Limits...
Profiling of the molecular weight and structural isomer abundance of macroalgae-derived phlorotannins.

Heffernan N, Brunton NP, FitzGerald RJ, Smyth TJ - Mar Drugs (2015)

Bottom Line: The majority of the low molecular weight (LMW) phlorotannins were found to have a molecular weight range equivalent to 4-12 monomers of phloroglucinol.The level of isomerization within the individual macroalgal species differed, resulting in substantially different numbers of phlorotannin isomers for particular molecular weights.These results highlight the complex nature of these extracts and emphasize the challenges involved in structural elucidation of these compounds.

View Article: PubMed Central - PubMed

Affiliation: Food Biosciences Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland. dick.fitzgerald@ul.ie.

ABSTRACT
Phlorotannins are a group of complex polymers of phloroglucinol (1,3,5-trihydroxybenzene) unique to macroalgae. These phenolic compounds are integral structural components of the cell wall in brown algae, but also play many secondary ecological roles such as protection from UV radiation and defense against grazing. This study employed Ultra Performance Liquid Chromatography (UPLC) with tandem mass spectrometry to investigate isomeric complexity and observed differences in phlorotannins derived from macroalgae harvested off the Irish coast (Fucus serratus, Fucus vesiculosus, Himanthalia elongata and Cystoseira nodicaulis). Antioxidant activity and total phenolic content assays were used as an index for producing phlorotannin fractions, enriched using molecular weight cut-off dialysis with subsequent flash chromatography to profile phlorotannin isomers in these macroalgae. These fractions were profiled using UPLC-MS with multiple reaction monitoring (MRM) and the level of isomerization for specific molecular weight phlorotannins between 3 and 16 monomers were determined. The majority of the low molecular weight (LMW) phlorotannins were found to have a molecular weight range equivalent to 4-12 monomers of phloroglucinol. The level of isomerization within the individual macroalgal species differed, resulting in substantially different numbers of phlorotannin isomers for particular molecular weights. F. vesiculosus had the highest number of isomers of 61 at one specific molecular mass, corresponding to 12 phloroglucinol units (PGUs). These results highlight the complex nature of these extracts and emphasize the challenges involved in structural elucidation of these compounds.

Show MeSH

Related in: MedlinePlus

Chemical structures of a selection of phlorotannins, illustrating varying bonding mechanisms between phloroglucinol units.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4306949&req=5

marinedrugs-13-00509-f002: Chemical structures of a selection of phlorotannins, illustrating varying bonding mechanisms between phloroglucinol units.

Mentions: The UPLC-QQQ-MS method employed in this study resulted in the detection of phlorotannins in the range of 374–1986 Daltons, using a tandem quadrupole MS in MRM mode for increased sensitivity and for isomer detection. By individually tuning the theoretical molecular weights of phlorotannins while infusing a complex phlorotannin mixture using the IntelliStart™ software, combined with the manual tuning, suitable MRM fragments for each phlorotannin up to 16 phloroglucinol units in negative ionization mode could be determined. It was apparent that the level of phlorotannin isomerization across species differed to some degree. This difference may be due to possible structural features of the phlorotannins present and the species-specific biosynthetic mechanisms. The monomeric units in phlorotannins are linked through aryl–aryl bonds and diaryl bonds, forming different subgroups of phlorotannins [43]. When aromatic rings are connected purely by aryl–aryl bonds, a group of fucols is formed (Figure 2). Phlorethols are formed solely by aryl ether bonds (Figure 2). Fuhalols are constructed by phloroglucinol units that are connected with para- and ortho-arranged ether bridges containing one additional OH-group in every third ring (Figure 2). When there exists at least one three-ring moiety with a dibenzodioxin elements substituted by a phenoxyl group at C-4, the group is named eckols (Figure 2). Carmalols are further derivatives of phlorethols containing a dibenzodioxin moiety. Endofucophlorethols and isofuhalols (Figure 2) are small, distinct, specialized groups. Figure 3 displays the MRM transitions of phlorotannins from F. serratus, highlighting the abundance of isomers present for individual deprotonated molecules. A high number of isomers were detected for individual molecular ions in the species F. serratus, with 178 isomers in total being detected between DP 6 and DP 14; DP 12 had the highest isomer number of 42. This would suggest a significant variation in branching positions is occurring between the monomeric units. Figure 4 presents a more detailed view of F. serratus MRM corresponding to DP of 12 (MS/MS 1489.5–229.1 transition) to show distinctly the complexity of isomerization occurring. Similar to what was observed for F. serratus, H. elongata has a large number of isomers between DP 7 and DP 12 units; 96 isomers in total were detected in this range, with DP 8 having the highest isomer content of 32. Unlike F. serratus and H. elongata, F. vesiculosus phlorotannins eluted at early retention times and the most prominent peaks were observed in the lower molecular range at DP 4 to DP 9. There were still a significant number of isomers observed in this range, with 144 isomers detected in total. C. nodicaulis was similar to F. vesiculosus in that the phlorotannins eluted in low percentages of organic solvent. The most prominent peaks are in the range DP 8–11 with a lower number of isomers observed for each deprotonated molecule [M − H]−. A total of 48 isomers were detected between the range of DP 4–11. Due to the low level of isomers detected, it may be assumed that there is limited variation in branching between monomeric units.


Profiling of the molecular weight and structural isomer abundance of macroalgae-derived phlorotannins.

Heffernan N, Brunton NP, FitzGerald RJ, Smyth TJ - Mar Drugs (2015)

Chemical structures of a selection of phlorotannins, illustrating varying bonding mechanisms between phloroglucinol units.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306949&req=5

marinedrugs-13-00509-f002: Chemical structures of a selection of phlorotannins, illustrating varying bonding mechanisms between phloroglucinol units.
Mentions: The UPLC-QQQ-MS method employed in this study resulted in the detection of phlorotannins in the range of 374–1986 Daltons, using a tandem quadrupole MS in MRM mode for increased sensitivity and for isomer detection. By individually tuning the theoretical molecular weights of phlorotannins while infusing a complex phlorotannin mixture using the IntelliStart™ software, combined with the manual tuning, suitable MRM fragments for each phlorotannin up to 16 phloroglucinol units in negative ionization mode could be determined. It was apparent that the level of phlorotannin isomerization across species differed to some degree. This difference may be due to possible structural features of the phlorotannins present and the species-specific biosynthetic mechanisms. The monomeric units in phlorotannins are linked through aryl–aryl bonds and diaryl bonds, forming different subgroups of phlorotannins [43]. When aromatic rings are connected purely by aryl–aryl bonds, a group of fucols is formed (Figure 2). Phlorethols are formed solely by aryl ether bonds (Figure 2). Fuhalols are constructed by phloroglucinol units that are connected with para- and ortho-arranged ether bridges containing one additional OH-group in every third ring (Figure 2). When there exists at least one three-ring moiety with a dibenzodioxin elements substituted by a phenoxyl group at C-4, the group is named eckols (Figure 2). Carmalols are further derivatives of phlorethols containing a dibenzodioxin moiety. Endofucophlorethols and isofuhalols (Figure 2) are small, distinct, specialized groups. Figure 3 displays the MRM transitions of phlorotannins from F. serratus, highlighting the abundance of isomers present for individual deprotonated molecules. A high number of isomers were detected for individual molecular ions in the species F. serratus, with 178 isomers in total being detected between DP 6 and DP 14; DP 12 had the highest isomer number of 42. This would suggest a significant variation in branching positions is occurring between the monomeric units. Figure 4 presents a more detailed view of F. serratus MRM corresponding to DP of 12 (MS/MS 1489.5–229.1 transition) to show distinctly the complexity of isomerization occurring. Similar to what was observed for F. serratus, H. elongata has a large number of isomers between DP 7 and DP 12 units; 96 isomers in total were detected in this range, with DP 8 having the highest isomer content of 32. Unlike F. serratus and H. elongata, F. vesiculosus phlorotannins eluted at early retention times and the most prominent peaks were observed in the lower molecular range at DP 4 to DP 9. There were still a significant number of isomers observed in this range, with 144 isomers detected in total. C. nodicaulis was similar to F. vesiculosus in that the phlorotannins eluted in low percentages of organic solvent. The most prominent peaks are in the range DP 8–11 with a lower number of isomers observed for each deprotonated molecule [M − H]−. A total of 48 isomers were detected between the range of DP 4–11. Due to the low level of isomers detected, it may be assumed that there is limited variation in branching between monomeric units.

Bottom Line: The majority of the low molecular weight (LMW) phlorotannins were found to have a molecular weight range equivalent to 4-12 monomers of phloroglucinol.The level of isomerization within the individual macroalgal species differed, resulting in substantially different numbers of phlorotannin isomers for particular molecular weights.These results highlight the complex nature of these extracts and emphasize the challenges involved in structural elucidation of these compounds.

View Article: PubMed Central - PubMed

Affiliation: Food Biosciences Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland. dick.fitzgerald@ul.ie.

ABSTRACT
Phlorotannins are a group of complex polymers of phloroglucinol (1,3,5-trihydroxybenzene) unique to macroalgae. These phenolic compounds are integral structural components of the cell wall in brown algae, but also play many secondary ecological roles such as protection from UV radiation and defense against grazing. This study employed Ultra Performance Liquid Chromatography (UPLC) with tandem mass spectrometry to investigate isomeric complexity and observed differences in phlorotannins derived from macroalgae harvested off the Irish coast (Fucus serratus, Fucus vesiculosus, Himanthalia elongata and Cystoseira nodicaulis). Antioxidant activity and total phenolic content assays were used as an index for producing phlorotannin fractions, enriched using molecular weight cut-off dialysis with subsequent flash chromatography to profile phlorotannin isomers in these macroalgae. These fractions were profiled using UPLC-MS with multiple reaction monitoring (MRM) and the level of isomerization for specific molecular weight phlorotannins between 3 and 16 monomers were determined. The majority of the low molecular weight (LMW) phlorotannins were found to have a molecular weight range equivalent to 4-12 monomers of phloroglucinol. The level of isomerization within the individual macroalgal species differed, resulting in substantially different numbers of phlorotannin isomers for particular molecular weights. F. vesiculosus had the highest number of isomers of 61 at one specific molecular mass, corresponding to 12 phloroglucinol units (PGUs). These results highlight the complex nature of these extracts and emphasize the challenges involved in structural elucidation of these compounds.

Show MeSH
Related in: MedlinePlus