Phaeodactylum tricornutum is a rich source of fucoxanthin, a carotenoid with several health benefits. In the present study, high performance countercurrent chromatography (HPCCC) was used to isolate fucoxanthin from an extract of P. tricornutum. A multiple sequential injection HPCCC method was developed combining two elution modes (reverse phase and extrusion). The lower phase of a biphasic solvent system (n-heptane, ethyl acetate, ethanol and water, ratio 5/5/6/3, v/v/v/v) was used as the mobile phase, while the upper phase was the stationary phase. Ten consecutive sample injections (240 mg of extract each) were performed leading to the separation of 38 mg fucoxanthin with purity of 97% and a recovery of 98%. The process throughput was 0.189 g/h, while the efficiency per gram of fucoxanthin was 0.003 g/h. Environmental risk and general process evaluation factors were used for assessment of the developed separation method and compared with existing fucoxanthin liquid-liquid isolation methods. The isolated fucoxanthin retained its well-described ability to induce nuclear translocation of transcription factor FOXO3. Overall, the developed isolation method may represent a useful model to produce biologically active fucoxanthin from diatom biomass.

Faculty of Science Department of Ecology Charles University Viničná 7 128 44 Prague 2 Czech Republic

Faculty of Science University of South Bohemia Branišovská 1760 370 05 České Budějovice Czech Republic

Laboratory of Algal Biotechnology Centre ALGATECH Institute of Microbiology of the Czech Academy of Sciences Opatovický Mlýn 379 81 Třeboň Czech Republic

Metabolic Syndrome Group BIOPROMET Madrid Institute for Advanced Studies IMDEA Food CEI UAM CSIC 28049 Madrid Spain

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Guler B.A., Deniz I., Demirel Z., Yesil-Celiktas O., Imamoglu E. A novel subcritical fucoxanthin extraction with a biorefinery approach. Biochem. Eng. J. 2020;153:107403. doi: 10.1016/j.bej.2019.107403. DOI

Zhang H., Tang Y., Zhang Y., Zhang S., Qu J., Wang X., Kong R., Han C., Liu Z. Fucoxanthin: A promising medicinal and nutritional ingredient. Evid. Based Complement. Alternat. Med. 2015:723515. doi: 10.1155/2015/723515. PubMed DOI PMC

Neumann U., Derwenskus F., Flaiz Flister V., Schmid-Staiger U., Hirth T., Bischoff S.C. Fucoxanthin, a carotenoid derived from Phaeodactylum tricornutum exerts antiproliferative and antioxidant activities in vitro. Antioxidants. 2019;8:183. doi: 10.3390/antiox8060183. PubMed DOI PMC

Maeda H., Hosokawa M., Sashima T., Funayama K., Miyashita K. Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues. Biochem. Biophys. Res. Commun. 2005;332:392–397. doi: 10.1016/j.bbrc.2005.05.002. PubMed DOI

Jeon S.M., Kim H.J., Woo M.N., Lee M.K., Shin Y.C., Park Y.B., Choi M.S. Fucoxanthin-rich seaweed extract suppresses body weight gain and improves lipid metabolism in high-fat-fed C57BL/6J mice. Biotechnol. J. 2010;5:961–969. doi: 10.1002/biot.201000215. PubMed DOI

Woo M.N., Jeon S.M., Shin Y.C., Lee M.K., Kang M.A., Choi M.S. Anti-obese property of fucoxanthin is partly mediated by altering lipid-regulating enzymes and uncoupling proteins of visceral adipose tissue in mice. Mol. Nutr. Food Res. 2009;53:1603–1611. doi: 10.1002/mnfr.200900079. PubMed DOI

Hosokawa M., Miyashita T., Nishikawa S., Emi S., Tsukui T., Beppu F., Okada T., Miyashita K. Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic/obese KK-Ay mice. Arch. Biochem. Biophys. 2010;1:17–25. doi: 10.1016/j.abb.2010.05.031. PubMed DOI

Maeda H., Hosokawa M., Sashima T., Murakami-Funayama K., Miyashita K. Anti-obesity and anti-diabetic effects of fucoxanthin on diet-induced obesity conditions in a murine model. Mol. Med. Rep. 2009;2:897–902. doi: 10.3892/mmr_00000189. PubMed DOI

Heo S.J., Yoon W.J., Kim K.N., Ahn G.N., Kang S.M., Kang D.H., Affan A., Oh C., Jung W.K., Jeon J.Y. Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown algae in lipopolysaccharide-stimulated RAW 264.7 macrophages. Food Chem. Toxicol. 2010;48:2045–2051. doi: 10.1016/j.fct.2010.05.003. PubMed DOI

Kim K.N., Heo S.J., Yoon W.J., Kang S.M., Ahn G., Yi T.H., Jeon Y.J. Fucoxanthin inhibits the inflammatory response by suppressing the activation of NF-κB and MAPKs in lipopolysaccharide-induced RAW 264.7 macrophages. Eur. J. Pharmacol. 2010;649:369–375. doi: 10.1016/j.ejphar.2010.09.032. PubMed DOI

Su J., Guo K., Huang M., Liu Y., Zhang J., Sun L., Li D., Pang K., Wang G., Chen L., et al. Fucoxanthin, a marine xanthophyll isolated from Conticribra weissflogii ND-8: Preventive anti-inflammatory effect in a mouse model of sepsis. Front. Pharmacol. 2019;10:906. doi: 10.3389/fphar.2019.00906. PubMed DOI PMC

Moghadamtousi S.Z., Karimian H., Khanabdali R., Razavi M., Firoozinia M., Zandi K., Kadir H.A. Anticancer and antitumor potential of fucoidan and fucoxanthin, two main metabolites isolated from brown algae. Sci. World J. 2014:768323. doi: 10.1155/2014/768323. PubMed DOI PMC

Ishikawa C., Tafuku S., Kadekaru T., Sawada S., Tomita M., Okudaira T., Nakazato T., Toda T., Uchihara J.N., Taira N., et al. Antiadult T-cell leukemia effects of brown algae fucoxanthin and its deacetylated product, fucoxanthinol. Int. J. Cancer. 2008;123:2702–2712. doi: 10.1002/ijc.23860. PubMed DOI

Das S.K., Hashimoto T., Kanazawa K. Growth inhibition of human hepatic carcinoma HepG2 cells by fucoxanthin is associated with down-regulation of cyclin D. Biochim. Biophys. 2008;1780:743–749. doi: 10.1016/j.bbagen.2008.01.003. PubMed DOI

Zhang Z., Zhang P., Hamada M., Takahashi S., Xing G., Liu J., Sugiura N. Potential chemoprevention effect of dietary fucoxanthin on urinary bladder cancer EJ-1 cell line. Oncol. Rep. 2008;20:1099–1103. doi: 10.3892/or_00000115. PubMed DOI

Sachindra N.M., Sato E., Maeda H., Hosokawa M., Niwano Y., Kohno M., Miyashita K. Radical scavenging and singlet oxygen quenching activity of marine carotenoid fucoxanthin and its metabolites. J. Agric. Food Chem. 2007;55:8516–8522. doi: 10.1021/jf071848a. PubMed DOI

Nomura T., Kikuchi M., Kubodera A., Kawakami Y. Proton-donative antioxidant activity of fucoxanthin with 1,1-diphenyl-2-picrylhydrazyl (DPPH) Biochem. Mol. Biol. Int. 1997;42:361–370. doi: 10.1080/15216549700202761. PubMed DOI

Yan X., Chuda Y., Suzuki M., Nagata T. Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed. Biosci. Biotechnol. Biochem. 1999;63:605–607. doi: 10.1271/bbb.63.605. PubMed DOI

Mise T., Ueda M., Yasumoto T. Production of fucoxanthin-rich powder from Cladosiphon okamuranus. Adv. J. Food Sci. Technol. 2011;3:73–76.

Sangeetha R.K., Bhaskar N., Baskaran V. Comparative effects of beta-carotene and fucoxanthin on retinol deficiency induced oxidative stress in rats. Mol. Cell Biochem. 2009;331:59–67. doi: 10.1007/s11010-009-0145-y. PubMed DOI

Yang G., Jin L., Zheng D., Tang X., Yang J., Fan L., Xie X. Fucoxanthin Alleviates Oxidative Stress through Akt/Sirt1/FoxO3α Signaling to Inhibit HG-Induced Renal Fibrosis in GMCs. Mar. Drugs. 2019;17:702. doi: 10.3390/md17120702. PubMed DOI PMC

Xiao H., Zhao J., Fang C., Cao Q., Xing M., Li X., Hou J., Ji A., Song S. Advances in Studies on the Pharmacological Activities of Fucoxanthin. Mar. Drugs. 2020;18:634. doi: 10.3390/md18120634. PubMed DOI PMC

Yang R., Wei D., Xie J. Diatoms as cell factories for high-value products: Chrysolaminarin, eicosapentaenoic acid, and fucoxanthin. Crit. Rev. Biotechnol. 2020;40:993–1009. doi: 10.1080/07388551.2020.1805402. PubMed DOI

BGG World [(accessed on 10 June 2021)]. Available online: https://bggworld.com/thinogentm-fucoxanthin/

Kim S.M., Jung Y.J., Kwon O.N., Cha K.H., Um B.H., Chung D., Pan C.H. A potential commercial source of fucoxanthin extracted from the microalga Phaeodactylum tricornutum. Appl. Biochem. Biotechnol. 2012;166:1843–1855. doi: 10.1007/s12010-012-9602-2. PubMed DOI

Derwenskus F., Metz F., Gille A., Schmid-Staiger U., Briviba K., Schließmann U., Hirth T. Pressurized extraction of unsaturated fatty acids and carotenoids from wet Chlorella vulgaris and Phaeodactylum tricornutum biomass using subcritical liquids. Glob. Bioenergy. 2019;11:335–344. doi: 10.1111/gcbb.12563. DOI

Global Fucoxanthin (CAS 3351-86-8) Market 2020 by Manufacturers, Regions, Type and Application, Forecast to 2025. [(accessed on 15 January 2021)]. Available online: https://www.360researchreports.com/global-fucoxanthin-cas-3351-86-8-market-16507769.

Noviendri D., Jaswir I., Salleh H.M., Taher M., Miyashita K., Ramli N. Fucoxanthin extraction and fatty acid analysis of Sargassum bindery and S. dulicatum. J. Med. Plants Res. 2011;5:2405–2412.

Jaswir I., Noviendri D., Salleh H.M., Muhammad T., Miyashita K. Isolation of fucoxanthin and fatty acids analysis of Padina australis and cytotoxic effect of fucoxanthin on human lung cancer (H1299) cell lines. Afr. J. Biotechnol. 2011;10:18855–18862. doi: 10.5897/AJB11.2765. DOI

Xia S., Wang K., Wan L., Li A., Hu Q., Zhang C. Production, characterization, and antioxidant activity of fucoxanthin from the marine diatom Odontella aurita. Mar. Drugs. 2013;11:2667–2681. doi: 10.3390/md11072667. PubMed DOI PMC

Jaswir I., Noviendri D., Salleh H.M., Taher M., Miyashita K., Ramli N. Analysis of fucoxanthin content and purification of all-trans-fucoxanthin from Turbinaria turbinate and Sargassum plagyophyllum by SiO2 open column chromatography and reversed phase-HPLC. J. Liq. Chromatogr. Relat. Technol. 2013;36:1340–1354. doi: 10.1080/10826076.2012.691435. DOI

Zhang W., Wang F., Gao B., Huang L., Zhang C. An integrated biorefinery process: Stepwise extraction of fucoxanthin, eicosapentaenoic acid and chrysolaminarin from the same Phaeodactylum tricornutum biomass. Algal Res. 2018;32:193–200. doi: 10.1016/j.algal.2018.04.002. DOI

Sun P., Wong C.C., Li Y., He Y., Mao X., Wu T., Ren Y., Chen F. A novel strategy for isolation and purification of fucoxanthinol and fucoxanthin from the diatom Nitzschia laevis. Food Chem. 2019;30:566–572. doi: 10.1016/j.foodchem.2018.10.133. PubMed DOI

Billakanti M.J., Catchpole O., Fenton T., Mitchell K. Enzyme-assisted extraction of fucoxanthin and lipids containing polyunsaturated fatty acids from Undaria pinnatifida using dimethyl ether and ethanol. Process. Biochem. 2013;48:1999–2008. doi: 10.1016/j.procbio.2013.09.015. DOI

Gómez-Loredo A., Benavides J., Rito-Palomares M. Partition behavior of fucoxanthin in ethanol-potassium phosphate two-phase systems. J. Chem. Technol. Biotechnol. 2014;89:1637–1645. doi: 10.1002/jctb.4514. DOI

Roh M.K., Uddin M.S., Chun B.S. Extraction of fucoxanthin and polyphenol from Undaria pinnatifida using supercritical carbon dioxide with co-solvent. Biotechnol Bioprocess. Eng. 2008;13:724–729. doi: 10.1007/s12257-008-0104-6. DOI

Shang Y.F., Kim S.M., Lee W.J., Um B.H. Pressurized liquid method for fucoxanthin extraction from Eisenia bicyclis (Kjellman) setchell. J. Biosci Bioeng. 2011;111:237–241. doi: 10.1016/j.jbiosc.2010.10.008. PubMed DOI

Gilbert-López B., Barranco A., Herrero M., Cifuentes A., Ibáñez E. Development of new green processes for the recovery of bioactives from Phaeodactylum tricornutum. Food Res. Int. 2017;99:1056–1065. doi: 10.1016/j.foodres.2016.04.022. PubMed DOI

Kuczynska P., Jemiola-Rzeminska M., Strzalka K. Photosynthetic pigments in diatoms. Mar. Drugs. 2015;13:5847–5881. doi: 10.3390/md13095847. PubMed DOI PMC

Ito Y. Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J. Chromatogr. A. 2005;1065:145–168. doi: 10.1016/j.chroma.2004.12.044. PubMed DOI

Michel T., Destandau E., Elfakir C. New advances in countercurrent chromatography and centrifugal partition chromatography: Focus on coupling strategy. Anal. Bioanal. Chem. 2014;406:957–969. doi: 10.1007/s00216-013-7017-8. PubMed DOI

Bárcenas-Pérez D., Lukeš M., Hrouzek P., Kubáč D., Kopecký J., Kaštánek P., Cheel J. A biorefinery approach to obtain docosahexaenoic acid and docosapentaenoic acid n-6 from Schizochytrium using high performance countercurrent chromatography. Algal Res. 2021;55:102241. doi: 10.1016/j.algal.2021.102241. DOI

Cheel J., Urajová P., Hájek J., Hrouzek P., Kuzma M., Bouju E., Faure K., Kopecký J. Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Anal. Bioanal. Chem. 2017;409:917–930. doi: 10.1007/s00216-016-0066-z. PubMed DOI

Cheel J., Hájek J., Kuzma M., Saurav K., Smýkalová I., Ondráčková E., Urajová P., Vu D.L., Faure K., Kopecký J., et al. Application of HPCCC combined with polymeric resins and HPLC for the separation of cyclic lipopeptides muscotoxins A–C and their antimicrobial activity. Molecules. 2018;23:2653. doi: 10.3390/molecules23102653. PubMed DOI PMC

Fábryová T., Cheel J., Kubac D., Hrouzek P., Vu D.L., Tůmová L., Kopecký J. Purification of lutein from the green microalgae Chlorella vulgaris by integrated use of a new extraction protocol and a multi-injection high performance counter-current chromatography (HPCCC) Algal Res. 2019;41:101574. doi: 10.1016/j.algal.2019.101574. DOI

Fábryová T., Tůmová L., da Silva D.C., Pereira D.M., Andrade P.B., Valentão P., Hrouzek P., Kopecký J., Cheel J. Isolation of astaxanthin monoesters from the microalgae Haematococcus pluvialis by high performance countercurrent chromatography (HPCCC) combined with high performance liquid chromatography (HPLC) Algal Res. 2020;49:101947. doi: 10.1016/j.algal.2020.101947. DOI

Nováková M., Fábryová T., Vokurková D., Dolečková I., Kopecký J., Hrouzek P., Tůmová L., Cheel J. Separation of the glycosylated carotenoid myxoxanthophyll from Synechocystis salina by HPCCC and evaluation of its antioxidant, tyrosinase inhibitory and immune-stimulating properties. Separations. 2020;7:73. doi: 10.3390/separations7040073. DOI

Fábryová T., Kubáč D., Kuzma M., Hrouzek P., Kopecký J., Tůmová L., Cheel J. High-performance countercurrent chromatography for lutein production from a chlorophyll-deficient strain of the microalgae Parachlorella kessleri HY1. J. Appl. Phycol. 2021 doi: 10.1007/s10811-021-02434-y. DOI

Sutherland I., Thickitt C., Douillet N., Freebairn K., Johns D., Mountain C., Wood P., Edwards N., Rooke D., Harris G., et al. Scalable technology for the extraction of pharmaceutics: Outcomes from a 3 year collaborative industry/academia research programme. J. Chromatogr. A. 2013;1282:84–94. doi: 10.1016/j.chroma.2013.01.049. PubMed DOI

Xiao X., Si X., Yuan Z., Xu X., Li G. Isolation of fucoxanthin from edible brown algae by microwave-assisted extraction coupled with high-speed countercurrent chromatography. J. Sep. Sci. 2012;35:2313–2317. doi: 10.1002/jssc.201200231. PubMed DOI

Kim S.M., Shang Y.F., Um B.H. A preparative method for isolation of fucoxanthin from Eisenia bicyclis by centrifugal partition chromatography. Phytochem Anal. 2011;22:322–329. doi: 10.1002/pca.1283. PubMed DOI

Gonçalves de Oliveira-Júnior R., Grougnet R., Bodet P.E., Bonnet A., Nicolau E., Jebali A., Rumin J., Picot L. 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. 2020;51:102035. doi: 10.1016/j.algal.2020.102035. DOI

Cheel J., Minceva M., Urajová P., Aslam R., Hrouzek P., Kopecký J. Separation of Aeruginosin -865 from cultivated soil cyanobacterium (Nostoc sp.) by centrifugal partitition chromatography combined with gel permeation chromatography. Nat. Prod. Commun. 2015;10:1719–1722. doi: 10.1177/1934578X1501001022. PubMed DOI

Ito Y., Conway W.D. Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography. III. Effects of physical properties of the solvent systems and operating temperature on the distribution of two-phase solvent systems. J. Chromatogr. A. 1984;301:405–414. doi: 10.1016/S0021-9673(01)89214-1. PubMed DOI

Berthod A., Maryutina T., Spivakov B., Shpigun O., Sutherland I. Countercurrent chromatography in analytical chemistry (IUPAC Technical Report) Pure Appl. Chem. 2009;81:355–387. doi: 10.1351/PAC-REP-08-06-05. DOI

Wood P., Ignatova S., Janaway L., Keay D., Hawes D., Garrard I., Sutherland I.A. Counter-current chromatography separation scaled up from an analytical column to a production column. J. Chromatogr. A. 2007;1151:25–30. doi: 10.1016/j.chroma.2007.02.014. PubMed DOI

Sutherland I.A. Liquid stationary phase retention and resolution in hydrodynamic CCC. In: Berthod A., editor. Comprehensive Analytical Chemistry. Volume 38. Elsevier Science B.V.; Amsterdam, The Netherlands: 2002. pp. 159–176.

Crupi P., Toci A.T., Mangini S., Wrubl F., Rodolfi L., Tredici M.R., Coletta A., Antonacci D. 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. 2013;15:1027–1035. doi: 10.1002/rcm.6531. PubMed DOI

Haugan J.A., Englert G., Glinz E., Liaaen-Jensen S. Algal Carotenoids. 48. Structural Assignments of Geometrical Isomers of Fucoxanthin. Acta Chem. Scand. 1992;46:389–395. doi: 10.3891/acta.chem.scand.46-0389. DOI

DeAmicis C., Edwards N.A., Giles M.B., Harris G.H., Hewitson P., Janaway L., Ignatova S. Comparison of preparative reversed phase liquid chromatography and countercurrent chromatography for the kilogram scale purification of crude spinetoram insecticide. J. Chromatogr. A. 2011;1218:6122–6127. doi: 10.1016/j.chroma.2011.06.073. PubMed DOI

Zhang M., Ignatova S., Liang Q., Wu Jun F., Sutherland I., Wang Y., Luo G. Rapid and high-throughput purification of salvianolic acid B from Salvia miltiorrhiza Bunge by high-performance counter-current chromatography. J. Chromatogr. A. 2009;18:3869–3873. doi: 10.1016/j.chroma.2009.02.067. PubMed DOI

Barradas M., Link W., Megias D., Fernandez-Marcos P.J. High-Throughput Image-Based Screening to Identify Chemical Compounds Capable of Activating FOXO. Methods Mol. Biol. 2019;1890:151–161. doi: 10.1007/978-1-4939-8900-3_13. PubMed DOI

Zanella F., Rosado A., Garcia B., Carnero A., Link W. Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators. BMC Cell Biol. 2009;10:14. doi: 10.1186/1471-2121-10-14. PubMed DOI PMC

Zanella F., Rosado A., García B., Carnero A., Link W. Chemical genetic analysis of FOXO nuclear-cytoplasmic shuttling by using image-based cell screening. Chembiochem. 2008;22:2229–2237. doi: 10.1002/cbic.200800255. PubMed DOI

Furet P., Guagnano V., Fairhurst R.A., Imbach-Weese P., Bruce I., Knapp M., Fritsch C., Blasco F., Blanz J., Aichholz R., et al. Discovery of NVP-BYL719 a potent and selective phosphatidylinositol-3 kinase alpha inhibitor selected for clinical evaluation. Bioorg. Med. Chem. Lett. 2013;23:3741–3748. doi: 10.1016/j.bmcl.2013.05.007. PubMed DOI

Garg S., Afzal S., Elwakeel A., Sharma D., Radhakrishnan N., Dhanjal J.K., Sundar D., Kaul S.C., Wadhwa R. Marine Carotenoid Fucoxanthin Possesses Anti-Metastasis Activity: Molecular Evidence. Mar. Drugs. 2019;17:338. doi: 10.3390/md17060338. PubMed DOI PMC

Starr R.C., Zeikus J.A. UTEX–The Culture Collection of Algae at the University of Texas at Austin 1993 List of Cultures. J. Phycol. 1993;29:106. doi: 10.1111/j.0022-3646.1993.00001.x. DOI

Bligh E.G., Dyer W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959;37:911–917. doi: 10.1139/o59-099. PubMed DOI