16
in Fig. 4, HPLC elution profiles of the iodine-catalyzed reaction gave diverse products of fucoxanthin isomers than those of the fucoxanthin fraction used in this study. A total of 8 peaks were separated, but 9 species of isomers were identified by subsequent several spectroscopic analyses. Peaks 1 and 4 to 8 were identified as di-cis, 13,9ʹ-di-cis, all-trans, 13ʹ-cis, 13-cis, and 9ʹ- cis in that order, while peaks 2 and 3 were a mixture of di-cis1 and 9ʹ,13ʹ-di-cis, and the rest peak 9 was 9-cis isomer. In this treatment, the transformation of isomers was enhanced 1.79-folds and 9-cis was newly found in this study. The summary of the results is presented in Table 3. As can be seen from the elution profile, six isomers were able to simultaneously separate and purify as similar to the main four isomers, especially, later eluted hydrophobic components such as 9ʹ-cis (peak 8) and 9-cis (peak 9) are likely to be much superior. However, di-cis isomers, 9ʹ,13ʹ-di-cis and 13,9ʹ- di-cis, could not be separated by this method. This technique was also applicable for the physical treatments by heat and irradiation which formed 4 and 8 species of isomers, respectively.
Accordingly, our method is applicable to a wide variety of structural and geometrical isomers of fucoxanthin and is versatile for the separation and simultaneous purification of the fucoxanthin isomers.
17
used routinely in our laboratory to study of fucoxanthin isomer transformation. This procedure assists the research and provides better understanding on the mechanism of fucoxanthin transformation and fucoxanthin study.
Credit authorship contribution statement
Arif Agung Wibowo: Formal analysis, Investigation, Curation, Writing—original draft preparation, Visualization; Heriyanto: Methodology, Formal analysis, Investigation, Curation, Writing—original draft preparation; Yuzo Shioi: Conceptualization, Methodology, Writing—
original draft preparation and review, Supervision; Leenawaty Limantara: Project administration; Tatas Hardo Panintingjati Brotosudarmo: Conceptualization, Methodology, Writing—review and editing, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
THPB acknowledged the financial support from the Directorate of Research and Community Services, under World Class Research scheme grant number 001/MACHUNG/LPPM/SP2H-LIT- MONO/IV/2021.
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18 Appendix A. Supplementary data
Supplementary data to this article can be found online at …
References
[1] T. Matsuno, Aquatic animal carotenoids, Fish. Sci. 67 (2001) 771–783.
https://doi.org/10.1046/j.1444-2906.2001.00323.x.
[2] J. Peng, J.P. Yuan, C.F. Wu, J.H. Wang, Fucoxanthin, a marine carotenoid present in brown seaweeds and diatoms: Metabolism and bioactivities relevant to human health, Mar. Drugs. 9 (2011) 1806–1828. https://doi.org/10.3390/md9101806.
[3] K. Mikami, M. Hosokawa, Biosynthetic pathway and health benefits of fucoxanthin, an algae-specific xanthophyll in brown seaweeds, Int. J. Mol. Sci. 14 (2013) 13763–13781.
https://doi.org/10.3390/ijms140713763.
[4] C. Lourenço-Lopes, P. Garcia-Oliveira, M. Carpena, M, Fraga-Corral, C. Jimenez-Lopez, A.G. Pereira, M.A. Prieto, J. Simal-Gandara, Foods 9 (2020) 1113.
https://doi.org/10.3390/foods9081113.
[5] S. Tachihana, N. Nagao, T. Katayama, M. Hirahara, F.Md. Yusoff, S. Banerjee, M.
Shariff, N. Kurosawa, T. Toda, K. Furuya, High productivity of eicosapentaenoic acid and fucoxanthin by a marine diatom Chaetoceros gracilis in a semi-continous culture, Front.
Bioeng. Biotechnol. 8 (2020) 602721. https://doi.org/10.3389/fbioe.2020.602721.
[6] W. Wang, L.-J. Yu, C. Xu, T. Tomizaki, S. Zhao, Y. Umena, X. Chen, X. Qin, Y. Xin, M.
Suga, G. Han, T. Kuang, J.-R. Shen, Structural basis for blue-green light harvesting and energy dissipation in diatoms, Science 363 (2019) 1–9.
https://doi.org/10.1126/science.aav0365.
[7] Y. Koyama, R. Fujii, Cis-trans carotenoids in photosynthesis: Configurations‚ excited- state properties and physiological functions, in: H.A. Frank, A.J. Young, G. Britton, R.J.
Cogdell (Eds.), The Photochemistry of Carotenoids, Springer Netherlands, Dordrecht, 2004, pp. 161-188.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
19
[8] L. Premvardhan, L. Bordes, A. Beer, C. Bu, B. Robert, Carotenoid structures and environments in trimeric and oligomeric fucoxanthin chlorophyll a/c2 proteins from resonance raman spectroscopy, J. Phys. Chem. B. 113 (2009) 12565–12574.
https://doi.org/10.1021/jp903029g.
[9] S. Méresse, M. Fodil, F. Fleury, B. Chénais, Fucoxanthin, a marine-derived carotenoid from brown seaweeds and microalgae: A promising bioactive compound for cancer therapy, Int. J. Mol. Sci. 21 (2020) 9273. https://doi.org/10.3390/ijms21239273.
[10] Y. Nakazawa, T. Sashima, M. Hosokawa, K. Miyashita, Comparative evaluation of growth inhibitory effect of stereoisomers of fucoxanthin in human cancer cell lines, J.
Funct. Foods. 1 (2009) 88–97. https://doi.org/10.1016/j.jff.2008.09.015.
[11] Global fucoxanthin market report 2018—Global Industry Reports (2018), https://www.
Businessindustryreports.Com/Report/102177/Global-Fucoxanthin-Market-Report-2018.
[12] G. Rajauria, N. Abu-Ghannam, Isolation and partial characterization of bioactive
fucoxanthin from Himanthalia elongata brown seaweed : A TLC-based approach, Int. J.
Anal. Chem. 1 (2013) 6. https://doi.org/10.1155/2013/802573.
[13] H. Kanazawa, M. Nishikawa, A. Mizutani, C. Sakamoto, Y. Nagata, A. Kikuchi, T.
Okano, Aqueous chromatographic system for separation of biomolecules using thermoresponsive polymer modified stationary phase, J. Chromatogr. A. 1191 (2008) 157–161. https://doi.org/10.1016/j.chroma.2008.01.056.
[14] T. Katoh, U. Nagashima, M. Mimuro, Fluorescence properties of the allenic carotenoid fucoxanthin: Implication for energy transfer in photosynthetic pigment systems,
Photosynth. Res. 27 (1991) 221–226. https://doi.org/10.1007/BF00035843.
[15] S.M. Kim, F. Shang, B. Um, A preparative method for isolation of fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography, Photochem. Anal. 22 (2011) 322–329. https://doi.org/10.1002/pca.1283.
[16] R.G. De Oliveira-Júnior, R. Grougnet, P.-E. Bodet, A. Bonnet, E. Nicolau, A. Jebali, J.
Rumin, L. Picot, Updated pigment composition of Tisochrysis lutea and purification of fucoxanthin using centrifugal partition chromatography coupled to flash chromatography for the chemosensitization of melanoma cells, Algal Res. 51 (2020) 102035.
https://doi.org/10.1016/j.algal.2020.102035.
[17] I. Jaswir, D. Noviendri, H.M. Salleh, M. Taher, K. Miyashita, N. Ramli, Analysis of
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
20
fucoxanthin content and purification of all-trans-fucoxanthin from Turbinaria turbinata and Sargassum plagyophyllum by SiO2 open column chromatography and reversed phase- HPLC, J. Liq. Chromatogr. Relat. Technol. 36 (2013) 1340–1354.
https://doi.org/10.1080/10826076.2012.691435.
[18] V. Rajendran, Y.S. Pu, B.H. Chen, An improved HPLC method for determination of carotenoids in human serum, J. Chromatogr. B. 824 (2005) 99–106.
https://doi.org/10.1016/j.jchromb.2005.07.004.L.C.
[19] F. Khachik, C.J. Spangler, J.C. Smith, L.M. Canfield, A. Steck, H. Pfander, Identification, quantification, and relative concentrations of carotenoids and their metabolites in human milk and serum, Anal. Chem. 69 (1997) 1873–1881. https://doi.org/10.1021/ac961085i.
[20] L.C. Sander, K.E. Sharpless, N.E. Craft, S.A. Wise, Development of engineered stationary phases for the separation of carotenoid isomers, Anal. Chem. 66 (1994) 1667–1674.
https://doi.org/10.1021/ac00082a012.
[21] L.C. Sander, K.E. Sharpless, M. Pursch, C30 Stationary phases for the analysis of food by liquid chromatography, J. Chromatogr. A. 880 (2000) 189–202.
https://doi.org/10.1016/S0021-9673(00)00121-7.
[22] J.S. Palmer, L.A. Lawton, R. Kindt, C. Edwards, Rapid analytical methods for the
microalgal and cyanobacterial biorefinery: Application on strains of industrial importance, MicrobiologyOpen 10 (2021) e1156. https://doi.org/10.1002/mbo3.1156.
[23] P. Crupi, A.T. Toci, S. Mangini, F. Wrubl, L. Rodolfi, M.R. Tredici, A. Coletta, D.
Antonacci, Determination of fucoxanthin isomers in microalgae (Isochrysis sp.) by high- performance liquid chromatography coupled with diode-array detector multistage mass spectrometry coupled with positive electrospray ionization, Rapid Commun. Mass Spectrom. 27 (2013) 1027–1035. https://doi.org/10.1002/rcm.6531.
[24] D. Zhao, S. Kim, C. Pan, D. Chung, Effects of heating, aerial exposure and illumination on stability of fucoxanthin in canola oil, Food Chem. 145 (2014) 505–513.
https://doi.org/10.1016/j.foodchem.2013.08.045.
[25] S.M. Rivera, P. Christou, R. Canela-Garayoa, Identification of carotenoids using mass spectrometry, Mass Spectrom. Rev. 33 (2014) 353–372.
https://doi.org/10.1002/mas.21390.
[26] M.M. Hegazi, A. Pérez-Ruzafa, L. Almela, M.E. Candela, Separation and identification of
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
21
chlorophylls and carotenoids from Caulerpa prolifera, Jania rubens and Padina pavonica by reversed-phase high-performance liquid chromatography, J. Chromatogr. A. 829 (1998) 153–159. https://doi.org/10.1016/S0021-9673(98)00803-6.
[27] Z. Zhang, Y. Xiao, D. Li, C. Liu, Identification and quantification of all-trans-zeaxanthin and its cis-isomers during illumination in a model system, Int. J. Food Prop. 19 (2016) 1282–1291. https://doi.org/10.1080/10942912.2015.1072209.
[28] A. Krieger-Liszkay, Singlet oxygen production in photosynthesis, J. Exp. Bot. 56 (2005) 337–346. https://doi.org/10.1093/jxb/erh237.
[29] H. Khoo, K.N. Prasad, K. Kong, Y. Jiang, A. Ismail, Carotenoids and their isomers: color pigments in fruits and vegetables, Molecules. 16 (2011) 1710–1738.
https://doi.org/10.3390/molecules16021710.
[30] S.S. Thayer, O. Bjrkman, Leaf xanthophyll content and composition in sun and shade determined by HPLC, Photosynth. Res. 23 (1990) 331–343.
https://doi.org/10.1007/BF00034864.
[31] J. Val, E. Monge, N.R. Baker, An improved HPLC method for rapid analysis of the xanthophyll cycle pigments, J. Chromatogr. Sci. 32 (1994) 286–289.
https://doi.org/10.1093/chromsci/32.7.286.
[32] F. Gritti, G. Guiochon, The van Deemter equation: Assumptions, limits, and adjustment to modern high performance liquid chromatography, J. Chromatogr. A. 1302 (2013) 1–13.
https://doi.org/10.1016/j.chroma.2013.06.032.
[33] J.A. Haugan, S. Liaaen-Jensen, Isolation and characterisation of four allenic (6ꞌS)-isomer of fucoxanthin, Tetrahedron Lett. 35 (1994) 2245–2248. https://doi.org/10.1016/S0040- 4039(00)76810-9.
[34] L. Fiedor, Heriyanto, J. Fiedor, M. Pilch, Effects of symetry on the electronic transitions in carotenoids, J. Phys. Chem. Lett. 7 (10) 1821–1829.
https://doi.org/10.1021/acs.jpclett.6b00637
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22 Figure caption
Fig. 1. Absorption spectra of the pigment extracts before and after silica-gel OCC. Absorption peaks were normalized at the highest peak.
Fig. 2. Chromatograms of fucoxanthin isomers separated by RP-HPLC on a YMC C30 column under the conditions of Methods III to VI shown in Table S4.
Fig. 3. Plots between Q-ratio and the number of carbon double bond in the shorter arm of fucoxanthin isomers.
Fig. 4. HPLC elution profiles of fucoxanthin isomers produced by iodine-catalysed stereomutation and separated by YMC C30 column with optimized Method IV (Table S4).
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
23 Table 1
Separation parameters obtained after eluting under the different chromatographic conditions, Methods I to VII described in Table S4, using a C30 column.
Method Fuco isomer
tR
(min)
% area
Separation parameters ± SE
Capacity factor (k') Selectivity (α) Resolution (Rs)
I
all-trans 13.25 64.92 6.14 ± 0.91 1.15 ± 0.01 1.86 ± 0.04 13'-cis 13.96 11.39 6.52 ± 0.95 1.06 ± 0.00 1.03 ± 0.03 13-cis 14.78 4.23 6.97 ± 1.00 1.07 ± 0.00 1.17 ± 0.06 9'-cis 17.52 14.57 8.44 ± 1.17 1.21 ± 0.01 4.88 ± 0.15 II
all-trans
11.27 74.48 5.08 ± 0.03 1.12 ± 0.04 1.09 ± 0.49 13'-cis
13-cis 12.33 5.02 5.65 ± 0.03 1.11 ± 0.00 2.32 ± 0.02 9'-cis 14.31 13.54 6.72 ± 0.03 1.04 ± 0.00 0.98 ± 0.07 III
all-trans 10.64 64.82 5.07 ± 0.40 1.15 ± 0.00 2.27 ± 0.04 13'-cis 11.66 11.02 5.66 ± 0.43 1.11 ± 0.00 2.31 ± 0.04 13-cis 12.23 4.05 5.98 ± 0.45 1.06 ± 0.00 1.25 ± 0.00 9'-cis 14.95 14.38 7.54 ± 0.56 1.03 ± 0.00 0.37 ± 0.08 IV
all-trans 13.68 64.82 6.64 ± 1.35 1.10 ± 0.04 2.03 ± 1.42 13'-cis 14.85 11.05 7.29 ± 1.44 1.10 ± 0.01 2.77 ± 0.09 13-cis 15.47 4.05 7.64 ± 1.48 1.05 ± 0.00 1.42 ± 0.05 9'-cis 17.43 14.90 8.73 ± 1.60 1.06 ± 0.00 2.83 ± 0.33 V
all-trans 5.78 63.56 2.04 ± 0.03 1.20 ± 0.00 1.79 ± 0.05 13'-cis 6.49 10.87 2.41 ± 0.03 1.18 ± 0.00 1.91 ± 0.03 13-cis 6.83 4.13 2.59 ± 0.04 1.07 ± 0.00 0.78 ± 0.02 9'-cis 8.89 14.63 3.67 ± 0.06 1.18 ± 0.11 2.58 ± 1.70 VI
all-trans 13.65 64.98 7.09 ± 0.60 1.12 ± 0.00 2.59 ± 0.11 13'-cis 14.83 11.00 7.78 ± 0.64 1.10 ± 0.00 2.66 ± 0.11 13-cis 15.45 4.07 8.15 ± 0.67 1.05 ± 0.00 1.36 ± 0.03 9'-cis 17.36 14.96 9.28 ± 0.76 1.05 ± 0.00 2.59 ± 0.21 VII
all-trans 11.53 64.90 5.57 ± 0.36 1.07 ± 0.01 1.69 ± 0.13 13'-cis 12.17 10.79 5.93 ± 0.36 1.07 ± 0.00 2.52 ± 0.02 13-cis 12.42 4.21 6.07 ± 0.36 1.02 ± 0.00 0.90 ± 0.07 9'-cis 13.27 14.99 6.56 ± 0.32 1.05 ± 0.01 1.76 ± 0.64 All separation parameters were obtained directly from LCsolution version 1.21 SP1 (Shimadzu).
Capacity factor, selectivity, and resolution of fucoxanthin isomers were calculated between the previous peak and each fucoxanthin isomer peak. The values are averages of three replications.
The values of standard error are calculated by using a confidence level value of 95%.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
24 Table 2
Properties of simultaneously purified fucoxanthin isomers by optimized HPLC with a C30 column and Method IV.
aPeak No. are assigned for convenience. They are the same as in Table 3, Figures 2 and 4.
bThe values are averages of 9 to 12 replications of 3 to 4 independent experiments.
c𝐼𝑓𝑟𝑎𝑔𝑚𝑒𝑛𝑡 𝑟𝑎𝑡𝑖𝑜 = 𝐼[𝑚/𝑧 641.5]
𝐼[𝑚/𝑧 641.5]+𝐼[𝑚/𝑧 581.4]× 100 Fucoxanthin
isomer
Peak No.a
tR
(min)
Purityb (%)
Yieldb
(%) λmax (nm)
Q-ratio
Precursor ion (m/z)
Main fragment ions (m/z)
Intensity ratio of main fragment ion
(% SD)c This
study
Ref.
[33]
all-trans 5 13.72 94.3 95.2 334, -, 451, - 0.08 0.07
659.5 [M+H]+
641.5 [M+H–H2O]+ 581.4 [M+H–H2O–
HOCOCH3]+
65.8 ± 0.4
13ʹ-cis 6 14.88 73.2 57.4 333, -, 445, - 0.48 0.52 69.7 ± 0.5
13-cis 7 15.55 72.2 51.7 332, -, 440, - 0.43 0.45 56.1 ± 3.2
9ʹ-cis 8 17.44 92.6 56.9 332, -, 448, - 0.12 0.12 17.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
25 Table 3
Summary of the separation and identification of fucoxanthin isomers after KI-catalyzed stereomutation by optimized HPLC using a C30 column and Method IV.