="[1307,1319,1267,1279]" fontSize="5" pageId="2" pageNumber="46">–</superScript>
|
||
</emphasis>
|
||
: [HT-Glc(Rha)- MeCA – H]
|
||
<emphasis id="B973404B0B55615DFF2EFF05FF5D70B1" box="[197,209,179,191]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFF2EFF05FF5D70B1" attach="none" box="[197,209,179,191]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
);
|
||
<emphasis id="B973404B0B55615DFF0CFF00FE8C70C7" bold="true" box="[231,256,182,201]" pageId="4" pageNumber="48">53</emphasis>
|
||
and
|
||
<emphasis id="B973404B0B55615DFED8FF00FEC070C7" bold="true" box="[307,332,182,201]" pageId="4" pageNumber="48">61</emphasis>
|
||
([M
|
||
<emphasis id="B973404B0B55615DFE93FF01FE0570C7" box="[376,393,183,201]" italics="true" pageId="4" pageNumber="48">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B55615DFE4AFF05FE2170B1" box="[417,429,179,191]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFE4AFF05FE2170B1" attach="none" box="[417,429,179,191]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
: [HT-Glc(Glc)-MeCA – H]
|
||
<emphasis id="B973404B0B55615DFD5BFF05FD3070B1" box="[688,700,179,191]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFD5BFF05FD3070B1" attach="none" box="[688,700,179,191]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
). The decay routes of these NPs were identical to that described in
|
||
<bibRefCitation id="EF96E1A80B55615DFFBCFF58FEE3710F" author="Kirmizibekmez, H. & Montoro, P. & Piacente, S. & Pizza, C. & Donmez, A. & Calis, I." box="[87,367,238,257]" pageId="4" pageNumber="48" pagination="1 - 6" refId="ref12044" refString="Kirmizibekmez, H., Montoro, P., Piacente, S., Pizza, C., Donmez, A., Calis, I., 2005. Identification by HPLC-PAD-MS and quantification by HPLC-PAD of phenylethanoid glycosides of five Phlomis species. Phytochem. Anal. 16, 1 - 6." type="journal article" year="2005">Kırmızıbekmez et al. (2005)</bibRefCitation>
|
||
. The proposed fragmentation schemes are shown in the Supplementary
|
||
<figureCitation id="133C80DC0B55615DFE51FEBCFE787113" box="[442,500,266,285]" captionStart="Fig" captionStartId="5.[113,139,1768,1782]" captionTargetBox="[205,1372,181,1738]" captionTargetId="figure-2@5.[197,1407,181,1738]" captionTargetPageId="5" captionText="Fig. 4. Principal component (PC) analysis score plot of the scaled, centered and per-composition averaged natural product abundance dataset. The plot shows the effect of media composition on the metabolome as a whole. The NP abundances were measured by LC–ESI–MS3 from MeOH extracts of P. lanceolata calli grown on media with different N source compositions. For more details, see text. Subplots: (a) Pseudo-3D score plot of PCs 1–3. PC2 and PC3 scores are plotted on axes x and y, respectively, while PC1 is made proportional to point size (see legend). Media codes are as follows: N concentration (mM), followed by NH + /NO – ratio (ratio of NH + in the total N source) in 4 3 4 parentheses. The NH + concentration in the media is coded by color (see legend). Abbreviations: NSC, N source concentration. (b), Loading plot for the first three PCs showing 4 contribution of the 89 individual metabolites to the PC scores. The order of NPs was obtained from hierarchical cluster analysis of the NP abundances." figureDoi="http://doi.org/10.5281/zenodo.10491354" httpUri="https://zenodo.org/record/10491354/files/figure.png" pageId="4" pageNumber="48">Fig. 4</figureCitation>
|
||
. The structures of these three NPs were supported by the presence of fragments
|
||
<emphasis id="B973404B0B55615DFD3BFE93FD6F7137" box="[720,739,293,313]" italics="true" pageId="4" pageNumber="48">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B55615DFD00FE93FD797137" box="[747,757,293,313]" italics="true" pageId="4" pageNumber="48">z</emphasis>
|
||
491[HT-Glc-MeCA – H]
|
||
<emphasis id="B973404B0B55615DFEA8FE88FEC37144" box="[323,335,318,330]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFEA8FE88FEC37144" attach="left" box="[323,335,318,330]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
and 315 [HT-Glc – H]
|
||
<emphasis id="B973404B0B55615DFDDAFE88FDB17144" box="[561,573,318,330]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFDDAFE88FDB17144" attach="none" box="[561,573,318,330]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
, indicating that it was not the HT aglycon, that carried the methyl group. The fragment ion
|
||
<emphasis id="B973404B0B55615DFF50FECEFF427182" box="[187,206,376,396]" italics="true" pageId="4" pageNumber="48">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B55615DFF3DFECEFF6C7182" box="[214,224,376,396]" italics="true" pageId="4" pageNumber="48">z</emphasis>
|
||
477 can be interpreted as [HT-Glc-Glc – H]
|
||
<emphasis id="B973404B0B55615DFD4BFEC0FD20718C" box="[672,684,374,386]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFD4BFEC0FD20718C" attach="left" box="[672,684,374,386]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
in this case, unlike in most other PGs. Methylated plantamajoside derivates have only been described from
|
||
<taxonomicName id="4C07E7DA0B55615DFE31FE06FD5B71CA" authority="(Jin et al., 2011)" baseAuthorityName="Jin" baseAuthorityYear="2011" box="[474,727,432,453]" class="Magnoliopsida" family="Plantaginaceae" genus="Digitalis" kingdom="Plantae" order="Lamiales" pageId="4" pageNumber="48" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B973404B0B55615DFE31FE06FDA571CA" box="[474,553,432,452]" italics="true" pageId="4" pageNumber="48">Digitalis</emphasis>
|
||
(
|
||
<bibRefCitation id="EF96E1A80B55615DFDD2FE07FD4271CA" author="Jin, Q. & Jin, H. - G. & Shin, J. - E. & Hong, J. - K. & Woo, E. - R." box="[569,718,433,453]" pageId="4" pageNumber="48" pagination="1721 - 1724" refId="ref11970" refString="Jin, Q., Jin, H. - G., Shin, J. - E., Hong, J. - K., Woo, E. - R., 2011. Phenylethanoid glycosides from Digitalis purpurea L. Bull. Korean Chem. Soc. 32, 1721 - 1724. http: // dx. doi. org / 10.5012 / bkcs. 2011.32.5.1721." type="journal article" year="2011">Jin et al., 2011</bibRefCitation>
|
||
)
|
||
</taxonomicName>
|
||
so far.
|
||
</paragraph>
|
||
<caption id="DF78CCD10B53615BFFBCF83CFB7C77D3" ID-DOI="http://doi.org/10.5281/zenodo.10491348" ID-Zenodo-Dep="10491348" httpUri="https://zenodo.org/record/10491348/files/figure.png" pageId="2" pageNumber="46" startId="2.[87,113,1930,1944]" targetBox="[209,1337,1371,1901]" targetPageId="2" targetType="figure">
|
||
<paragraph id="8BB89C590B53615BFFBCF83CFB7C77D3" blockId="2.[87,1476,1930,2013]" pageId="2" pageNumber="46">
|
||
<emphasis id="B973404B0B53615BFFBCF83CFF047796" bold="true" box="[87,136,1930,1944]" pageId="2" pageNumber="46">Fig. 2.</emphasis>
|
||
LC–ESI–MS chromatogram of the MeOH extract of a
|
||
<taxonomicName id="4C07E7DA0B53615BFDDEF83CFD197797" box="[565,661,1930,1945]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="2" pageNumber="46" phylum="Tracheophyta" rank="species" species="lanceolata">
|
||
<emphasis id="B973404B0B53615BFDDEF83CFD197797" box="[565,661,1930,1945]" italics="true" pageId="2" pageNumber="46">P. lanceolata</emphasis>
|
||
</taxonomicName>
|
||
callus grown on medium 10(0.33) (25 mg dry wt./ml, total ion chromatogram in negative ion mode). Note that in this injection, the area under curve of plantamajoside was beyond the linear range of determination, and thus, major NPs were quantified in the 100-fold dilution, and not from the chromatogram presented here. Main peaks are marked with the metabolite numbers used in text, see Table 1 for putative identification. NPs compared with standards are marked with an asterisk. For
|
||
<emphasis id="B973404B0B53615BFE51F878FE4577D3" box="[442,457,1998,2013]" italics="true" pageId="2" pageNumber="46">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B53615BFE24F878FE5B77D3" box="[463,471,1998,2013]" italics="true" pageId="2" pageNumber="46">z</emphasis>
|
||
values, and putative identification, see Table 1.
|
||
<emphasis id="B973404B0B53615BFC8BF878FC4377D3" box="[864,975,1998,2013]" italics="true" pageId="2" pageNumber="46">Abbreviations:</emphasis>
|
||
AC, acteoside; PM, plantamajoside.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF78CCD10B52615AFF9AFA9EFD197542" ID-DOI="http://doi.org/10.5281/zenodo.10491350" ID-Zenodo-Dep="10491350" httpUri="https://zenodo.org/record/10491350/files/figure.png" pageId="3" pageNumber="47" startId="3.[113,139,1320,1334]" targetBox="[348,1255,182,1289]" targetPageId="3" targetType="figure">
|
||
<paragraph id="8BB89C590B52615AFF9AFA9EFD197542" blockId="3.[113,1500,1316,1357]" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF9AFA9EFF287538" bold="true" box="[113,164,1320,1334]" pageId="3" pageNumber="47">Fig. 3.</emphasis>
|
||
Proposed fragmentation pattern of plantamajoside, the main phenylethanoid glycoside of the
|
||
<taxonomicName id="4C07E7DA0B52615AFC59FA91FB997538" box="[946,1045,1319,1334]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="3" pageNumber="47" phylum="Tracheophyta" rank="species" species="lanceolata">
|
||
<emphasis id="B973404B0B52615AFC59FA91FB997538" box="[946,1045,1319,1334]" italics="true" pageId="3" pageNumber="47">P. lanceolata</emphasis>
|
||
</taxonomicName>
|
||
callus extract in negative mode LC–ESI–MS
|
||
<superScript id="7C7231110B52615AFA96FA92FA087520" attach="left" box="[1405,1412,1316,1326]" fontSize="4" pageId="3" pageNumber="47">3</superScript>
|
||
. The main fragmentation route is highlighted with bold arrows and bold font.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF78CCD10B52615AFF99FA3EFC3D75CD" ID-Table-UUID="DF78CCD10B52615AFF99FA3EFC3D75CD" httpUri="http://table.plazi.org/id/DF78CCD10B52615AFF99FA3EFC3D75CD" pageId="3" pageNumber="47" startId="3.[114,158,1416,1430]" targetBox="[136,1478,1496,1870]" targetIsTable="true" targetPageId="3" targetType="table">
|
||
<paragraph id="8BB89C590B52615AFF99FA3EFC3D75CD" blockId="3.[113,1501,1416,1476]" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF99FA3EFF227598" bold="true" box="[114,174,1416,1430]" pageId="3" pageNumber="47">Table 1</emphasis>
|
||
Fragmentation pattern of putatively identified major peaks obtained by LC–ESI–MS
|
||
<superScript id="7C7231110B52615AFCE9FA2DFC8575AB" attach="left" box="[770,777,1435,1445]" fontSize="4" pageId="3" pageNumber="47">3</superScript>
|
||
analysis of defatted
|
||
<taxonomicName id="4C07E7DA0B52615AFC46FA28FBCD75A3" box="[941,1089,1438,1453]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="3" pageNumber="47" phylum="Tracheophyta" rank="species" species="lanceolata">
|
||
<emphasis id="B973404B0B52615AFC46FA28FBCD75A3" box="[941,1089,1438,1453]" italics="true" pageId="3" pageNumber="47">Plantago lanceolata</emphasis>
|
||
</taxonomicName>
|
||
calli MeOH extracts in negative ion mode. MS
|
||
<superScript id="7C7231110B52615AFA44FA2DFA3A75AB" attach="left" box="[1455,1462,1435,1445]" fontSize="4" pageId="3" pageNumber="47">2</superScript>
|
||
ions are CID products of the [M
|
||
<emphasis id="B973404B0B52615AFEADFA00FEDF75CA" box="[326,339,1462,1476]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B52615AFE8DFA04FEFC75B5" box="[358,368,1458,1467]" italics="true" pageId="3" pageNumber="47">
|
||
<superScript id="7C7231110B52615AFE8DFA04FEFC75B5" attach="none" box="[358,368,1458,1467]" fontSize="4" pageId="3" pageNumber="47">–</superScript>
|
||
</emphasis>
|
||
, while MS
|
||
<superScript id="7C7231110B52615AFE2FFA07FE4775B5" attach="left" box="[452,459,1457,1467]" fontSize="4" pageId="3" pageNumber="47">3</superScript>
|
||
ions are CID products of the most abundant MS
|
||
<superScript id="7C7231110B52615AFCA6FA07FCD875B5" attach="left" box="[845,852,1457,1467]" fontSize="4" pageId="3" pageNumber="47">2</superScript>
|
||
ion (MS
|
||
<superScript id="7C7231110B52615AFC7CFA07FC2A75B5" attach="left" box="[919,934,1457,1467]" fontSize="4" pageId="3" pageNumber="47">2a</superScript>
|
||
).
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8BB89C590B52615AFF63FA6AFA497745" pageId="3" pageNumber="47">
|
||
<table id="F9076EF90B529EA6FF63FA6EFA4A7740" box="[136,1478,1496,1870]" gridcols="8" gridrows="16" pageId="3" pageNumber="47">
|
||
<tr id="35379E1B0B529EA6FF63FA6EFA4A75E4" box="[136,1478,1496,1514]" gridrow="0" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63FA6EFF1075E4" box="[136,156,1496,1514]" gridcol="0" gridrow="0" pageId="3" pageNumber="47">#</th>
|
||
<th id="76E6F7670B529EA6FF58FA6EFF6C75E4" box="[179,224,1496,1514]" gridcol="1" gridrow="0" pageId="3" pageNumber="47">Rt</th>
|
||
<th id="76E6F7670B529EA6FF1CFA6EFDA875E4" box="[247,548,1496,1514]" gridcol="2" gridrow="0" pageId="3" pageNumber="47">Putative identification</th>
|
||
<th id="76E6F7670B529EA6FDD1FA6EFCB975E4" box="[570,821,1496,1514]" gridcol="3" gridrow="0" pageId="3" pageNumber="47">
|
||
Structure of [M
|
||
<emphasis id="B973404B0B52615AFD5CFA6AFD4875E4" box="[695,708,1500,1514]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B52615AFD33FA6EFD6E75EF" box="[728,738,1496,1505]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</th>
|
||
<th id="76E6F7670B529EA6FCA0FA6EFC0175E4" box="[843,909,1496,1514]" gridcol="4" gridrow="0" pageId="3" pageNumber="47">
|
||
[M
|
||
<emphasis id="B973404B0B52615AFC89FA6AFCE375E4" box="[866,879,1500,1514]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B52615AFC68FA6EFC0175EF" box="[899,909,1496,1505]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</th>
|
||
<th id="76E6F7670B529EA6FC48FA6EFC4775E4" box="[931,971,1496,1514]" gridcol="5" gridrow="0" pageId="3" pageNumber="47">MS2a</th>
|
||
<th id="76E6F7670B529EA6FC0AFA6EFB4575E4" box="[993,1225,1496,1514]" gridcol="6" gridrow="0" pageId="3" pageNumber="47">MS2 b</th>
|
||
<th id="76E6F7670B529EA6FB34FA6EFA4A75E4" box="[1247,1478,1496,1514]" gridcol="7" gridrow="0" pageId="3" pageNumber="47">MS3</th>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63FA4CFA4A7602" box="[136,1478,1530,1548]" gridrow="1" pageId="3" pageNumber="47" rowspan-6="1">
|
||
<th id="76E6F7670B529EA6FF63FA4CFF107602" box="[136,156,1530,1548]" gridcol="0" gridrow="1" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63FA4BFF1E7605" bold="true" box="[136,146,1533,1547]" pageId="3" pageNumber="47">8</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58FA4CFF6C7602" box="[179,224,1530,1548]" gridcol="1" gridrow="1" pageId="3" pageNumber="47">4.85</td>
|
||
<td id="76E6F7670B529EA6FF1CFA4CFDA87602" box="[247,548,1530,1548]" gridcol="2" gridrow="1" pageId="3" pageNumber="47">Plantamajoside hexoside</td>
|
||
<td id="76E6F7670B529EA6FDD1FA4CFCB97602" box="[570,821,1530,1548]" gridcol="3" gridrow="1" pageId="3" pageNumber="47">
|
||
[HT-Glc(Glc)(Glc)-CA – H]
|
||
<emphasis id="B973404B0B52615AFCE7FA4CFC9A760D" box="[780,790,1530,1539]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0FA4CFC017602" box="[843,909,1530,1548]" gridcol="4" gridrow="1" pageId="3" pageNumber="47">801</td>
|
||
<td id="76E6F7670B529EA6FC48FA4CFC477602" box="[931,971,1530,1548]" gridcol="5" gridrow="1" pageId="3" pageNumber="47">639</td>
|
||
<td id="76E6F7670B529EA6FB34FA4CFA4A7602" box="[1247,1478,1530,1548]" gridcol="7" gridrow="1" pageId="3" pageNumber="47">477</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F9A6FA4A762C" box="[136,1478,1552,1570]" gridrow="2" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F9A6FF10762C" box="[136,156,1552,1570]" gridcol="0" gridrow="2" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F9A2FF10762C" bold="true" box="[136,156,1556,1570]" pageId="3" pageNumber="47">18</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F9A6FF6C762C" box="[179,224,1552,1570]" gridcol="1" gridrow="2" pageId="3" pageNumber="47">8.08</td>
|
||
<td id="76E6F7670B529EA6FF1CF9A6FDA8762C" box="[247,548,1552,1570]" gridcol="2" gridrow="2" pageId="3" pageNumber="47">
|
||
Plantamajoside
|
||
<emphasis id="B973404B0B52615AFE99F9A7FEF57617" bold="true" box="[370,377,1553,1561]" italics="true" pageId="3" pageNumber="47">⁄</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FDD1F9A6FCB9762C" box="[570,821,1552,1570]" gridcol="3" gridrow="2" pageId="3" pageNumber="47">
|
||
[HT-Glc(Glc)-CA – H]
|
||
<emphasis id="B973404B0B52615AFD0DF9A6FD7C7617" box="[742,752,1552,1561]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F9A6FC01762C" box="[843,909,1552,1570]" gridcol="4" gridrow="2" pageId="3" pageNumber="47">639</td>
|
||
<td id="76E6F7670B529EA6FC48F9A6FC47762C" box="[931,971,1552,1570]" gridcol="5" gridrow="2" pageId="3" pageNumber="47">477</td>
|
||
<td id="76E6F7670B529EA6FC0AF9A6FB45762C" box="[993,1225,1552,1570]" gridcol="6" gridrow="2" pageId="3" pageNumber="47">315</td>
|
||
<td id="76E6F7670B529EA6FB34F9A6FA4A762C" box="[1247,1478,1552,1570]" gridcol="7" gridrow="2" pageId="3" pageNumber="47">135, 143, 161, 179, 297, 315</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F991FA4A7637" box="[136,1478,1575,1593]" gridrow="3" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F991FF107637" box="[136,156,1575,1593]" gridcol="0" gridrow="3" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F99DFF107637" bold="true" box="[136,156,1579,1593]" pageId="3" pageNumber="47">19</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F991FF6C7637" box="[179,224,1575,1593]" gridcol="1" gridrow="3" pageId="3" pageNumber="47">8.16</td>
|
||
<td id="76E6F7670B529EA6FF1CF991FDA87637" box="[247,548,1575,1593]" gridcol="2" gridrow="3" pageId="3" pageNumber="47">Lavandulifolioside</td>
|
||
<td id="76E6F7670B529EA6FDD1F991FCB97637" box="[570,821,1575,1593]" gridcol="3" gridrow="3" pageId="3" pageNumber="47">
|
||
[HT-Glc(Rha(Ara))-CA – H]
|
||
<emphasis id="B973404B0B52615AFCF9F991FC90763E" box="[786,796,1575,1584]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F991FC017637" box="[843,909,1575,1593]" gridcol="4" gridrow="3" pageId="3" pageNumber="47">755</td>
|
||
<td id="76E6F7670B529EA6FC48F991FC477637" box="[931,971,1575,1593]" gridcol="5" gridrow="3" pageId="3" pageNumber="47">593</td>
|
||
<td id="76E6F7670B529EA6FC0AF991FB457637" box="[993,1225,1575,1593]" gridcol="6" gridrow="3" pageId="3" pageNumber="47">443, 447, 461, 575, 609, 623</td>
|
||
<td id="76E6F7670B529EA6FB34F991FA4A7637" box="[1247,1478,1575,1593]" gridcol="7" gridrow="3" pageId="3" pageNumber="47">461, 447</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F988FA4A765E" box="[136,1478,1598,1616]" gridrow="4" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F988FF10765E" box="[136,156,1598,1616]" gridcol="0" gridrow="4" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F9F4FF10765E" bold="true" box="[136,156,1602,1616]" pageId="3" pageNumber="47">22</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F988FF6C765E" box="[179,224,1598,1616]" gridcol="1" gridrow="4" pageId="3" pageNumber="47">9.07</td>
|
||
<td id="76E6F7670B529EA6FF1CF988FDA8765E" box="[247,548,1598,1616]" gridcol="2" gridrow="4" pageId="3" pageNumber="47">Desrhamnosylacteoside</td>
|
||
<td id="76E6F7670B529EA6FDD1F988FCB9765E" box="[570,821,1598,1616]" gridcol="3" gridrow="4" pageId="3" pageNumber="47">
|
||
[HT-Glc-CA – H]
|
||
<emphasis id="B973404B0B52615AFD54F988FD457649" box="[703,713,1598,1607]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F988FC01765E" box="[843,909,1598,1616]" gridcol="4" gridrow="4" pageId="3" pageNumber="47">477</td>
|
||
<td id="76E6F7670B529EA6FC48F988FC47765E" box="[931,971,1598,1616]" gridcol="5" gridrow="4" pageId="3" pageNumber="47">161</td>
|
||
<td id="76E6F7670B529EA6FC0AF988FB45765E" box="[993,1225,1598,1616]" gridcol="6" gridrow="4" pageId="3" pageNumber="47">135, 179, 203, 315, 323, 341</td>
|
||
<td id="76E6F7670B529EA6FB34F988FA4A765E" box="[1247,1478,1598,1616]" gridcol="7" gridrow="4" pageId="3" pageNumber="47">133</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F9E3FA4A7669" box="[136,1478,1621,1639]" gridrow="5" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F9E3FF107669" box="[136,156,1621,1639]" gridcol="0" gridrow="5" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F9EEFF107668" bold="true" box="[136,156,1624,1638]" pageId="3" pageNumber="47">27</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F9E3FF6C7669" box="[179,224,1621,1639]" gridcol="1" gridrow="5" pageId="3" pageNumber="47">9.31</td>
|
||
<td id="76E6F7670B529EA6FF1CF9E3FDA87669" box="[247,548,1621,1639]" gridcol="2" gridrow="5" pageId="3" pageNumber="47">
|
||
Acteoside
|
||
<emphasis id="B973404B0B52615AFEAFF9E0FEC77650" bold="true" box="[324,331,1622,1630]" italics="true" pageId="3" pageNumber="47">⁄</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FDD1F9E3FCB97669" box="[570,821,1621,1639]" gridcol="3" gridrow="5" pageId="3" pageNumber="47">
|
||
[HT-Glc(Rha)-CA – H]
|
||
<emphasis id="B973404B0B52615AFD01F9E3FD787650" box="[746,756,1621,1630]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F9E3FC017669" box="[843,909,1621,1639]" gridcol="4" gridrow="5" pageId="3" pageNumber="47">623</td>
|
||
<td id="76E6F7670B529EA6FC48F9E3FC477669" box="[931,971,1621,1639]" gridcol="5" gridrow="5" pageId="3" pageNumber="47">461</td>
|
||
<td id="76E6F7670B529EA6FC0AF9E3FB457669" box="[993,1225,1621,1639]" gridcol="6" gridrow="5" pageId="3" pageNumber="47">315, 443, 477</td>
|
||
<td id="76E6F7670B529EA6FB34F9E3FA4A7669" box="[1247,1478,1621,1639]" gridcol="7" gridrow="5" pageId="3" pageNumber="47">135, 161, 297, 315</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F9DAFA4A7670" box="[136,1478,1644,1662]" gridrow="6" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F9DAFF107670" box="[136,156,1644,1662]" gridcol="0" gridrow="6" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F9D9FF107673" bold="true" box="[136,156,1647,1661]" pageId="3" pageNumber="47">30</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F9DAFF6C7670" box="[179,224,1644,1662]" gridcol="1" gridrow="6" pageId="3" pageNumber="47">9.61</td>
|
||
<td id="76E6F7670B529EA6FF1CF9DAFDA87670" box="[247,548,1644,1662]" gridcol="2" gridrow="6" pageId="3" pageNumber="47">Desrhamnosylacteoside 2</td>
|
||
<td id="76E6F7670B529EA6FDD1F9DAFCB97670" box="[570,821,1644,1662]" gridcol="3" gridrow="6" pageId="3" pageNumber="47">
|
||
[HT-Glc-CA – H]
|
||
<emphasis id="B973404B0B52615AFD54F9DAFD45767B" box="[703,713,1644,1653]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F9DAFC017670" box="[843,909,1644,1662]" gridcol="4" gridrow="6" pageId="3" pageNumber="47">477</td>
|
||
<td id="76E6F7670B529EA6FC48F9DAFC477670" box="[931,971,1644,1662]" gridcol="5" gridrow="6" pageId="3" pageNumber="47">161</td>
|
||
<td id="76E6F7670B529EA6FC0AF9DAFB457670" box="[993,1225,1644,1662]" gridcol="6" gridrow="6" pageId="3" pageNumber="47">179, 251, 281, 315</td>
|
||
<td id="76E6F7670B529EA6FB34F9DAFA4A7670" box="[1247,1478,1644,1662]" gridcol="7" gridrow="6" pageId="3" pageNumber="47">133</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F935FA4A769B" box="[136,1478,1667,1685]" gridrow="7" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F935FF10769B" box="[136,156,1667,1685]" gridcol="0" gridrow="7" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F930FF10769A" bold="true" box="[136,156,1670,1684]" pageId="3" pageNumber="47">37</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F935FF6C769B" box="[179,224,1667,1685]" gridcol="1" gridrow="7" pageId="3" pageNumber="47">9.88</td>
|
||
<td id="76E6F7670B529EA6FF1CF935FDA8769B" box="[247,548,1667,1685]" gridcol="2" gridrow="7" pageId="3" pageNumber="47">Plantamajoside 2</td>
|
||
<td id="76E6F7670B529EA6FDD1F935FCB9769B" box="[570,821,1667,1685]" gridcol="3" gridrow="7" pageId="3" pageNumber="47">
|
||
[HT-Glc(Glc)-CA – H]
|
||
<emphasis id="B973404B0B52615AFD0DF935FD7C7682" box="[742,752,1667,1676]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F935FC01769B" box="[843,909,1667,1685]" gridcol="4" gridrow="7" pageId="3" pageNumber="47">639</td>
|
||
<td id="76E6F7670B529EA6FC48F935FC47769B" box="[931,971,1667,1685]" gridcol="5" gridrow="7" pageId="3" pageNumber="47">477</td>
|
||
<td id="76E6F7670B529EA6FC0AF935FB45769B" box="[993,1225,1667,1685]" gridcol="6" gridrow="7" pageId="3" pageNumber="47">315</td>
|
||
<td id="76E6F7670B529EA6FB34F935FA4A769B" box="[1247,1478,1667,1685]" gridcol="7" gridrow="7" pageId="3" pageNumber="47">135, 143, 161, 179, 297, 315</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F92FFA4A76A5" box="[136,1478,1689,1707]" gridrow="8" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F92FFF1076A5" box="[136,156,1689,1707]" gridcol="0" gridrow="8" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F92BFF1076A5" bold="true" box="[136,156,1693,1707]" pageId="3" pageNumber="47">43</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F92FFF6C76A5" box="[179,224,1689,1707]" gridcol="1" gridrow="8" pageId="3" pageNumber="47">10.45</td>
|
||
<td id="76E6F7670B529EA6FF1CF92FFDA876A5" box="[247,548,1689,1707]" gridcol="2" gridrow="8" pageId="3" pageNumber="47">Acteoside 2</td>
|
||
<td id="76E6F7670B529EA6FDD1F92FFCB976A5" box="[570,821,1689,1707]" gridcol="3" gridrow="8" pageId="3" pageNumber="47">
|
||
[HT-Glc(Rha)-CA – H]
|
||
<emphasis id="B973404B0B52615AFD01F92FFD7876AC" box="[746,756,1689,1698]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F92FFC0176A5" box="[843,909,1689,1707]" gridcol="4" gridrow="8" pageId="3" pageNumber="47">623</td>
|
||
<td id="76E6F7670B529EA6FC48F92FFC4776A5" box="[931,971,1689,1707]" gridcol="5" gridrow="8" pageId="3" pageNumber="47">461</td>
|
||
<td id="76E6F7670B529EA6FC0AF92FFB4576A5" box="[993,1225,1689,1707]" gridcol="6" gridrow="8" pageId="3" pageNumber="47">315, 443, 477</td>
|
||
<td id="76E6F7670B529EA6FB34F92FFA4A76A5" box="[1247,1478,1689,1707]" gridcol="7" gridrow="8" pageId="3" pageNumber="47">135, 161, 297, 315</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F906FA4A76CC" box="[136,1478,1712,1730]" gridrow="9" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F906FF1076CC" box="[136,156,1712,1730]" gridcol="0" gridrow="9" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F902FF1076CC" bold="true" box="[136,156,1716,1730]" pageId="3" pageNumber="47">49</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F906FF6C76CC" box="[179,224,1712,1730]" gridcol="1" gridrow="9" pageId="3" pageNumber="47">10.54</td>
|
||
<td id="76E6F7670B529EA6FF1CF906FDA876CC" box="[247,548,1712,1730]" gridcol="2" gridrow="9" pageId="3" pageNumber="47">Plantamajoside 3</td>
|
||
<td id="76E6F7670B529EA6FDD1F906FCB976CC" box="[570,821,1712,1730]" gridcol="3" gridrow="9" pageId="3" pageNumber="47">
|
||
[HT-Glc(Glc)-CA – H]
|
||
<emphasis id="B973404B0B52615AFD0DF906FD7C76B7" box="[742,752,1712,1721]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F906FC0176CC" box="[843,909,1712,1730]" gridcol="4" gridrow="9" pageId="3" pageNumber="47">639</td>
|
||
<td id="76E6F7670B529EA6FC48F906FC4776CC" box="[931,971,1712,1730]" gridcol="5" gridrow="9" pageId="3" pageNumber="47">477</td>
|
||
<td id="76E6F7670B529EA6FC0AF906FB4576CC" box="[993,1225,1712,1730]" gridcol="6" gridrow="9" pageId="3" pageNumber="47">251, 315</td>
|
||
<td id="76E6F7670B529EA6FB34F906FA4A76CC" box="[1247,1478,1712,1730]" gridcol="7" gridrow="9" pageId="3" pageNumber="47">161, 315</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F971FA4A76D7" box="[136,1478,1735,1753]" gridrow="10" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F971FF1076D7" box="[136,156,1735,1753]" gridcol="0" gridrow="10" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F97DFF1076D7" bold="true" box="[136,156,1739,1753]" pageId="3" pageNumber="47">53</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F971FF6C76D7" box="[179,224,1735,1753]" gridcol="1" gridrow="10" pageId="3" pageNumber="47">11.00</td>
|
||
<td id="76E6F7670B529EA6FF1CF971FDA876D7" box="[247,548,1735,1753]" gridcol="2" gridrow="10" pageId="3" pageNumber="47">Methyl-plantamajoside</td>
|
||
<td id="76E6F7670B529EA6FDD1F971FCB976D7" box="[570,821,1735,1753]" gridcol="3" gridrow="10" pageId="3" pageNumber="47">
|
||
[HT-Glc(Glc)-MeCA – H]
|
||
<emphasis id="B973404B0B52615AFD14F971FC8576DE" box="[767,777,1735,1744]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F971FC0176D7" box="[843,909,1735,1753]" gridcol="4" gridrow="10" pageId="3" pageNumber="47">653</td>
|
||
<td id="76E6F7670B529EA6FC48F971FC4776D7" box="[931,971,1735,1753]" gridcol="5" gridrow="10" pageId="3" pageNumber="47">607</td>
|
||
<td id="76E6F7670B529EA6FC0AF971FB4576D7" box="[993,1225,1735,1753]" gridcol="6" gridrow="10" pageId="3" pageNumber="47">491</td>
|
||
<td id="76E6F7670B529EA6FB34F971FA4A76D7" box="[1247,1478,1735,1753]" gridcol="7" gridrow="10" pageId="3" pageNumber="47">315, 443, 461</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F968FA4A76FE" box="[136,1478,1758,1776]" gridrow="11" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F968FF1076FE" box="[136,156,1758,1776]" gridcol="0" gridrow="11" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F954FF1076FE" bold="true" box="[136,156,1762,1776]" pageId="3" pageNumber="47">59</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F968FF6C76FE" box="[179,224,1758,1776]" gridcol="1" gridrow="11" pageId="3" pageNumber="47">11.63</td>
|
||
<td id="76E6F7670B529EA6FF1CF968FDA876FE" box="[247,548,1758,1776]" gridcol="2" gridrow="11" pageId="3" pageNumber="47">Methyl acteoside (leucosceptoside A)</td>
|
||
<td id="76E6F7670B529EA6FDD1F968FCB976FE" box="[570,821,1758,1776]" gridcol="3" gridrow="11" pageId="3" pageNumber="47">
|
||
[HT-Glc(Rha)-MeCA – H]
|
||
<emphasis id="B973404B0B52615AFCE8F968FC8176E9" box="[771,781,1758,1767]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F968FC0176FE" box="[843,909,1758,1776]" gridcol="4" gridrow="11" pageId="3" pageNumber="47">637</td>
|
||
<td id="76E6F7670B529EA6FC48F968FC4776FE" box="[931,971,1758,1776]" gridcol="5" gridrow="11" pageId="3" pageNumber="47">461</td>
|
||
<td id="76E6F7670B529EA6FC0AF968FB4576FE" box="[993,1225,1758,1776]" gridcol="6" gridrow="11" pageId="3" pageNumber="47">443, 475, 491</td>
|
||
<td id="76E6F7670B529EA6FB34F968FA4A76FE" box="[1247,1478,1758,1776]" gridcol="7" gridrow="11" pageId="3" pageNumber="47">135, 315</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F943FA4A7709" box="[136,1478,1781,1799]" gridrow="12" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F943FF107709" box="[136,156,1781,1799]" gridcol="0" gridrow="12" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F94EFF107708" bold="true" box="[136,156,1784,1798]" pageId="3" pageNumber="47">61</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F943FF6C7709" box="[179,224,1781,1799]" gridcol="1" gridrow="12" pageId="3" pageNumber="47">11.84</td>
|
||
<td id="76E6F7670B529EA6FF1CF943FDA87709" box="[247,548,1781,1799]" gridcol="2" gridrow="12" pageId="3" pageNumber="47">Methyl-plantamajoside 2</td>
|
||
<td id="76E6F7670B529EA6FDD1F943FCB97709" box="[570,821,1781,1799]" gridcol="3" gridrow="12" pageId="3" pageNumber="47">
|
||
[HT-Glc(Glc)-MeCA – H]
|
||
<emphasis id="B973404B0B52615AFD14F943FC8576F0" box="[767,777,1781,1790]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F943FC017709" box="[843,909,1781,1799]" gridcol="4" gridrow="12" pageId="3" pageNumber="47">653</td>
|
||
<td id="76E6F7670B529EA6FC48F943FC477709" box="[931,971,1781,1799]" gridcol="5" gridrow="12" pageId="3" pageNumber="47">477</td>
|
||
<td id="76E6F7670B529EA6FC0AF943FB457709" box="[993,1225,1781,1799]" gridcol="6" gridrow="12" pageId="3" pageNumber="47">459, 491</td>
|
||
<td id="76E6F7670B529EA6FB34F943FA4A7709" box="[1247,1478,1781,1799]" gridcol="7" gridrow="12" pageId="3" pageNumber="47">315</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F8BAFA4A7710" box="[136,1478,1804,1822]" gridrow="13" pageId="3" pageNumber="47">
|
||
<th id="76E6F7670B529EA6FF63F8BAFF107710" box="[136,156,1804,1822]" gridcol="0" gridrow="13" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F8B9FF107713" bold="true" box="[136,156,1807,1821]" pageId="3" pageNumber="47">64</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F8BAFF6C7710" box="[179,224,1804,1822]" gridcol="1" gridrow="13" pageId="3" pageNumber="47">11.97</td>
|
||
<td id="76E6F7670B529EA6FF1CF8BAFDA87710" box="[247,548,1804,1822]" gridcol="2" gridrow="13" pageId="3" pageNumber="47">Dimethyl-plantamajoside</td>
|
||
<td id="76E6F7670B529EA6FDD1F8BAFCB97710" box="[570,821,1804,1822]" gridcol="3" gridrow="13" pageId="3" pageNumber="47">
|
||
[MeHT-Glc(Glc)-MeCA – H]
|
||
<emphasis id="B973404B0B52615AFCF3F8BAFCAE771B" box="[792,802,1804,1813]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F8BAFC017710" box="[843,909,1804,1822]" gridcol="4" gridrow="13" pageId="3" pageNumber="47">667</td>
|
||
<td id="76E6F7670B529EA6FC48F8BAFC477710" box="[931,971,1804,1822]" gridcol="5" gridrow="13" pageId="3" pageNumber="47">473</td>
|
||
<td id="76E6F7670B529EA6FC0AF8BAFB457710" box="[993,1225,1804,1822]" gridcol="6" gridrow="13" pageId="3" pageNumber="47">193, 491, 505</td>
|
||
<td id="76E6F7670B529EA6FB34F8BAFA4A7710" box="[1247,1478,1804,1822]" gridcol="7" gridrow="13" pageId="3" pageNumber="47">311</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F895FA4A7739" box="[136,1478,1827,1847]" gridrow="14" pageId="3" pageNumber="47" rowspan-6="1">
|
||
<th id="76E6F7670B529EA6FF63F895FF107739" box="[136,156,1827,1847]" gridcol="0" gridrow="14" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F890FF10773A" bold="true" box="[136,156,1830,1844]" pageId="3" pageNumber="47">65</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F895FF6C7739" box="[179,224,1827,1847]" gridcol="1" gridrow="14" pageId="3" pageNumber="47">12.30</td>
|
||
<td id="76E6F7670B529EA6FF1CF895FDA87739" box="[247,548,1827,1847]" gridcol="2" gridrow="14" pageId="3" pageNumber="47">Dimethyl-acetyl-acteoside</td>
|
||
<td id="76E6F7670B529EA6FDD1F895FCB97739" box="[570,821,1827,1847]" gridcol="3" gridrow="14" pageId="3" pageNumber="47">
|
||
[HT-Glc(Ac)(Rha)-Me CA – H]
|
||
<emphasis id="B973404B0B52615AFCC0F895FCB97722" box="[811,821,1827,1836]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
2
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F895FC017739" box="[843,909,1827,1847]" gridcol="4" gridrow="14" pageId="3" pageNumber="47">693</td>
|
||
<td id="76E6F7670B529EA6FC48F895FC477739" box="[931,971,1827,1847]" gridcol="5" gridrow="14" pageId="3" pageNumber="47">649</td>
|
||
<td id="76E6F7670B529EA6FB34F895FA4A7739" box="[1247,1478,1827,1847]" gridcol="7" gridrow="14" pageId="3" pageNumber="47">443, 461, 485, 503, 589, 607</td>
|
||
</tr>
|
||
<tr id="35379E1B0B529EA6FF63F88FFA4A7740" box="[136,1478,1849,1870]" gridrow="15" pageId="3" pageNumber="47" rowspan-6="1">
|
||
<th id="76E6F7670B529EA6FF63F88FFF107740" box="[136,156,1849,1870]" gridcol="0" gridrow="15" pageId="3" pageNumber="47">
|
||
<emphasis id="B973404B0B52615AFF63F88BFF107745" bold="true" box="[136,156,1853,1867]" pageId="3" pageNumber="47">71</emphasis>
|
||
</th>
|
||
<td id="76E6F7670B529EA6FF58F88FFF6C7740" box="[179,224,1849,1870]" gridcol="1" gridrow="15" pageId="3" pageNumber="47">12.64</td>
|
||
<td id="76E6F7670B529EA6FF1CF88FFDA87740" box="[247,548,1849,1870]" gridcol="2" gridrow="15" pageId="3" pageNumber="47">Dimethyl-acetyl-acteoside 2</td>
|
||
<td id="76E6F7670B529EA6FDD1F88FFCB97740" box="[570,821,1849,1870]" gridcol="3" gridrow="15" pageId="3" pageNumber="47">
|
||
[HT-Glc(Ac)(Rha)-Me CA – H]
|
||
<emphasis id="B973404B0B52615AFCC0F88FFCB9774C" box="[811,821,1849,1858]" italics="true" pageId="3" pageNumber="47">–</emphasis>
|
||
2
|
||
</td>
|
||
<td id="76E6F7670B529EA6FCA0F88FFC017740" box="[843,909,1849,1870]" gridcol="4" gridrow="15" pageId="3" pageNumber="47">693</td>
|
||
<td id="76E6F7670B529EA6FC48F88FFC477740" box="[931,971,1849,1870]" gridcol="5" gridrow="15" pageId="3" pageNumber="47">649</td>
|
||
<td id="76E6F7670B529EA6FB34F88FFA4A7740" box="[1247,1478,1849,1870]" gridcol="7" gridrow="15" pageId="3" pageNumber="47">443, 461, 485, 503, 589, 607</td>
|
||
</tr>
|
||
</table>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B52615AFF9AF8D5FE2D77B8" blockId="3.[113,1501,1891,1975]" pageId="3" pageNumber="47">
|
||
<tableNote id="76E19DD70B52615AFF9AF8D5FE2D77B8" pageId="3" pageNumber="47" targetBox="[136,1478,1496,1870]" targetPageId="3">
|
||
<emphasis id="B973404B0B52615AFF9AF8D5FF6C777C" box="[113,224,1891,1906]" italics="true" pageId="3" pageNumber="47">Abbreviations:</emphasis>
|
||
Ara, arabinose; CA, caffeic acid; Glc, glucose; HT, hydroxytyrosol; MeCA, methyl caffeic acid; Me
|
||
<subScript id="17839E1C0B52615AFC0FF8DCFC67777A" attach="both" box="[996,1003,1898,1908]" fontSize="4" pageId="3" pageNumber="47">2</subScript>
|
||
CA, dimethyl caffeic acid; MeHT, methyl-hydroxytyrosol; Rha, rhamnose, Rt, retention time (minutes). Metabolites that were identical with standards, are marked with an asterisk. For identification and references, see text.
|
||
<superScript id="7C7231110B52615AFF6AF838FF047799" attach="right" box="[129,136,1934,1943]" fontSize="4" pageId="3" pageNumber="47">a</superScript>
|
||
Most intensive MS
|
||
<superScript id="7C7231110B52615AFEC3F838FEA37796" attach="left" box="[296,303,1934,1944]" fontSize="4" pageId="3" pageNumber="47">2</superScript>
|
||
ion, subjected to CID, with daughter ions shown in column ‘MS
|
||
<superScript id="7C7231110B52615AFCD2F838FCCC7796" attach="left" box="[825,832,1934,1944]" fontSize="4" pageId="3" pageNumber="47">3</superScript>
|
||
’.
|
||
<superScript id="7C7231110B52615AFF6BF813FF0B77A1" attach="right" box="[128,135,1957,1967]" fontSize="4" pageId="3" pageNumber="47">b</superScript>
|
||
MS
|
||
<superScript id="7C7231110B52615AFF40F813FF3E77A1" attach="left" box="[171,178,1957,1967]" fontSize="4" pageId="3" pageNumber="47">2</superScript>
|
||
ions not fragmented further.
|
||
</tableNote>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFF9DFE5FFECC7289" blockId="4.[87,757,179,1205]" pageId="4" pageNumber="48">
|
||
Even less polar, dimethyl- and/or acetylated PGs were also found.
|
||
<emphasis id="B973404B0B55615DFF4AFDB3FF367216" bold="true" box="[161,186,517,536]" pageId="4" pageNumber="48">64</emphasis>
|
||
was putatively identified as dimethyl-plantamajoside, both aglyca (HT and CA) were found to be methlyated, [M
|
||
<emphasis id="B973404B0B55615DFD53FD97FD45723D" box="[696,713,545,563]" italics="true" pageId="4" pageNumber="48">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B55615DFD0AFDABFD617227" box="[737,749,541,553]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFD0AFDABFD617227" attach="none" box="[737,749,541,553]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
: [MeHT-Glc(Glc)-MeCA – H]
|
||
<emphasis id="B973404B0B55615DFE80FD8FFEFB724B" box="[363,375,569,581]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFE80FD8FFEFB724B" attach="none" box="[363,375,569,581]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
. The fragmentation pattern fully supports this structure, the most specific fragment was [MeHT-Glc- MeCA – H]
|
||
<emphasis id="B973404B0B55615DFF2DFDC7FF5E7273" box="[198,210,625,637]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFF2DFDC7FF5E7273" attach="left" box="[198,210,625,637]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
(
|
||
<emphasis id="B973404B0B55615DFF0AFDC2FF787286" box="[225,244,628,648]" italics="true" pageId="4" pageNumber="48">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B55615DFF17FDC2FE8A7286" box="[252,262,628,648]" italics="true" pageId="4" pageNumber="48">z</emphasis>
|
||
505).
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFF9DFD27FEC2738D" blockId="4.[87,757,179,1205]" pageId="4" pageNumber="48">
|
||
In the case of
|
||
<emphasis id="B973404B0B55615DFECCFD26FECC72AD" bold="true" box="[295,320,656,675]" pageId="4" pageNumber="48">71</emphasis>
|
||
and
|
||
<emphasis id="B973404B0B55615DFE6EFD26FE1272AD" bold="true" box="[389,414,656,675]" pageId="4" pageNumber="48">65</emphasis>
|
||
, the two, completely identical fragmentation patterns indicated the structure [HT-Glc(Ac)(Rha) Me
|
||
<subScript id="17839E1C0B55615DFF9CFD67FF0C72D3" attach="both" box="[119,128,721,733]" fontSize="5" pageId="4" pageNumber="48">2</subScript>
|
||
CA – H]
|
||
<emphasis id="B973404B0B55615DFF3CFD73FF6F72DF" box="[215,227,709,721]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFF3CFD73FF6F72DF" attach="none" box="[215,227,709,721]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
. The
|
||
<collectingCountry id="F310DCC90B55615DFEC8FD7EFECC72D5" box="[291,320,712,731]" name="Switzerland" pageId="4" pageNumber="48">CH</collectingCountry>
|
||
<subScript id="17839E1C0B55615DFEAAFD67FEC672D3" attach="right" box="[321,330,721,733]" fontSize="5" pageId="4" pageNumber="48">3</subScript>
|
||
CHO loss from the dimethyl CA aglycon was the main fragmentation route resulting in [MeHT-Glc(Ac) (Rha)-pCoumA – H]
|
||
<emphasis id="B973404B0B55615DFEC9FD4AFEA27306" box="[290,302,764,776]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFEC9FD4AFEA27306" attach="left" box="[290,302,764,776]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
(
|
||
<emphasis id="B973404B0B55615DFED4FD49FEDE731D" box="[319,338,767,787]" italics="true" pageId="4" pageNumber="48">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B55615DFEB2FD49FEEF731D" box="[345,355,767,787]" italics="true" pageId="4" pageNumber="48">z</emphasis>
|
||
649), by conversion of the CA aglycon into p-coumaric acid (pCoumA). The ion
|
||
<emphasis id="B973404B0B55615DFDEBFCADFD9F7321" box="[512,531,795,815]" italics="true" pageId="4" pageNumber="48">m</emphasis>
|
||
/
|
||
<emphasis id="B973404B0B55615DFDF1FCADFDA87321" box="[538,548,795,815]" italics="true" pageId="4" pageNumber="48">z</emphasis>
|
||
649 yielded several fragments in MS
|
||
<superScript id="7C7231110B55615DFF10FC82FE88734E" attach="left" box="[251,260,820,832]" fontSize="5" pageId="4" pageNumber="48">3</superScript>
|
||
, all supporting the proposed structure. Identification of fragments, and pathways of decay in MS/MS is summarized in Supplementary
|
||
<figureCitation id="133C80DC0B55615DFEF8FCC6FEC5738D" box="[275,329,880,899]" captionStart="Fig" captionStartId="6.[87,113,1291,1305]" captionTargetBox="[255,1300,182,1261]" captionTargetId="figure-460@6.[254,1306,181,1262]" captionTargetPageId="6" captionText="Fig. 5. Abundance of selected natural products from extracts of P. lanceolata calli grown on media with different N source concentrations and NH + /NO – ratios (ratio of NH + in 4 3 4 the total N source).Color is proportional to the abundance (area under curve) for the given NPs, quantified in extracted ion chromatograms (a, b, c, e), or content of dry weight expressed as percent (d, f) Subplots: (a), 22, putatively identified as desrhamnosyl-acteoside, cluster 1; (b), 49, a plantamajoside isomer, cluster 2; (c), 8, putatively identified as a plantamajoside hexoside, cluster 3; (d), 18, plantamajoside (plantamajoside), cluster 4; (e), 65, putatively identified as a dimethyl-acetyl acteoside, cluster 4; (f), 27, acteoside, cluster 6." figureDoi="http://doi.org/10.5281/zenodo.10491356" httpUri="https://zenodo.org/record/10491356/files/figure.png" pageId="4" pageNumber="48">Fig. 5</figureCitation>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFF9DFC3AFF3274BB" blockId="4.[87,757,179,1205]" pageId="4" pageNumber="48">
|
||
Many unidentified peaks were also detected. These showed characteristic fragments common to the identified PGs. Several of them also showed
|
||
<collectingCountry id="F310DCC90B55615DFEF1FC75FEBB73D8" box="[282,311,963,982]" name="Switzerland" pageId="4" pageNumber="48">CH</collectingCountry>
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|
||
CO loss, indicating possible presence of an acetyl group, but most had insufficient fragments to deduce their structures, or the pattern led to conflicting evidence on substituent positions. Identification of these molecules was beyond the scope of our study. As most of the found PGs yielded MS
|
||
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||
ions between 40 and 70% efficacy (ratio of abundance of main MS
|
||
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ion and [M
|
||
<emphasis id="B973404B0B55615DFF9FFBDDFF097473" box="[116,133,1131,1149]" italics="true" pageId="4" pageNumber="48">–</emphasis>
|
||
H]
|
||
<emphasis id="B973404B0B55615DFF76FBD1FF25747D" box="[157,169,1127,1139]" italics="true" pageId="4" pageNumber="48">
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</emphasis>
|
||
), the lack of MS
|
||
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||
ions was considered to be indicative of a substantially different structure. These molecules could not be identified.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFFBCFB6FFD927507" blockId="4.[87,711,1241,1289]" pageId="4" pageNumber="48">
|
||
<emphasis id="B973404B0B55615DFFBCFB6FFD927507" italics="true" pageId="4" pageNumber="48">
|
||
2.3. Effects of N source composition on phenylethanoid glycoside pattern and concentration in
|
||
<taxonomicName id="4C07E7DA0B55615DFE9EFB43FE7C7507" box="[373,496,1269,1289]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="4" pageNumber="48" phylum="Tracheophyta" rank="subSpecies" species="lanceolata" subSpecies="calli">P. lanceolata</taxonomicName>
|
||
calli
|
||
</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFFBCFA9BFE6C7553" blockId="4.[87,757,1325,1793]" pageId="4" pageNumber="48">
|
||
<heading id="D0F02B350B55615DFFBCFA9BFE6C7553" fontSize="8" level="3" pageId="4" pageNumber="48" reason="8">
|
||
<emphasis id="B973404B0B55615DFFBCFA9BFE6C7553" italics="true" pageId="4" pageNumber="48">2.3.1. Effects of N source composition on phenylethanoid glycoside pattern, and the metabolome as a whole</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFF9DFAD0FDEC76F1" blockId="4.[87,757,1325,1793]" pageId="4" pageNumber="48">
|
||
Of the many peaks found in the set of total ion chromatograms, 89 were selected for a more in-depth study (see Section 4.8. for filtering, and data processing). As the dataset had a high degree of multicollinearity, the scaled, centered abundance data of the 89 NPs were subjected to dimensionality reduction with principle component analysis (PCA). The first three PC scores were visualized in a pseudo-3D plot shown in
|
||
<figureCitation id="133C80DC0B55615DFE68F9BBFE49762F" box="[387,453,1549,1569]" captionStart="Fig" captionStartId="5.[113,139,1768,1782]" captionTargetBox="[205,1372,181,1738]" captionTargetId="figure-2@5.[197,1407,181,1738]" captionTargetPageId="5" captionText="Fig. 4. Principal component (PC) analysis score plot of the scaled, centered and per-composition averaged natural product abundance dataset. The plot shows the effect of media composition on the metabolome as a whole. The NP abundances were measured by LC–ESI–MS3 from MeOH extracts of P. lanceolata calli grown on media with different N source compositions. For more details, see text. Subplots: (a) Pseudo-3D score plot of PCs 1–3. PC2 and PC3 scores are plotted on axes x and y, respectively, while PC1 is made proportional to point size (see legend). Media codes are as follows: N concentration (mM), followed by NH + /NO – ratio (ratio of NH + in the total N source) in 4 3 4 parentheses. The NH + concentration in the media is coded by color (see legend). Abbreviations: NSC, N source concentration. (b), Loading plot for the first three PCs showing 4 contribution of the 89 individual metabolites to the PC scores. The order of NPs was obtained from hierarchical cluster analysis of the NP abundances." figureDoi="http://doi.org/10.5281/zenodo.10491354" httpUri="https://zenodo.org/record/10491354/files/figure.png" pageId="4" pageNumber="48">Fig. 4a</figureCitation>
|
||
. It is visible, that high and low NH
|
||
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||
content media are separated along the axis PC2. The media without NH
|
||
<subScript id="17839E1C0B55615DFF38F9F8FF507654" attach="left" box="[211,220,1614,1626]" fontSize="5" pageId="4" pageNumber="48">4</subScript>
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<superScript id="7C7231110B55615DFF38F9F7FF507643" attach="left" box="[211,220,1601,1613]" fontSize="5" pageId="4" pageNumber="48">+</superScript>
|
||
had very similar scores suggesting highly similar metabolome. It can also be seen, that the metabolome response seemed to be non-linear, as no other clear trends can be observed in the PCA plot. In the loading plots (
|
||
<figureCitation id="133C80DC0B55615DFE31F92FFD9276A2" box="[474,542,1689,1708]" captionStart="Fig" captionStartId="5.[113,139,1768,1782]" captionTargetBox="[205,1372,181,1738]" captionTargetId="figure-2@5.[197,1407,181,1738]" captionTargetPageId="5" captionText="Fig. 4. Principal component (PC) analysis score plot of the scaled, centered and per-composition averaged natural product abundance dataset. The plot shows the effect of media composition on the metabolome as a whole. The NP abundances were measured by LC–ESI–MS3 from MeOH extracts of P. lanceolata calli grown on media with different N source compositions. For more details, see text. Subplots: (a) Pseudo-3D score plot of PCs 1–3. PC2 and PC3 scores are plotted on axes x and y, respectively, while PC1 is made proportional to point size (see legend). Media codes are as follows: N concentration (mM), followed by NH + /NO – ratio (ratio of NH + in the total N source) in 4 3 4 parentheses. The NH + concentration in the media is coded by color (see legend). Abbreviations: NSC, N source concentration. (b), Loading plot for the first three PCs showing 4 contribution of the 89 individual metabolites to the PC scores. The order of NPs was obtained from hierarchical cluster analysis of the NP abundances." figureDoi="http://doi.org/10.5281/zenodo.10491354" httpUri="https://zenodo.org/record/10491354/files/figure.png" pageId="4" pageNumber="48">Fig. 4b</figureCitation>
|
||
) it is obvious, that a small group of metabolites is responsible for most of the variance in PC2. We can state that the NP pattern as a whole was indicative of both N source concentration and NH
|
||
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||
/NO
|
||
<subScript id="17839E1C0B55615DFDFCF943FDAC770F" attach="left" box="[535,544,1781,1793]" fontSize="5" pageId="4" pageNumber="48">3</subScript>
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<emphasis id="B973404B0B55615DFDFCF95FFDAF76FB" box="[535,547,1769,1781]" italics="true" pageId="4" pageNumber="48">
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<superScript id="7C7231110B55615DFDFCF95FFDAF76FB" attach="left" box="[535,547,1769,1781]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
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||
</emphasis>
|
||
ratio.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFFBCF895FF74775D" blockId="4.[87,757,1827,2015]" pageId="4" pageNumber="48">
|
||
<heading id="D0F02B350B55615DFFBCF895FF74775D" fontSize="8" level="3" pageId="4" pageNumber="48" reason="8">
|
||
<emphasis id="B973404B0B55615DFFBCF895FF74775D" italics="true" pageId="4" pageNumber="48">2.3.2. Effects of N source composition on abundance of individual natural products</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFF9DF8EAFCF871A7" blockId="4.[87,757,1827,2015]" lastBlockId="4.[805,1475,182,1792]" pageId="4" pageNumber="48">
|
||
As we wanted to evaluate the effects of the treatments on the NPs directly, every metabolite abundance was subjected to ANOVA separately, the N source concentration and the NH
|
||
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/NO
|
||
<subScript id="17839E1C0B55615DFD46F82BFD3A77A7" attach="left" box="[685,694,1949,1961]" fontSize="5" pageId="4" pageNumber="48">3</subScript>
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<emphasis id="B973404B0B55615DFD46F826FD357792" box="[685,697,1936,1948]" italics="true" pageId="4" pageNumber="48">
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<superScript id="7C7231110B55615DFD46F826FD357792" attach="left" box="[685,697,1936,1948]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
ratio being the independent variables. The highest abundances of individual NPs were compared to those found on the most frequently used TC medium, MSM (60(0.33)), and the 20(0) medium used for the culturing of
|
||
<taxonomicName id="4C07E7DA0B55615DFBEFFF67FB3770EB" box="[1028,1211,209,229]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="4" pageNumber="53" phylum="Tracheophyta" rank="subSpecies" species="lanceolata" subSpecies="calli">
|
||
<emphasis id="B973404B0B55615DFBEFFF67FB0970EB" box="[1028,1157,209,229]" italics="true" pageId="4" pageNumber="48">P. lanceolata</emphasis>
|
||
calli
|
||
</taxonomicName>
|
||
by
|
||
<bibRefCitation id="EF96E1A80B55615DFB06FF64FCEB710F" author="Budzianowska, A. & Skrzypczak, L. & Budzianowski, J." pageId="4" pageNumber="48" pagination="834 - 840" refId="ref11162" refString="Budzianowska, A., Skrzypczak, L., Budzianowski, J., 2004. Phenylethanoid glucosides from in vitro propagated plants and callus cultures of Plantago lanceolata. Planta Med. 70, 834 - 840. http: // dx. doi. org / 10.1055 / s- 2004 - 827232." type="journal article" year="2004">Budzianowska et al. (2004)</bibRefCitation>
|
||
. Overall, we can state, that the N source composition had a very significant effect on the abundance of individual NPs: the N source concentration significantly influenced the abundance of 42 metabolites (
|
||
<emphasis id="B973404B0B55615DFC2CFEF7FC5F715B" box="[967,979,321,341]" italics="true" pageId="4" pageNumber="48">p</emphasis>
|
||
<0.00011), while the NH
|
||
<subScript id="17839E1C0B55615DFB3BFEFDFB557159" attach="left" box="[1232,1241,331,343]" fontSize="5" pageId="4" pageNumber="48">4</subScript>
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<superScript id="7C7231110B55615DFB3BFE88FB557144" attach="left" box="[1232,1241,318,330]" fontSize="5" pageId="4" pageNumber="48">+</superScript>
|
||
/NO
|
||
<subScript id="17839E1C0B55615DFAEAFEFDFA867159" attach="left" box="[1281,1290,331,343]" fontSize="5" pageId="4" pageNumber="48">3</subScript>
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||
<emphasis id="B973404B0B55615DFAEAFE88FA817144" box="[1281,1293,318,330]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFAEAFE88FA817144" attach="left" box="[1281,1293,318,330]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
ratio significantly altered concentrations of 10 metabolites (of 89 NPs examined) (
|
||
<emphasis id="B973404B0B55615DFCC6FECEFCB57182" box="[813,825,376,396]" italics="true" pageId="4" pageNumber="48">p</emphasis>
|
||
<0.00011). These results are summarized in Supplementary
|
||
<tableCitation id="C685A9E20B55615DFCCEFE23FCE371A7" box="[805,879,405,425]" captionStart="Table 1" captionStartId="3.[114,158,1416,1430]" captionTargetPageId="3" captionText="Table 1 Fragmentation pattern of putatively identified major peaks obtained by LC–ESI–MS3 analysis of defatted Plantago lanceolata calli MeOH extracts in negative ion mode. MS2 ions are CID products of the [M–H] –, while MS3 ions are CID products of the most abundant MS2 ion (MS2a)." httpUri="http://table.plazi.org/id/DF78CCD10B52615AFF99FA3EFC3D75CD" pageId="4" pageNumber="48" tableUuid="DF78CCD10B52615AFF99FA3EFC3D75CD">Table 1</tableCitation>
|
||
.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFCAFFE07FC4173D8" blockId="4.[805,1475,182,1792]" pageId="4" pageNumber="48">
|
||
If we take a closer look at the abundance gains of individual NPs compared to MSM, 60(0.33) in Supplementary
|
||
<tableCitation id="C685A9E20B55615DFAE6FE7BFAD571EE" box="[1293,1369,461,480]" captionStart="Table 1" captionStartId="3.[114,158,1416,1430]" captionTargetPageId="3" captionText="Table 1 Fragmentation pattern of putatively identified major peaks obtained by LC–ESI–MS3 analysis of defatted Plantago lanceolata calli MeOH extracts in negative ion mode. MS2 ions are CID products of the [M–H] –, while MS3 ions are CID products of the most abundant MS2 ion (MS2a)." httpUri="http://table.plazi.org/id/DF78CCD10B52615AFF99FA3EFC3D75CD" pageId="4" pageNumber="48" tableUuid="DF78CCD10B52615AFF99FA3EFC3D75CD">Table 1</tableCitation>
|
||
, it can be observed that the conventional medium was one of the worst for production of NPs. For most NPs, the best media were those containing six times less (10 mM) N source only. Most metabolites were synthesized at highest rate, when the NH
|
||
<superScript id="7C7231110B55615DFAF2FD8FFAAE724B" attach="left" box="[1305,1314,569,581]" fontSize="5" pageId="4" pageNumber="48">+</superScript>
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||
/NO
|
||
<subScript id="17839E1C0B55615DFAA1FDF0FADF725C" attach="left" box="[1354,1363,582,594]" fontSize="5" pageId="4" pageNumber="48">3</subScript>
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<emphasis id="B973404B0B55615DFAA1FD8FFADA724B" box="[1354,1366,569,581]" italics="true" pageId="4" pageNumber="48">
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<superScript id="7C7231110B55615DFAA1FD8FFADA724B" attach="left" box="[1354,1366,569,581]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
ratio was kept at 0.33, including the chief compound, plantamajoside. The best medium was therefore 10(0.33) for overall production. But, in case the yield of a specific NP would be the aim to be optimized, the optimal medium can be a very different composition (Supplementary
|
||
<tableCitation id="C685A9E20B55615DFC6AFD7EFC4072D5" box="[897,972,712,731]" captionStart="Table 1" captionStartId="3.[114,158,1416,1430]" captionTargetPageId="3" captionText="Table 1 Fragmentation pattern of putatively identified major peaks obtained by LC–ESI–MS3 analysis of defatted Plantago lanceolata calli MeOH extracts in negative ion mode. MS2 ions are CID products of the [M–H] –, while MS3 ions are CID products of the most abundant MS2 ion (MS2a)." httpUri="http://table.plazi.org/id/DF78CCD10B52615AFF99FA3EFC3D75CD" pageId="4" pageNumber="48" tableUuid="DF78CCD10B52615AFF99FA3EFC3D75CD">Table 1</tableCitation>
|
||
). In many of the studies regarding the optimization of medium N source for high NP yield and growth, media with lower NH
|
||
<superScript id="7C7231110B55615DFC53FD4AFC4D7306" attach="left" box="[952,961,764,776]" fontSize="5" pageId="4" pageNumber="48">+</superScript>
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<subScript id="17839E1C0B55615DFC53FCBFFC4D731B" attach="left" box="[952,961,777,789]" fontSize="5" pageId="4" pageNumber="48">4</subScript>
|
||
content than that in MSM was found to be optimal for the production of secondary metabolites, for example in a study on
|
||
<taxonomicName id="4C07E7DA0B55615DFCAFFC81FC287345" box="[836,932,823,843]" class="Magnoliopsida" family="Asteraceae" genus="Echinacea" kingdom="Plantae" order="Asterales" pageId="4" pageNumber="48" phylum="Tracheophyta" rank="genus">
|
||
<emphasis id="B973404B0B55615DFCAFFC81FC287345" box="[836,932,823,843]" italics="true" pageId="4" pageNumber="48">Echinacea</emphasis>
|
||
</taxonomicName>
|
||
adventitious shoots (
|
||
<bibRefCitation id="EF96E1A80B55615DFB96FC8EFA997345" author="Wu, C. - H. & Dewir, Y. H. & Hahn, E. - J. & Paek, K. - Y." box="[1149,1301,824,843]" pageId="4" pageNumber="48" pagination="193 - 199" refId="ref12843" refString="Wu, C. - H., Dewir, Y. H., Hahn, E. - J., Paek, K. - Y., 2006. Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots of Echinacea angustifolia. J. Plant Biol. 49, 193 - 199. http: // dx. doi. org / 10.1007 / BF 03030532." type="journal article" year="2006">Wu et al., 2006</bibRefCitation>
|
||
). In our case, the 10(0.33) or 10(0) media were the best for production of most of the different NPs (Supplementary
|
||
<tableCitation id="C685A9E20B55615DFB68FCC6FB42738D" box="[1155,1230,880,899]" captionStart="Table 1" captionStartId="3.[114,158,1416,1430]" captionTargetPageId="3" captionText="Table 1 Fragmentation pattern of putatively identified major peaks obtained by LC–ESI–MS3 analysis of defatted Plantago lanceolata calli MeOH extracts in negative ion mode. MS2 ions are CID products of the [M–H] –, while MS3 ions are CID products of the most abundant MS2 ion (MS2a)." httpUri="http://table.plazi.org/id/DF78CCD10B52615AFF99FA3EFC3D75CD" pageId="4" pageNumber="48" tableUuid="DF78CCD10B52615AFF99FA3EFC3D75CD">Table 1</tableCitation>
|
||
). Despite several of the tested NPs were identified as phenylethanoid glycosides, very different responses to NH
|
||
<superScript id="7C7231110B55615DFBC3FC12FBBD73BE" attach="left" box="[1064,1073,932,944]" fontSize="5" pageId="4" pageNumber="48">+</superScript>
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||
/NO
|
||
<subScript id="17839E1C0B55615DFBB2FC07FBEE73B3" attach="left" box="[1113,1122,945,957]" fontSize="5" pageId="4" pageNumber="48">3</subScript>
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||
<emphasis id="B973404B0B55615DFBB2FC12FBE973BE" box="[1113,1125,932,944]" italics="true" pageId="4" pageNumber="48">
|
||
<superScript id="7C7231110B55615DFBB2FC12FBE973BE" attach="left" box="[1113,1125,932,944]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
</emphasis>
|
||
ratio and N source were detected, as detailed later.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFCAFFC69FC157504" blockId="4.[805,1475,182,1792]" pageId="4" pageNumber="48">
|
||
After obtaining the correlation matrix of the scaled and centered matrix of metabolite abundances, and clustering of the metabolites with respect to their relative abundances in calli grown on different media, a heatmap was generated in R (Supplementary Fig. 6). A relatively strong multi-correlation can be observed for most of the metabolites. The clustering of the metabolites resulted in six groups (
|
||
<figureCitation id="133C80DC0B55615DFBBDFB31FB117494" box="[1110,1181,1159,1178]" captionStart="Fig" captionStartId="5.[113,139,1768,1782]" captionTargetBox="[205,1372,181,1738]" captionTargetId="figure-2@5.[197,1407,181,1738]" captionTargetPageId="5" captionText="Fig. 4. Principal component (PC) analysis score plot of the scaled, centered and per-composition averaged natural product abundance dataset. The plot shows the effect of media composition on the metabolome as a whole. The NP abundances were measured by LC–ESI–MS3 from MeOH extracts of P. lanceolata calli grown on media with different N source compositions. For more details, see text. Subplots: (a) Pseudo-3D score plot of PCs 1–3. PC2 and PC3 scores are plotted on axes x and y, respectively, while PC1 is made proportional to point size (see legend). Media codes are as follows: N concentration (mM), followed by NH + /NO – ratio (ratio of NH + in the total N source) in 4 3 4 parentheses. The NH + concentration in the media is coded by color (see legend). Abbreviations: NSC, N source concentration. (b), Loading plot for the first three PCs showing 4 contribution of the 89 individual metabolites to the PC scores. The order of NPs was obtained from hierarchical cluster analysis of the NP abundances." figureDoi="http://doi.org/10.5281/zenodo.10491354" httpUri="https://zenodo.org/record/10491354/files/figure.png" pageId="4" pageNumber="48">Fig. 4b</figureCitation>
|
||
, Supplementary Fig. 6), that reacted relatively similarly to different media compositions. Interestingly, the NPs putatively identified as PGs were sporadically spread across the clusters with different response to the treatments.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFCAFFAA4FB4576F1" blockId="4.[805,1475,182,1792]" pageId="4" pageNumber="48">
|
||
While the abundance of NPs in clusters 1 and 2 was the highest on 10(0), maximal abundances for NPs in clusters 3, 4 or 5 could be achieved on 10(0.33). As 10 mM N media are substantially different from the reference media, the production of these metabolites could be increased to a high extent (Supplementary
|
||
<tableCitation id="C685A9E20B55615DFAA0FA34FA14759B" box="[1355,1432,1410,1429]" captionStart="Table 1" captionStartId="3.[114,158,1416,1430]" captionTargetPageId="3" captionText="Table 1 Fragmentation pattern of putatively identified major peaks obtained by LC–ESI–MS3 analysis of defatted Plantago lanceolata calli MeOH extracts in negative ion mode. MS2 ions are CID products of the [M–H] –, while MS3 ions are CID products of the most abundant MS2 ion (MS2a)." httpUri="http://table.plazi.org/id/DF78CCD10B52615AFF99FA3EFC3D75CD" pageId="4" pageNumber="48" tableUuid="DF78CCD10B52615AFF99FA3EFC3D75CD">Table 1</tableCitation>
|
||
). In many cases, threshold-like responses were found, that were for example described for anthocyanin production response to N source concentration by
|
||
<bibRefCitation id="EF96E1A80B55615DFBCEFA63FA8475E6" author="Hirasuna, T. & Shuler, M. & Lackney, V. & Spanswick, R." box="[1061,1288,1493,1513]" pageId="4" pageNumber="48" pagination="107 - 120" refId="ref11843" refString="Hirasuna, T., Shuler, M., Lackney, V., Spanswick, R., 1991. Enhanced anthocyanin production in grape cell-cultures. Plant Sci. 78, 107 - 120. http: // dx. doi. org / 10.1016 / 0168 - 9452 (91) 90167 - 7." type="journal article" year="1991">Hirasuna et al. (1991)</bibRefCitation>
|
||
in grape cell-cultures. The NP with the highest abundance in the sample was plantamajoside (
|
||
<emphasis id="B973404B0B55615DFC4AF9BBFC36762E" bold="true" box="[929,954,1549,1568]" pageId="4" pageNumber="48">18</emphasis>
|
||
) in cluster 4. For
|
||
<emphasis id="B973404B0B55615DFB81F9BBFB0F762E" bold="true" box="[1130,1155,1549,1568]" pageId="4" pageNumber="48">18</emphasis>
|
||
, the best yield was obtained on medium 10(0.33), where 3.54 ± 0.83% (dry wt.) content was found. The least content was 1.04 ± 0.63% (dry wt.), found on medium 40(0.11). Interestingly, the metabolite with the second highest abundance fell in a different cluster; both the optimal medium and the response to the treatments was found to be substantially different – as it can be seen by comparing the response surfaces (
|
||
<figureCitation id="133C80DC0B55615DFCC6F966FC2376ED" box="[813,943,1744,1763]" captionStart="Fig" captionStartId="6.[87,113,1291,1305]" captionTargetBox="[255,1300,182,1261]" captionTargetId="figure-460@6.[254,1306,181,1262]" captionTargetPageId="6" captionText="Fig. 5. Abundance of selected natural products from extracts of P. lanceolata calli grown on media with different N source concentrations and NH + /NO – ratios (ratio of NH + in 4 3 4 the total N source).Color is proportional to the abundance (area under curve) for the given NPs, quantified in extracted ion chromatograms (a, b, c, e), or content of dry weight expressed as percent (d, f) Subplots: (a), 22, putatively identified as desrhamnosyl-acteoside, cluster 1; (b), 49, a plantamajoside isomer, cluster 2; (c), 8, putatively identified as a plantamajoside hexoside, cluster 3; (d), 18, plantamajoside (plantamajoside), cluster 4; (e), 65, putatively identified as a dimethyl-acetyl acteoside, cluster 4; (f), 27, acteoside, cluster 6." figureDoi="http://doi.org/10.5281/zenodo.10491356" httpUri="https://zenodo.org/record/10491356/files/figure.png" pageId="4" pageNumber="48">Fig. 5d and f</figureCitation>
|
||
). Acteoside content varied from 0.54 ± 0.29% (of dry wt.) on 20(0) to 1.30 ± 0.40% on 40(0.33).
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615DFCCEF895FA137739" blockId="4.[805,1475,1824,2015]" box="[805,1439,1824,1850]" pageId="4" pageNumber="48">
|
||
<emphasis id="B973404B0B55615DFCCEF895FA137739" box="[805,1439,1824,1850]" italics="true" pageId="4" pageNumber="48">
|
||
2.3.3. Statistical interaction between N source and NH
|
||
<superScript id="7C7231110B55615DFADBF896FAB57723" attach="left" box="[1328,1337,1824,1837]" fontSize="5" pageId="4" pageNumber="48">+</superScript>
|
||
<subScript id="17839E1C0B55615DFADBF89BFAB57734" attach="left" box="[1328,1337,1837,1850]" fontSize="5" pageId="4" pageNumber="48">4</subScript>
|
||
/NO
|
||
<subScript id="17839E1C0B55615DFAB5F89BFAEB7734" attach="left" box="[1374,1383,1837,1850]" fontSize="5" pageId="4" pageNumber="48">3</subScript>
|
||
<superScript id="7C7231110B55615DFAB5F897FAE67723" attach="left" box="[1374,1386,1825,1837]" fontSize="5" pageId="4" pageNumber="48">–</superScript>
|
||
ratio
|
||
</emphasis>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B55615FFCAFF8F6FF4675D0" blockId="4.[805,1475,1824,2015]" lastBlockId="6.[87,757,1451,2006]" lastPageId="6" lastPageNumber="50" pageId="4" pageNumber="48">
|
||
Most of the NPs responded in a non-linear fashion to the combinations of these two factors tested. Responses are plotted as heatmaps for better evaluation possibility of this interaction (
|
||
<figureCitation id="133C80DC0B55615DFCC6F822FCEF77A8" box="[813,867,1939,1959]" captionStart="Fig" captionStartId="6.[87,113,1291,1305]" captionTargetBox="[255,1300,182,1261]" captionTargetId="figure-460@6.[254,1306,181,1262]" captionTargetPageId="6" captionText="Fig. 5. Abundance of selected natural products from extracts of P. lanceolata calli grown on media with different N source concentrations and NH + /NO – ratios (ratio of NH + in 4 3 4 the total N source).Color is proportional to the abundance (area under curve) for the given NPs, quantified in extracted ion chromatograms (a, b, c, e), or content of dry weight expressed as percent (d, f) Subplots: (a), 22, putatively identified as desrhamnosyl-acteoside, cluster 1; (b), 49, a plantamajoside isomer, cluster 2; (c), 8, putatively identified as a plantamajoside hexoside, cluster 3; (d), 18, plantamajoside (plantamajoside), cluster 4; (e), 65, putatively identified as a dimethyl-acetyl acteoside, cluster 4; (f), 27, acteoside, cluster 6." figureDoi="http://doi.org/10.5281/zenodo.10491356" httpUri="https://zenodo.org/record/10491356/files/figure.png" pageId="4" pageNumber="48">Fig. 5</figureCitation>
|
||
). As stated before, there were many response
|
||
<typeStatus id="54BC22FB0B55615DFADCF822FAE277A9" box="[1335,1390,1940,1959]" pageId="4" pageNumber="48">types</typeStatus>
|
||
, despite many NPs were putatively identified as phenylethanoid glycosides. The presence of local maxima and/or minima renders the independent optimization of
|
||
<specimenCode id="DBA134220B57615FFEDDFA19FED375CA" box="[310,351,1455,1476]" collectionCode="NH" country="South Africa" lsid="urn:lsid:biocol.org:col:14911" name="South African National Biodiversity Institute" pageId="6" pageNumber="50" type="Herbarium">
|
||
NH
|
||
<subScript id="17839E1C0B57615FFEBDFA0EFED375CA" attach="left" box="[342,351,1464,1476]" fontSize="5" pageId="6" pageNumber="50">4</subScript>
|
||
</specimenCode>
|
||
<superScript id="7C7231110B57615FFEBDFA1DFED375B9" attach="left" box="[342,351,1451,1463]" fontSize="5" pageId="6" pageNumber="50">+</superScript>
|
||
/
|
||
<specimenCode id="DBA134220B57615FFE8CFA19FE1C75CA" box="[359,400,1455,1476]" collectionCode="NO" country="USA" lsid="urn:lsid:biocol.org:col:15617" name="Tulane University Herbarium" pageId="6" pageNumber="50" type="Herbarium">
|
||
NO
|
||
<subScript id="17839E1C0B57615FFE6CFA0EFE1C75CA" attach="left" box="[391,400,1464,1476]" fontSize="5" pageId="6" pageNumber="50">3</subScript>
|
||
</specimenCode>
|
||
<emphasis id="B973404B0B57615FFE6CFA1DFE1F75B9" box="[391,403,1451,1463]" italics="true" pageId="6" pageNumber="50">
|
||
<superScript id="7C7231110B57615FFE6CFA1DFE1F75B9" attach="left" box="[391,403,1451,1463]" fontSize="5" pageId="6" pageNumber="50">–</superScript>
|
||
</emphasis>
|
||
ratio and
|
||
<collectionCode id="ED16049C0B57615FFDEFFA19FD9875CC" box="[516,532,1455,1474]" country="China" lsid="urn:lsid:biocol.org:col:13092" name="Nanjing University" pageId="6" pageNumber="50" type="Herbarium">N</collectionCode>
|
||
source concentration impossible.
|
||
</paragraph>
|
||
<caption id="DF78CCD10B54615CFF9AF95EFAB17766" ID-DOI="http://doi.org/10.5281/zenodo.10491354" ID-Zenodo-Dep="10491354" httpUri="https://zenodo.org/record/10491354/files/figure.png" pageId="5" pageNumber="49" startId="5.[113,139,1768,1782]" targetBox="[205,1372,181,1738]" targetPageId="5" targetType="figure">
|
||
<paragraph id="8BB89C590B54615CFF9AF95EFAB17766" blockId="5.[113,1501,1768,1897]" pageId="5" pageNumber="49">
|
||
<emphasis id="B973404B0B54615CFF9AF95EFF2F76F8" bold="true" box="[113,163,1768,1782]" pageId="5" pageNumber="49">Fig. 4.</emphasis>
|
||
Principal component (PC) analysis score plot of the scaled, centered and per-composition averaged natural product abundance dataset. The plot shows the effect of media composition on the metabolome as a whole. The NP abundances were measured by LC–ESI–MS
|
||
<superScript id="7C7231110B54615CFC28F94DFC46770B" attach="left" box="[963,970,1787,1797]" fontSize="4" pageId="5" pageNumber="49">3</superScript>
|
||
from MeOH extracts of
|
||
<taxonomicName id="4C07E7DA0B54615CFB7DF948FAAD7703" box="[1174,1313,1790,1805]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="5" pageNumber="53" phylum="Tracheophyta" rank="subSpecies" species="lanceolata" subSpecies="calli">
|
||
<emphasis id="B973404B0B54615CFB7DF948FB757703" box="[1174,1273,1790,1805]" italics="true" pageId="5" pageNumber="49">P. lanceolata</emphasis>
|
||
calli
|
||
</taxonomicName>
|
||
grown on media with different N source compositions. For more details, see text.
|
||
<emphasis id="B973404B0B54615CFDBBF8A2FD1A772A" box="[592,662,1812,1828]" italics="true" pageId="5" pageNumber="49">Subplots:</emphasis>
|
||
(a) Pseudo-3D score plot of PCs 1–3. PC2 and PC3 scores are plotted on axes
|
||
<emphasis id="B973404B0B54615CFAE8F8A3FA80772A" box="[1283,1292,1813,1828]" italics="true" pageId="5" pageNumber="49">x</emphasis>
|
||
and
|
||
<emphasis id="B973404B0B54615CFAD8F8A3FAB0772A" box="[1331,1340,1813,1828]" italics="true" pageId="5" pageNumber="49">y</emphasis>
|
||
, respectively, while PC1 is made proportional to point size (see legend). Media codes are as follows: N concentration (mM), followed by NH
|
||
<superScript id="7C7231110B54615CFBADF89FFBC1773C" attach="left" box="[1094,1101,1833,1842]" fontSize="4" pageId="5" pageNumber="49">+</superScript>
|
||
/NO
|
||
<emphasis id="B973404B0B54615CFB86F89FFBFB773C" box="[1133,1143,1833,1842]" italics="true" pageId="5" pageNumber="49">
|
||
<superScript id="7C7231110B54615CFB86F89FFBFB773C" attach="left" box="[1133,1143,1833,1842]" fontSize="4" pageId="5" pageNumber="49">–</superScript>
|
||
</emphasis>
|
||
ratio (ratio of NH
|
||
<superScript id="7C7231110B54615CFAE1F89FFA9D773C" attach="left" box="[1290,1297,1833,1842]" fontSize="4" pageId="5" pageNumber="49">+</superScript>
|
||
in the total N source) in
|
||
<subScript id="17839E1C0B54615CFBADF885FA9D7733" attach="left" box="[1094,1297,1843,1853]" fontSize="4" pageId="5" pageNumber="49">4 3 4</subScript>
|
||
parentheses. The NH
|
||
<superScript id="7C7231110B54615CFEF3F8F6FE937747" attach="left" box="[280,287,1856,1865]" fontSize="4" pageId="5" pageNumber="49">+</superScript>
|
||
concentration in the media is coded by color (see legend).
|
||
<emphasis id="B973404B0B54615CFD17F8F5FCE7775C" box="[764,875,1859,1874]" italics="true" pageId="5" pageNumber="49">Abbreviations:</emphasis>
|
||
NSC, N source concentration. (b), Loading plot for the first three PCs showing
|
||
<subScript id="17839E1C0B54615CFEF3F8FCFE93775A" attach="left" box="[280,287,1866,1876]" fontSize="4" pageId="5" pageNumber="49">4</subScript>
|
||
contribution of the 89 individual metabolites to the PC scores. The order of NPs was obtained from hierarchical cluster analysis of the NP abundances.
|
||
</paragraph>
|
||
</caption>
|
||
<caption id="DF78CCD10B57615FFFBCFABDFF74757A" ID-DOI="http://doi.org/10.5281/zenodo.10491356" ID-Zenodo-Dep="10491356" httpUri="https://zenodo.org/record/10491356/files/figure.png" pageId="6" pageNumber="50" startId="6.[87,113,1291,1305]" targetBox="[255,1300,182,1261]" targetPageId="6" targetType="figure">
|
||
<paragraph id="8BB89C590B57615FFFBCFABDFF74757A" blockId="6.[87,1474,1287,1397]" pageId="6" pageNumber="50">
|
||
<emphasis id="B973404B0B57615FFFBCFABDFF047517" bold="true" box="[87,136,1291,1305]" pageId="6" pageNumber="50">Fig. 5.</emphasis>
|
||
Abundance of selected natural products from extracts of
|
||
<taxonomicName id="4C07E7DA0B57615FFDBCFABCFD517517" box="[599,733,1290,1305]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="6" pageNumber="53" phylum="Tracheophyta" rank="subSpecies" species="lanceolata" subSpecies="calli">
|
||
<emphasis id="B973404B0B57615FFDBCFABCFD347517" box="[599,696,1290,1305]" italics="true" pageId="6" pageNumber="50">P. lanceolata</emphasis>
|
||
calli
|
||
</taxonomicName>
|
||
grown on media with different N source concentrations and NH
|
||
<superScript id="7C7231110B57615FFB0BFAB1FB6B751E" attach="left" box="[1248,1255,1287,1296]" fontSize="4" pageId="6" pageNumber="50">+</superScript>
|
||
/NO
|
||
<emphasis id="B973404B0B57615FFAECFAB1FA9D751E" box="[1287,1297,1287,1296]" italics="true" pageId="6" pageNumber="50">
|
||
<superScript id="7C7231110B57615FFAECFAB1FA9D751E" attach="left" box="[1287,1297,1287,1296]" fontSize="4" pageId="6" pageNumber="50">–</superScript>
|
||
</emphasis>
|
||
ratios (ratio of NH
|
||
<superScript id="7C7231110B57615FFA4DFAB1FA21751E" attach="left" box="[1446,1453,1287,1296]" fontSize="4" pageId="6" pageNumber="50">+</superScript>
|
||
in
|
||
<subScript id="17839E1C0B57615FFB0BFAA4FA217512" attach="left" box="[1248,1453,1298,1308]" fontSize="4" pageId="6" pageNumber="50">4 3 4</subScript>
|
||
the total N source). Color is proportional to the abundance (area under curve) for the given NPs, quantified in extracted ion chromatograms (a, b, c, e), or content of dry weight expressed as percent (d, f)
|
||
<emphasis id="B973404B0B57615FFEC4FA81FEF97549" box="[303,373,1335,1351]" italics="true" pageId="6" pageNumber="50">Subplots:</emphasis>
|
||
(a),
|
||
<emphasis id="B973404B0B57615FFE73FA8FFE207549" bold="true" box="[408,428,1337,1351]" pageId="6" pageNumber="50">22</emphasis>
|
||
, putatively identified as desrhamnosyl-acteoside, cluster 1; (b),
|
||
<emphasis id="B973404B0B57615FFC5AFA8FFC497549" bold="true" box="[945,965,1337,1351]" pageId="6" pageNumber="50">49</emphasis>
|
||
, a plantamajoside isomer, cluster 2; (c), 8, putatively identified as a plantamajoside hexoside, cluster 3; (d),
|
||
<emphasis id="B973404B0B57615FFE2DFAE6FE567550" bold="true" box="[454,474,1360,1374]" pageId="6" pageNumber="50">18</emphasis>
|
||
, plantamajoside (plantamajoside), cluster 4; (e),
|
||
<emphasis id="B973404B0B57615FFC86FAE6FC0D7550" bold="true" box="[877,897,1360,1374]" pageId="6" pageNumber="50">65</emphasis>
|
||
, putatively identified as a dimethyl-acetyl acteoside, cluster 4; (f),
|
||
<emphasis id="B973404B0B57615FFA41FAE6FA327550" bold="true" box="[1450,1470,1360,1374]" pageId="6" pageNumber="50">27</emphasis>
|
||
, acteoside, cluster 6.
|
||
</paragraph>
|
||
</caption>
|
||
<paragraph id="8BB89C590B57615FFF9DFA51FDD976FB" blockId="6.[87,757,1451,2006]" pageId="6" pageNumber="50">
|
||
This statistical interaction means that the response to the N concentration at different levels of the NH
|
||
<superScript id="7C7231110B57615FFE38FA49FE507605" attach="left" box="[467,476,1535,1547]" fontSize="5" pageId="6" pageNumber="50">+</superScript>
|
||
<subScript id="17839E1C0B57615FFE38F9BAFE507616" attach="left" box="[467,476,1548,1560]" fontSize="5" pageId="6" pageNumber="50">4</subScript>
|
||
/NO
|
||
<subScript id="17839E1C0B57615FFDEFF9BAFD817616" attach="left" box="[516,525,1548,1560]" fontSize="5" pageId="6" pageNumber="50">3</subScript>
|
||
<emphasis id="B973404B0B57615FFDEFFA49FD9C7605" box="[516,528,1535,1547]" italics="true" pageId="6" pageNumber="50">
|
||
<superScript id="7C7231110B57615FFDEFFA49FD9C7605" attach="left" box="[516,528,1535,1547]" fontSize="5" pageId="6" pageNumber="50">–</superScript>
|
||
</emphasis>
|
||
ratio was different: for example in the case of
|
||
<emphasis id="B973404B0B57615FFEBEF9A8FEE2763F" bold="true" box="[341,366,1566,1585]" pageId="6" pageNumber="50">27</emphasis>
|
||
(
|
||
<figureCitation id="133C80DC0B57615FFE6AF9A8FE49763F" box="[385,453,1566,1585]" captionStart="Fig" captionStartId="6.[87,113,1291,1305]" captionTargetBox="[255,1300,182,1261]" captionTargetId="figure-460@6.[254,1306,181,1262]" captionTargetPageId="6" captionText="Fig. 5. Abundance of selected natural products from extracts of P. lanceolata calli grown on media with different N source concentrations and NH + /NO – ratios (ratio of NH + in 4 3 4 the total N source).Color is proportional to the abundance (area under curve) for the given NPs, quantified in extracted ion chromatograms (a, b, c, e), or content of dry weight expressed as percent (d, f) Subplots: (a), 22, putatively identified as desrhamnosyl-acteoside, cluster 1; (b), 49, a plantamajoside isomer, cluster 2; (c), 8, putatively identified as a plantamajoside hexoside, cluster 3; (d), 18, plantamajoside (plantamajoside), cluster 4; (e), 65, putatively identified as a dimethyl-acetyl acteoside, cluster 4; (f), 27, acteoside, cluster 6." figureDoi="http://doi.org/10.5281/zenodo.10491356" httpUri="https://zenodo.org/record/10491356/files/figure.png" pageId="6" pageNumber="50">Fig. 5f</figureCitation>
|
||
), at 0.33 NH
|
||
<subScript id="17839E1C0B57615FFDA5F99EFDDB763A" attach="right" box="[590,599,1576,1588]" fontSize="5" pageId="6" pageNumber="50">4</subScript>
|
||
<superScript id="7C7231110B57615FFDA5F9ADFDDB7629" attach="right" box="[590,599,1563,1575]" fontSize="5" pageId="6" pageNumber="50">+</superScript>
|
||
/NO
|
||
<subScript id="17839E1C0B57615FFD95F99EFD0B763A" attach="left" box="[638,647,1576,1588]" fontSize="5" pageId="6" pageNumber="50">3</subScript>
|
||
<emphasis id="B973404B0B57615FFD95F9ADFD067629" box="[638,650,1563,1575]" italics="true" pageId="6" pageNumber="50">
|
||
<superScript id="7C7231110B57615FFD95F9ADFD067629" attach="left" box="[638,650,1563,1575]" fontSize="5" pageId="6" pageNumber="50">–</superScript>
|
||
</emphasis>
|
||
ratio, the response to the N concentration showed an optimum curve: the highest abundance was detected at 40 mM N, while at 0 NH
|
||
<superScript id="7C7231110B57615FFD53F9E5FD4D7651" attach="left" box="[696,705,1619,1631]" fontSize="5" pageId="6" pageNumber="50">+</superScript>
|
||
<subScript id="17839E1C0B57615FFD53F9E9FD4D7665" attach="left" box="[696,705,1631,1643]" fontSize="5" pageId="6" pageNumber="50">4</subScript>
|
||
/NO
|
||
<subScript id="17839E1C0B57615FFD03F9E9FD7D7665" attach="left" box="[744,753,1631,1643]" fontSize="5" pageId="6" pageNumber="50">3</subScript>
|
||
<emphasis id="B973404B0B57615FFD03F9E5FD787651" box="[744,756,1619,1631]" italics="true" pageId="6" pageNumber="50">
|
||
<superScript id="7C7231110B57615FFD03F9E5FD787651" attach="left" box="[744,756,1619,1631]" fontSize="5" pageId="6" pageNumber="50">–</superScript>
|
||
</emphasis>
|
||
ratio, a threshold-like response was observed, with low abundance except at 10 mM N. This means that if we would have done the yield optimization for
|
||
<emphasis id="B973404B0B57615FFEAFF91CFED176B3" bold="true" box="[324,349,1706,1725]" pageId="6" pageNumber="50">27</emphasis>
|
||
using the one-factor at a time design, we might have ended up in a suboptimal medium composition, as shown later by
|
||
<emphasis id="B973404B0B57615FFF1FF957FEB176FB" box="[244,317,1761,1781]" italics="true" pageId="6" pageNumber="50">in silico</emphasis>
|
||
OFAT optimization studies.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B57615FFF9DF948FAE07792" blockId="6.[87,757,1451,2006]" lastBlockId="6.[805,1474,1455,2004]" pageId="6" pageNumber="50">
|
||
The studies on TCs from
|
||
<taxonomicName id="4C07E7DA0B57615FFE9DF94BFE77771F" box="[374,507,1789,1809]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="6" pageNumber="50" phylum="Tracheophyta" rank="species" species="undetermined">
|
||
<emphasis id="B973404B0B57615FFE9DF94BFE46771F" box="[374,458,1789,1809]" italics="true" pageId="6" pageNumber="50">Plantago</emphasis>
|
||
spp.
|
||
</taxonomicName>
|
||
almost exclusively used the original MS, supplemented with various hormone concentrations (
|
||
<bibRefCitation id="EF96E1A80B57615FFF70F880FECB7747" author="Fons, F. & Gargadennec, A. & Rapior, S." box="[155,327,1846,1865]" pageId="6" pageNumber="50" pagination="277 - 300" refId="ref11243" refString="Fons, F., Gargadennec, A., Rapior, S., 2008. Culture of Plantago species as bioactive components resources: a 20 - year review and recent applications. Acta Bot. Gallica 155, 277 - 300." type="journal article" year="2008">Fons et al., 2008</bibRefCitation>
|
||
). A few studies used modified MS, these were either diluted variants (e.g. ½ strength) (
|
||
<bibRefCitation id="EF96E1A80B57615FFDD6F8E4FD6B776B" author="Fons, F. & Gargadennec, A. & Rapior, S." box="[573,743,1873,1893]" pageId="6" pageNumber="50" pagination="277 - 300" refId="ref11243" refString="Fons, F., Gargadennec, A., Rapior, S., 2008. Culture of Plantago species as bioactive components resources: a 20 - year review and recent applications. Acta Bot. Gallica 155, 277 - 300." type="journal article" year="2008">Fons et al., 2008</bibRefCitation>
|
||
), or reduction in some inorganic components were used, as in
|
||
<bibRefCitation id="EF96E1A80B57615FFFBCF83FFEBB7792" author="Mederos, S. & Martin, C. & Navarro, E. & Ayuso, M. J." box="[87,311,1929,1949]" pageId="6" pageNumber="50" pagination="465 - 468" refId="ref12176" refString="Mederos, S., Martin, C., Navarro, E., Ayuso, M. J., 1997. Micropropagation of a medicinal plant, Plantago major L. Biol. Plant. 40, 465 - 468. http: // dx. doi. org / 10.1023 / A: 1001190603295." type="journal article" year="1997">Mederos et al. (1997)</bibRefCitation>
|
||
. The effects of the N source concentration and the NH
|
||
<subScript id="17839E1C0B57615FFF39F818FF5777B4" attach="left" box="[210,219,1966,1978]" fontSize="5" pageId="6" pageNumber="50">4</subScript>
|
||
<superScript id="7C7231110B57615FFF39F817FF5777A3" attach="left" box="[210,219,1953,1965]" fontSize="5" pageId="6" pageNumber="50">+</superScript>
|
||
/NO
|
||
<subScript id="17839E1C0B57615FFEE8F818FE8077B4" attach="left" box="[259,268,1966,1978]" fontSize="5" pageId="6" pageNumber="50">3</subScript>
|
||
<emphasis id="B973404B0B57615FFEE8F817FE8377A3" box="[259,271,1953,1965]" italics="true" pageId="6" pageNumber="50">
|
||
<superScript id="7C7231110B57615FFEE8F817FE8377A3" attach="left" box="[259,271,1953,1965]" fontSize="5" pageId="6" pageNumber="50">–</superScript>
|
||
</emphasis>
|
||
ratio on phenylethanoid glycoside production was investigated for the first time in our study. NH
|
||
<subScript id="17839E1C0B57615FFD8FF87CFDE177D8" attach="left" box="[612,621,1994,2006]" fontSize="5" pageId="6" pageNumber="50">4</subScript>
|
||
<superScript id="7C7231110B57615FFD8FF80BFDE177C7" attach="left" box="[612,621,1981,1993]" fontSize="5" pageId="6" pageNumber="50">+</superScript>
|
||
and NO
|
||
<subScript id="17839E1C0B57615FFD2AF87CFD4677D8" attach="left" box="[705,714,1994,2006]" fontSize="5" pageId="6" pageNumber="50">3</subScript>
|
||
<emphasis id="B973404B0B57615FFD2AF80BFD4177C7" box="[705,717,1981,1993]" italics="true" pageId="6" pageNumber="50">
|
||
<superScript id="7C7231110B57615FFD2AF80BFD4177C7" attach="left" box="[705,717,1981,1993]" fontSize="5" pageId="6" pageNumber="50">–</superScript>
|
||
</emphasis>
|
||
are usually thought to modulate metabolism via enhancing growth and driving the TC towards more enhanced growth at the cost of production of less secondary metabolites. High carbon source to N source ratios in plant TCs usually lead to higher metabolite biosynthesis. As the growth speed decreases with the reduction of the relatively available N (or other limiting nutrient), the excess carbon pool is driven towards synthesis of secondary metabolites to a higher extent – thus, secondary metabolite accumulation often occurs after the period of maximum growth (
|
||
<bibRefCitation id="EF96E1A80B57615FFB1CF938FAE376AF" author="Collin, H." box="[1271,1391,1678,1697]" pageId="6" pageNumber="50" pagination="119 - 134" refId="ref11218" refString="Collin, H., 2001. Secondary product formation in plant tissue cultures. Plant Growth Regul. 34, 119 - 134." type="journal article" year="2001">Collin, 2001</bibRefCitation>
|
||
). In our case, however, none of the metabolite abundances were shown to be significantly influenced by GI itself (as shown by a linear model). If the N source would have been limiting in any of the media, GI would have likely been higher at higher N concentrations, but GI was unaffected by N concentration (
|
||
<emphasis id="B973404B0B57615FFAD8F8AFFAB37723" box="[1331,1343,1817,1837]" italics="true" pageId="6" pageNumber="50">p</emphasis>
|
||
= 0.54). This suggests that the availability of NO
|
||
<subScript id="17839E1C0B57615FFB7EF889FB127745" attach="left" box="[1173,1182,1855,1867]" fontSize="5" pageId="6" pageNumber="50">3</subScript>
|
||
<emphasis id="B973404B0B57615FFB7EF884FB2D7730" box="[1173,1185,1842,1854]" italics="true" pageId="6" pageNumber="50">
|
||
<superScript id="7C7231110B57615FFB7EF884FB2D7730" attach="left" box="[1173,1185,1842,1854]" fontSize="5" pageId="6" pageNumber="50">–</superScript>
|
||
</emphasis>
|
||
and NH
|
||
<superScript id="7C7231110B57615FFB12F884FA8E7730" attach="left" box="[1273,1282,1842,1854]" fontSize="5" pageId="6" pageNumber="50">+</superScript>
|
||
<quantity id="4CFF31BC0B57615FFB12F889FAAC7747" box="[1273,1312,1846,1867]" metricMagnitude="-1" metricUnit="m" metricValue="1.016" pageId="6" pageNumber="50" unit="in" value="4.0">
|
||
<subScript id="17839E1C0B57615FFB12F889FA8E7745" attach="left" box="[1273,1282,1855,1867]" fontSize="5" pageId="6" pageNumber="50">4</subScript>
|
||
in
|
||
</quantity>
|
||
different ratios and net concentrations exerted more direct effects on biosynthesis of the examined NPs. These direct effects may include NH
|
||
<subScript id="17839E1C0B57615FFA85F8C0FAFB778C" attach="left" box="[1390,1399,1910,1922]" fontSize="5" pageId="6" pageNumber="50">4</subScript>
|
||
<superScript id="7C7231110B57615FFA85F8DCFAFB7778" attach="left" box="[1390,1399,1898,1910]" fontSize="5" pageId="6" pageNumber="50">+</superScript>
|
||
acidity stress (
|
||
<bibRefCitation id="EF96E1A80B57615FFC86F83FFBA77792" author="George, E. F. & Hall, M. A. & Klerk, G. - J. D." box="[877,1067,1929,1949]" pageId="6" pageNumber="50" pagination="186" refId="ref11390" refString="George, E. F., Hall, M. A., Klerk, G. - J. D., 2007. Plant Propagation by Tissue Culture: Volume 1. The Background, first ed. Springer, pp. 71, 73, 186." type="journal article" year="2007">George et al., 2007</bibRefCitation>
|
||
), or other, unknown pathways.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B57615EFCAFF810FE9870EB" blockId="6.[805,1474,1455,2004]" lastBlockId="7.[113,783,182,229]" lastPageId="7" lastPageNumber="51" pageId="6" pageNumber="50">We can state, that while the widely used MSM is suboptimal with respect to NP abundance in the TCs, careful experimental design has to be implemented when reoptimizing the N source in the TC medium.</paragraph>
|
||
<paragraph id="8BB89C590B56615EFF99FE94FF24715C" blockId="7.[113,783,290,1568]" pageId="7" pageNumber="51">
|
||
<heading id="D0F02B350B56615EFF99FE94FF24715C" centered="true" fontSize="8" level="3" pageId="7" pageNumber="51" reason="8">
|
||
<emphasis id="B973404B0B56615EFF99FE94FF24715C" italics="true" pageId="7" pageNumber="51">2.3.4. Simulated one-factor at a time optimization of natural product yields</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFF7AFEEAFF3D72B3" blockId="7.[113,783,290,1568]" pageId="7" pageNumber="51">
|
||
As the abundance of several of the NPs were very good examples of the statistical interaction between the N source concentration and the NH
|
||
<superScript id="7C7231110B56615EFEFDFE39FE937195" attach="left" box="[278,287,399,411]" fontSize="5" pageId="7" pageNumber="51">+</superScript>
|
||
<subScript id="17839E1C0B56615EFEFDFE2AFE9371A6" attach="left" box="[278,287,412,424]" fontSize="5" pageId="7" pageNumber="51">4</subScript>
|
||
/NO
|
||
<subScript id="17839E1C0B56615EFEACFE2AFEDC71A6" attach="left" box="[327,336,412,424]" fontSize="5" pageId="7" pageNumber="51">3</subScript>
|
||
<emphasis id="B973404B0B56615EFEACFE39FEDF7195" box="[327,339,399,411]" italics="true" pageId="7" pageNumber="51">
|
||
<superScript id="7C7231110B56615EFEACFE39FEDF7195" attach="left" box="[327,339,399,411]" fontSize="5" pageId="7" pageNumber="51">–</superScript>
|
||
</emphasis>
|
||
ratio, OFAT experiments were simulated
|
||
<emphasis id="B973404B0B56615EFD17FE24FF2D71CC" italics="true" pageId="7" pageNumber="51">in silico</emphasis>
|
||
to assess the risk of ending up with a NP suboptimal yield. The inability to independently optimize media components was also suggested by
|
||
<bibRefCitation id="EF96E1A80B56615EFED0FE51FDAE71F4" author="Amdoun, R. & Khelifi, L. & Khelifi-Slaoui, M. & Amroune, S. & Benyoussef, E. - H. & Thi, D. V. & Assaf-Ducrocq, C. & Gontier, E." box="[315,546,487,506]" pageId="7" pageNumber="51" pagination="81 - 87" refId="ref10876" refString="Amdoun, R., Khelifi, L., Khelifi-Slaoui, M., Amroune, S., Benyoussef, E. - H., Thi, D. V., Assaf-Ducrocq, C., Gontier, E., 2009. Influence of minerals and elicitation on Datura stramonium L. tropane alkaloid production: modelization of the in vitro biochemical response. Plant Sci. 177, 81 - 87." type="journal article" year="2009">Amdoun et al. (2009)</bibRefCitation>
|
||
(regarding NO
|
||
<subScript id="17839E1C0B56615EFD28FE46FD4071F2" attach="left" box="[707,716,496,508]" fontSize="5" pageId="7" pageNumber="51">3</subScript>
|
||
<emphasis id="B973404B0B56615EFD28FE55FD4371E1" box="[707,719,483,495]" italics="true" pageId="7" pageNumber="51">
|
||
<superScript id="7C7231110B56615EFD28FE55FD4371E1" attach="left" box="[707,719,483,495]" fontSize="5" pageId="7" pageNumber="51">–</superScript>
|
||
</emphasis>
|
||
/Ca
|
||
<superScript id="7C7231110B56615EFD1BFE55FC8E71E1" attach="right" box="[752,770,483,495]" fontSize="5" pageId="7" pageNumber="51">2+</superScript>
|
||
). Other studies also found, that NP accumulation depended on media N source, but the interactions between the NH
|
||
<subScript id="17839E1C0B56615EFD71FD9EFD2F723A" attach="left" box="[666,675,552,564]" fontSize="5" pageId="7" pageNumber="51">4</subScript>
|
||
<superScript id="7C7231110B56615EFD71FDADFD2F7229" attach="left" box="[666,675,539,551]" fontSize="5" pageId="7" pageNumber="51">+</superScript>
|
||
/NO
|
||
<subScript id="17839E1C0B56615EFD20FD9EFD58723A" attach="left" box="[715,724,552,564]" fontSize="5" pageId="7" pageNumber="51">3</subScript>
|
||
<emphasis id="B973404B0B56615EFD20FDADFD5B7229" box="[715,727,539,551]" italics="true" pageId="7" pageNumber="51">
|
||
<superScript id="7C7231110B56615EFD20FDADFD5B7229" attach="left" box="[715,727,539,551]" fontSize="5" pageId="7" pageNumber="51">–</superScript>
|
||
</emphasis>
|
||
ratio and the N source concentrations could not be assessed for effects on the metabolome in most cases, because most studies used one-factor at a time strategy (
|
||
<bibRefCitation id="EF96E1A80B56615EFE5FFDC4FDED728B" author="Guo, X. & Gao, W. & Xiao, P." box="[436,609,626,646]" pageId="7" pageNumber="51" pagination="133 - 139" refId="ref11688" refString="Guo, X., Gao, W., Xiao, P., 2005. Factors affecting root growth and metabolite production in Salvia miltiorrhiza adventitious root cultures. Minerva Biotechnol. 17, 133 - 139." type="journal article" year="2005">Guo et al., 2005</bibRefCitation>
|
||
;
|
||
<bibRefCitation id="EF96E1A80B56615EFD9BFDC5FF2F72AF" author="Wang, J. & Tan, R." pageId="7" pageNumber="51" pagination="1153 - 1156" refId="ref12777" refString="Wang, J., Tan, R., 2002. Artemisinin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium. Biotechnol. Lett. 24, 1153 - 1156." type="journal article" year="2002">Wang and Tan, 2002</bibRefCitation>
|
||
), or did not use a factorial approach (
|
||
<bibRefCitation id="EF96E1A80B56615EFDD9FD38FF2F72B3" author="Jacob, A. & Malpathak, N." pageId="7" pageNumber="51" pagination="247 - 257" refId="ref11902" refString="Jacob, A., Malpathak, N., 2005. Manipulation of MS and B 5 components for enhancement of growth and solasodine production in hairy root cultures of Solanum khasianum Clarke. Plant Cell, Tissue Organ Cult. 80, 247 - 257. http: // dx. doi. org / 10.1007 / s 11240 - 004 - 0740 - 2." type="journal article" year="2005">Jacob and Malpathak, 2005</bibRefCitation>
|
||
).
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFF7AFD70FE3E7610" blockId="7.[113,783,290,1568]" pageId="7" pageNumber="51">
|
||
To show the advantage of the FF strategy, four virtual optimization studies were carried out
|
||
<emphasis id="B973404B0B56615EFE77FD57FE6972FB" box="[412,485,737,757]" italics="true" pageId="7" pageNumber="51">in silico</emphasis>
|
||
. The interaction between the tested parameters and the consequence of using OFAT is perhaps best presented this way. The composition of the media was optimized in different OFAT regimes for each of the 89 NPs individually. The virtual experiments either started from MSM (60(0.33)) or 20(0). The optimization of the NH
|
||
<superScript id="7C7231110B56615EFE00FCDCFE787378" attach="left" box="[491,500,874,886]" fontSize="5" pageId="7" pageNumber="51">+</superScript>
|
||
<subScript id="17839E1C0B56615EFE00FCC0FE78738C" attach="left" box="[491,500,886,898]" fontSize="5" pageId="7" pageNumber="51">4</subScript>
|
||
/NO
|
||
<subScript id="17839E1C0B56615EFDF7FCC0FDA9738C" attach="left" box="[540,549,886,898]" fontSize="5" pageId="7" pageNumber="51">3</subScript>
|
||
<emphasis id="B973404B0B56615EFDF7FCDCFDA47378" box="[540,552,874,886]" italics="true" pageId="7" pageNumber="51">
|
||
<superScript id="7C7231110B56615EFDF7FCDCFDA47378" attach="left" box="[540,552,874,886]" fontSize="5" pageId="7" pageNumber="51">–</superScript>
|
||
</emphasis>
|
||
ratio was followed by the optimization of the N source concentration or vice versa. In strategy #1, the starting medium was MSM and the virtual optimization began with the NH
|
||
<superScript id="7C7231110B56615EFE9FFC0BFEF173C7" attach="left" box="[372,381,957,969]" fontSize="5" pageId="7" pageNumber="51">+</superScript>
|
||
<subScript id="17839E1C0B56615EFE9FFC7CFEF173D8" attach="left" box="[372,381,970,982]" fontSize="5" pageId="7" pageNumber="51">4</subScript>
|
||
/NO
|
||
<subScript id="17839E1C0B56615EFE4EFC7CFE2273D8" attach="left" box="[421,430,970,982]" fontSize="5" pageId="7" pageNumber="51">3</subScript>
|
||
<emphasis id="B973404B0B56615EFE4EFC0BFE3D73C7" box="[421,433,957,969]" italics="true" pageId="7" pageNumber="51">
|
||
<superScript id="7C7231110B56615EFE4EFC0BFE3D73C7" attach="left" box="[421,433,957,969]" fontSize="5" pageId="7" pageNumber="51">–</superScript>
|
||
</emphasis>
|
||
ratio, followed by the virtual optimization of the N source concentration. In this scenario, 53 of the 89 NP abundances were suboptimal, with the suboptimal NPs showing a mean 1.56-fold abundance advantage in FF compared to the OFAT optima. The list of suboptimal media obtained by the different OFAT simulations for each NP is shown in Supplementary Table 2. Starting with medium 20(0) in strategy #2 resulted in similar results: 44 NP abundances were sub-optimal with the FF optima being 1.28-fold better on average. When the scheme was reversed to the experimental setup less common in the literature (N source concentration was optimized first in strategies #3–4), the OFAT performance substantially increased. Despite this, there were still 12 and 19 NPs sub-optimal abundance NPs, when starting from MSM or 20(0), respectively. The average fold advantage of the FF for these NPs was 1.30 and 1.37. For some NPs, the difference between the best FF and OFAT medium was greater, than 2-fold (Supplementary Table 2). It is also noteworthy, that the abundances of 6 NPs did not reach the FF optimum, regardless of the starting medium. Thus, if NP abundance optima are reached with OFAT, it can also simply be by chance. This chance can be increased however, by optimizing the N source concentration first, followed by the NH
|
||
<subScript id="17839E1C0B56615EFED3F9A2FECD762E" attach="left" box="[312,321,1556,1568]" fontSize="5" pageId="7" pageNumber="51">4</subScript>
|
||
<superScript id="7C7231110B56615EFED3F9B1FECD761D" attach="left" box="[312,321,1543,1555]" fontSize="5" pageId="7" pageNumber="51">+</superScript>
|
||
/NO
|
||
<subScript id="17839E1C0B56615EFE82F9A2FEFE762E" attach="left" box="[361,370,1556,1568]" fontSize="5" pageId="7" pageNumber="51">3</subScript>
|
||
<emphasis id="B973404B0B56615EFE82F9B1FEF9761D" box="[361,373,1543,1555]" italics="true" pageId="7" pageNumber="51">
|
||
<superScript id="7C7231110B56615EFE82F9B1FEF9761D" attach="left" box="[361,373,1543,1555]" fontSize="5" pageId="7" pageNumber="51">–</superScript>
|
||
</emphasis>
|
||
ratio.
|
||
</paragraph>
|
||
</subSubSection>
|
||
<subSubSection id="C31DCFD20B56615EFF99F9D7FBF47632" pageId="7" pageNumber="51" type="discussion">
|
||
<paragraph id="8BB89C590B56615EFF99F9D7FE86767A" blockId="7.[114,266,1633,1652]" box="[114,266,1633,1652]" pageId="7" pageNumber="51">
|
||
<heading id="D0F02B350B56615EFF99F9D7FE86767A" bold="true" box="[114,266,1633,1652]" fontSize="36" level="1" pageId="7" pageNumber="51" reason="1">
|
||
<emphasis id="B973404B0B56615EFF99F9D7FE86767A" bold="true" box="[114,266,1633,1652]" pageId="7" pageNumber="51">3. Conclusions</emphasis>
|
||
</heading>
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFF7AF92FFC9C76F1" blockId="7.[113,784,1685,2015]" pageId="7" pageNumber="51">
|
||
In this study, we successfully applied LC–ESI–
|
||
<collectionCode id="ED16049C0B56615EFD89F92FFD0C76A2" box="[610,640,1689,1708]" country="Italy" lsid="urn:lsid:biocol.org:col:14396" name="Herbarium Messanaensis, Università di Messina" pageId="7" pageNumber="51" type="Herbarium">MS</collectionCode>
|
||
<superScript id="7C7231110B56615EFD6BF923FD0576AF" attach="left" box="[640,649,1685,1697]" fontSize="5" pageId="7" pageNumber="51">3</superScript>
|
||
for the characterization of tissue cultures of a medicinal plant,
|
||
<taxonomicName id="4C07E7DA0B56615EFD96F905FF2676EA" authority="L. Many" authorityName="L. Many" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="7" pageNumber="51" phylum="Tracheophyta" rank="species" species="lanceolata">
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||
<emphasis id="B973404B0B56615EFD96F905FD7476C9" box="[637,760,1715,1735]" italics="true" pageId="7" pageNumber="51">P. lanceolata</emphasis>
|
||
L. Many
|
||
</taxonomicName>
|
||
phenylethanoid glycosides previously not found in these calli were detected, and their structures were putatively identified.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFF7AF8BEFF7E77D0" blockId="7.[113,784,1685,2015]" pageId="7" pageNumber="51">
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The main goal of the study was to examine the effects of the
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||
composition of the medium on the production of phenylethanoid glycosides and other NPs in
|
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|
||
<emphasis id="B973404B0B56615EFE72F889FD98775D" box="[409,532,1855,1875]" italics="true" pageId="7" pageNumber="51">P. lanceolata</emphasis>
|
||
calli
|
||
</taxonomicName>
|
||
. Four
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||
concentrations and four levels of the
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</emphasis>
|
||
ratio were tested in a full-factorial experiment enabling the estimation of interactions between these two parameters. Many conclusions could be drawn with regard to secondary metabolite production in medicinal plant tissue cultures.
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||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFCB5FF00FC1A715B" blockId="7.[831,1501,182,1596]" pageId="7" pageNumber="51">
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||
The metabolomic approach was shown to be a powerful one when evaluating the results. The data processing and visualization procedures provided high throughput and highlighted most of the phenomena. The presented approach is therefore strongly encouraged when optimizing secondary metabolite production in
|
||
<emphasis id="B973404B0B56615EFA7DFE93FA507137" box="[1430,1500,293,313]" italics="true" pageId="7" pageNumber="51">in vitro</emphasis>
|
||
cultures.
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||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFCB5FEE8FB7B71EE" blockId="7.[831,1501,182,1596]" pageId="7" pageNumber="51">
|
||
The original Murashige Skoog medium was found unsuitable for high yield production of phenylethanoid glycosides. In fact, it was one of the media with least NP abundances. The medium proposed by
|
||
<bibRefCitation id="EF96E1A80B56615EFCB5FE07FBFD71CA" author="Budzianowska, A. & Skrzypczak, L. & Budzianowski, J." box="[862,1137,433,453]" pageId="7" pageNumber="51" pagination="834 - 840" refId="ref11162" refString="Budzianowska, A., Skrzypczak, L., Budzianowski, J., 2004. Phenylethanoid glucosides from in vitro propagated plants and callus cultures of Plantago lanceolata. Planta Med. 70, 834 - 840. http: // dx. doi. org / 10.1055 / s- 2004 - 827232." type="journal article" year="2004">Budzianowska et al. (2004)</bibRefCitation>
|
||
for
|
||
<taxonomicName id="4C07E7DA0B56615EFB71FE06FA9971CA" box="[1178,1301,432,452]" class="Magnoliopsida" family="Plantaginaceae" genus="Plantago" kingdom="Plantae" order="Lamiales" pageId="7" pageNumber="51" phylum="Tracheophyta" rank="species" species="lanceolata">
|
||
<emphasis id="B973404B0B56615EFB71FE06FA9971CA" box="[1178,1301,432,452]" italics="true" pageId="7" pageNumber="51">P. lanceolata</emphasis>
|
||
</taxonomicName>
|
||
tissue cultures was optimal for growth, but not NP production.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFCB5FE5FFC20738D" blockId="7.[831,1501,182,1596]" pageId="7" pageNumber="51">
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||
However, with the manipulation of the
|
||
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/
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<superScript id="7C7231110B56615EFABBFE50FAD071FC" attach="left" box="[1360,1372,486,498]" fontSize="5" pageId="7" pageNumber="51">–</superScript>
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</emphasis>
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ratio and
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||
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||
source concentration, very significant increase in the natural product accumulation could be achieved. Compared to the reference media, 1.5–3-fold increases in abundance were achieved for most metabolites. The highest NP abundance media were those with low
|
||
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|
||
source concentrations (10 mM
|
||
<collectionCode id="ED16049C0B56615EFB5EFDC3FB447286" box="[1205,1224,629,648]" country="China" lsid="urn:lsid:biocol.org:col:13092" name="Nanjing University" pageId="7" pageNumber="51" type="Herbarium">N</collectionCode>
|
||
). Thus, we have shown that a very simple and inexpensive modification of the medium can dramatically increase phenylethanoid glycoside yields. What is more, the manipulation of these parameters can also be easily implemented in industrial scale cultures, and is compatible with elicitations as well. The results suggest that
|
||
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|
||
source composition must be re-optimized for optimal production of these natural products and that the two-stage culture strategy is to be preferred when tissue cultures are used for production of phenylethanoid glycosides.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFCB5FC3AFA837424" blockId="7.[831,1501,182,1596]" pageId="7" pageNumber="51">For major metabolites, the maximum yields could be observed on different media: optimal plantamajoside production was achieved on 10(0.33), 3.54 ± 0.83% (dry wt.), while, in the case of acteoside, the best yield was 1.30 ± 0.40% (dry wt.) on 40(0.33). For most metabolites, the medium 10(0.33) was found to be optimal, for most of the others, 10(0) or 40(0.33).</paragraph>
|
||
<paragraph id="8BB89C590B56615EFCB5FB85FC5B759B" blockId="7.[831,1501,182,1596]" pageId="7" pageNumber="51">
|
||
Comparing the optimization strategies also led us to interesting conclusions. Interactions were detected between the two parameters for many metabolites – the response to the
|
||
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<emphasis id="B973404B0B56615EFA85FBD1FAF6747D" box="[1390,1402,1127,1139]" italics="true" pageId="7" pageNumber="51">
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||
</emphasis>
|
||
ratio was not the same at different
|
||
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|
||
source concentrations. The manipulation of the
|
||
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/
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</emphasis>
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ratio and
|
||
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|
||
source concentrations has led to sub-optimal yields in case of
|
||
<emphasis id="B973404B0B56615EFB8AFB0BFB2574DF" box="[1121,1193,1213,1233]" italics="true" pageId="7" pageNumber="51">in silico</emphasis>
|
||
simulated one-factor at a time experimental designs, for many natural products. Therefore, factorial experiments with less repetitions must be preferred to one-factor at a time experiments with higher repetition counts. If
|
||
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|
||
is unavoidable due to some constraint, the
|
||
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|
||
source concentration must be optimized first, as it was shown to lead to suboptimal yields much less often than
|
||
<collectionCode id="ED16049C0B56615EFB8CFAD0FB107577" box="[1127,1180,1382,1401]" pageId="7" pageNumber="51">OFAT</collectionCode>
|
||
protocols optimizing the
|
||
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<superScript id="7C7231110B56615EFA20FAD4FA587560" attach="left" box="[1483,1492,1378,1390]" fontSize="5" pageId="7" pageNumber="51">+</superScript>
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/
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<emphasis id="B973404B0B56615EFCB5FAC8FCE67584" box="[862,874,1406,1418]" italics="true" pageId="7" pageNumber="51">
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<superScript id="7C7231110B56615EFCB5FAC8FCE67584" attach="left" box="[862,874,1406,1418]" fontSize="5" pageId="7" pageNumber="51">–</superScript>
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</emphasis>
|
||
ratio first.
|
||
</paragraph>
|
||
<paragraph id="8BB89C590B56615EFCB5FA28FBF47632" blockId="7.[831,1501,182,1596]" pageId="7" pageNumber="51">As the overall polyphenolic metabolite concentration was also heavily influenced by the tested parameters, our suggestions should be considered to be adopted when optimizing yields of other natural products in tissue cultures. These results are likely to apply for other metabolites, that are biosynthesized through the phenylpropanoid pathway.</paragraph>
|
||
</subSubSection>
|
||
</treatment>
|
||
</document> |