A novel strategy for isolation and purification of fucoxanthinol and fucoxanthin from the diatom Nitzschia laevis

Accepted Manuscript A novel strategy for isolation and purification of fucoxanthinol and fucoxanthin from the diatom Nitzschia laevis Peipei Sun, Chi-Chun Wong, Yuelian Li, Yongjin He, Xuemei Mao, Tao Wu, Yuanyuan Ren, Feng Chen PII: DOI: Reference:

S0308-8146(18)31913-7 https://doi.org/10.1016/j.foodchem.2018.10.133 FOCH 23794

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

24 April 2018 22 October 2018 28 October 2018

Please cite this article as: 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 Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.10.133

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A novel strategy for isolation and purification of fucoxanthinol and fucoxanthin from the diatom Nitzschia laevis Peipei Suna,b,c, Chi-Chun Wongd, Yuelian Lia,c, Yongjin Hea,c, Xuemei Maoa,c, Tao Wua,c, Yuanyuan Rena,c, Feng Chen*a,c a

BIC-ESAT, College of Engineering, Peking University, Beijing 100871, China

b

School of Food Science and Engineering, South China University of Technology,

Guangzhou 510641, China c

Institute for Food and Bioresource Engineering, College of Engineering,

Peking University, Beijing 100871, China d

Department of Medicine and Therapeutics, Chinese University of Hong Kong，

Shatin, NT, Hong Kong

*

Corresponding author E-mail: [email protected] (F. Chen) 1

Abstract: In this study, the microalga Nitzschia laevis (N. laevis) can accumulate a marine carotenoid fucoxanthinol. In particular, fucoxanthinol was firstly isolated from microalgae, accompanied by its derivative fucoxanthin. The identification and quantification of fucoxanthinol and fucoxanthin were determined by ultra-performance liquid chromatography coupled to photodiode array detector-quadrupole/ travelling-wave ion mobility mass spectrometry/time-of-flight mass spectrometry (UPLC-PDA-TWIMS-QTOF-MS). Furthermore, a cost-effective approach mediated with solid-phase extraction (SPE) and thin-layer chromatography (TLC) technique was used to isolate and purify fucoxanthinol and fucoxanthin from the extracts of N. laevis. This two-step method can obtain 98% fucoxanthinol and 95% fucoxanthin, with the recovery efficiencies of around 85% for fucoxanthinol and 70% for fucoxanthin, respectively. Moreover, 1H and 13C nuclear magnetic resonance (NMR) techniques were adopted to record the purified compounds for supporting the above results. In all, the developed method has a promising potential to purify fucoxanthinol and fucoxanthin of microalgae for food and pharmaceutical applications.

Keywords: Nitzschia laevis, fucoxanthinol, fucoxanthin, solid-phase extraction, thin-layer chromatography

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1 Introduction Fucoxanthin is a marine xanthophyll carotenoid with health beneficial properties, including anticancer, anti-inflammation, antidiabetic, and antiobesity (Hoang Van & Eun, 2017; Yen et al., 2013). In mammals, dietary fucoxanthin is metabolized into fucoxanthinol by digestive enzymes from the gastrointestinal tract, and is then directly absorbed in the intestine and transported into the circulation (Yamamoto et al., 2011). Fucoxanthin and its deacetylated derivative fucoxanthinol have similar extraordinary structures with an allelic bond, a conjugated carbonyl, and a 5, 6-monoepoxide. Moreover, emerging evidence has shown that fucoxanthinol has superior functional effects compared to fucoxanthin. For example, fucoxanthinol demonstrated a stronger anti-proliferative effect on 3T3-L1 cells (Maeda et al., 2006) and human T-cell leukemia virus type 1 cells (Ishikawa et al., 2008). The pharmacokinetics of fucoxanthin in mice revealed that a single orally administered fucoxanthin was not detectable in all tissues, and fucoxanthinol was a major gastrointestinal metabolite (Beppu et al., 2009; Hashimoto et al., 2009). Based on these findings, it is now recognized that the key biological functions of fucoxanthin could be attributed to its hydrolzyed derivative fucoxanthinol (Zhang et al., 2015). As far as we know, whilst fucoxanthinol exhibits excellent bioactivity and potential as nutraceutical, there are few reports about natural sources for the preparation of this compound. Chemical and enzymatic approaches have been mainly used to produce fucoxanthinol. After treatment of fucoxanthin with NaBH4, LiAlH4, and oxidation (p-chloroanil) in benzene, fucoxanthinol was isolated. Chemical methods involved the use of toxic solvents and environmentally unfriendly catalysts; and fucoxanthinol yield is very low (Nitsche, 1974). Enzymatic method involves the enzymatic hydrolysis of fucoxanthin to form fucoxanthinol. The 3

reaction of fucoxanthin, sodium taurocholate and porcine pancreatic lipase was incubated at 37 °C for 3 h, then fucoxanthinol was purified by column chromatography, liquid-liquid partition and recrystallization(Yamamoto et al., 2011). This process has many disadvantages, such as complex processing, high cost of fucoxanthin substrate and the low hydrolysis yield, limiting its application. As a consequence, fucoxanthinol is commercially much more expensive than fucoxanthin. Thus, the natural sources offer an attractive alternative. Fucoxanthinol is found in marine animals such as Dreissena bugensis (Bridoux et al., 2017), Echinozoa subphylum (Mamelona & Brion, 2015) and Mactra chinensis (Maoka, 2011; Maoka et al., 2007). However, there are major bottlenecks to the extraction of fucoxanthinol from marine animals, such as high cost of animal, time-consuming procedures and the low extraction efficiency (Konishi, et al., 2006). Moreover, these animals grow slowly, and their fucoxanthinol contents are frequently low (Mamelona & Brion, 2015). The acquisition from marine animals is not thus economic and unrealistic, that might damage the oceanic ecology. On the basis of the marine food chain, it is postulated that marine animals obtain fucoxanthinol directly via the consumption of marine microalgae. The microalgae constitute the largest group of living organisms, and are important primary producers. Previous studies have shown that fucoxanthinol exists in Prymnesiophyceae, Chrysophyceae and Phaeophyceae (Young, 1993), such as Ochromonas sp., Poterioochromonas malhamensis, Olisthodiscus luteus, Sarcinochrysis marina, etc (Withers, et al.,1981). In our preliminary study, we showed that the diatom Nitzschia laevis not only could accumulate fucoxanthin, but also large amounts of fucoxanthinol. In the aqueous acetone extracts, the contents of fucoxanthin and fucoxanthinol were determined by high performance liquid chromatography coupled with diode array detector (HPLC-DAD), reaching 2.24 and 4.40 mg/g 4

dry weight respectively in N. laevis (Sun et al., 2018). In this respect, N. laevis offers a promising alternative for fucoxanthinol and fucoxanthin production. To our best knowledge, there have been no reports on the isolation and purification of fucoxanthinol and fucoxanthin from microalgae simultaneously. Most studies focused on the purification of fucoxanthin. The purification methods were reported such as column chromatography, thin layer chromatography or high speed counter current chromatography (HSCCC) (Gong & Bassi, 2016). Conventional silica gel column chromatography has been applied in the purification of fucoxanthin, using n-hexane and acetone as the eluting solvent system. In one previous report (Xia et al., 2013), fucoxanthin of crude purity (86.7%) was obtained after the pigmented extract from the diatom Odontella aurita was eluted with n-hexane/acetone (6:4, v/v). Further preparative HPLC was needed to obtain fucoxanthin with 97% purity. Other described studies also typically require multi-step processes to purify fucoxanthin (Gómez-Loredo et al., 2015; Kim et al., 2012). These methods were inefficient and time consuming. Xiao et al. (Xiao, et al., 2012) reported that fucoxanthin was isolated from edible brown algae by HSCCC with a two-phase solvent system consisting of hexane-ethyl acetate-ethanol-water (5:5:6:4, v/v/v/v), and the purity of fucoxanthin determined by HPLC was  90%. The mobile phase system was complex and water was also more difficult to remove than organic solvents. Then, a cost effective simple process is desirable for high purity fucoxanthinol and fucoxanthin production. Mass spectrometry recognized as a common approach was used for the identification of fucoxanthinol and fucoxanthin (Sun, et al., 2018). Nevertheless, a traveling wave ion mobility mass spectrometer (TWIM-MS) was investigated to be useful as a promising technique that could 5

provide further identification criterion by collision cross section (CCS) values. Especially, the coupling of TWIMS with time-of-flight mass spectrometry (TOF-MS) allows the detection of compounds separated by ion mobility (Pacini, et al., 2015). In the present study, a simple two-step method for efficient isolation and purification of fucoxanthinol and fucoxanthin from the diatom N. laevis was described. Silica gel column chromatographic method used in this study enables purification of fucoxanthinol and fucoxanthin with HPLC-purity of > 95%. Final purification by preparative-TLC enables us to obtain pure fucoxanthinol and fucoxanthin. On the basis of above steps, the techniques of UPLC-PDA-TWIMS-QTOF-MS and NMR were employed to determine these two purified compounds.

2 Materials and Methods 2.1 Chemicals and Reagents Fucoxanthinol and fucoxanthin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All MS-grade formic acid, methanol, and acetonitrile were obtained from Merck KGaA (Darmstadt, Germany). Analytical reagents n-hexane, diethyl ether (Et2O) and acetone were purchased from Sinopharm (Beijing, China). Ultrapure water was produced by a Millipore Milli-Q system (Billerica, MA, USA). 2.2 Strains and Culture Conditions The diatom Nitzschia laevis (UTEX 2047) was obtained from University of Texas Culture Collection (Texas, USA). Cells were cultured in modified Lewins marine diatom medium (Wen & Chen, 2001), supplemented with 5 g/L glucose and 60 mg/L NaSiO39H2O. LDM medium consisted of (per litre) 1 g tryptone, 1 g NaNO3, 892 mL artificial seawater, 100 mL Bristol solution, 6 mL PIV metal solution, 1 mL stock solutions of biotin (25.010-5 g/L) and 1 mL 6

vitamin B12 (15.010-5 g/L) without yeast extract. The medium pH was adjusted to 8.6 prior to autoclaving at 121 °C for 20 min. Algal cells were incubated in 700 mL bubble column photobioreactors ( 4.5  60 cm) with a working volume of 600 mL medium under continuous illumination of 80 μmol photons m-2s-1 at 23 °C. 2.3 Determination of Biomass Concentration Biomass concentration was measured by cell dry weight. Five milliliters cell suspension were centrifuged at 5000 rpm for 3 min. The pellets were washed twice with distilled water and filtered using pre-weighted glass microfiber filter paper (Whatman GF/C). The loaded filter paper was dried over vacuum overnight at 80 °C until reaching constant weight. In the present study, algal cells of 8 d were used for later experiments with cell dry weight of 3.04 g/L. To obtain microalgal freeze-dried powders, cells were harvested by centrifugation at 5000 rpm for 3 min, and then lyophilized. Lyophilized powders were stored at -20 °C for later analysis. 2.4 Preparation of Pigments from Microalgae Freeze-dried powders (4 g) were weighted and ground in liquid nitrogen to break the cell walls, followed by extraction in methanol (120 mL) for 20 min under vortex. Methanol extracts were centrifuged for 10 min at 5000 rpm at 4 °C in the centrifuge (Thermo Scientific, Sunnyvale, USA) and the supernatants were collected. Residues were re-extracted with methanol (120 mL) until their supernatants became colourless. The supernatants were then combined, vacuum concentrated at 20 °C by a rotary evaporator (EYELA Ltd., Tokyo, Japan), lyophilized at -50 °C and weighted. All procedures were carried out under darkness to avoid photo-oxidation. Lyophilized extracts were used in the following assays. 2.5 Isolation of Fucoxanthinol and Fucoxanthin by Solid-Phase Extraction (SPE) 7

Methanol extracts (1.44 g) were re-dissolved in methanol (30 mL), absorbed with silica gel (2.88 g) and then vacuum evaporated at 20 °C. Six hundred milligrams (60 mg  10) of the mixtures were subjected to silica gel chromatography (600 mg, Waters) and eluted using eluents n-hexane/Et2O 80:20, v/v (80 mL each), n-hexane/Et2O 65:35, v/v (80 mL each), n-hexane/Et2O 50:50, v/v (80 mL each), Et2O (80 mL each), and methanol (20 mL each). The fractions eluted with n-hexane/Et2O (50:50, v/v) and Et2O, namely, fraction 1 (fucoxanthin) and fraction 2 (fucoxanthinol), were collected, vacuum evaporated to remove the solvent at 20 °C and then respectively suspended in acetone (1 mL  4). Fraction 1 and 2 were freezed in -80 °C for 6 hours and then centrifuged at 14000 rpm in -10 °C for 15 min. The supernatants of fraction 1 and 2 were analyzed by UPLC-PDA to determine the HPLC-purity of fucoxanthinol (2) and fucoxanthin (1), and then they were dried in the nitrogen, weighted and re-dissolved in chloroform with the concentration of 50 mg/mL for TLC purification. 2.6 Purification of Fraction 2 (Fucoxanthinol) and Fraction 1 (Fucoxanthin) by Thin-Layer Chromatography (TLC) In our preliminary experiments, a small amount of lipids were co-eluted in the aforementioned fractions. To acquire pure fucoxanthinol and fucoxanthin, the fractions were dissolved in chloroform (50 mg/mL) and applied on a preparative TLC plates (silica gel G, 250 m, Merck, USA). The plate was developed in hexane/diethyl ether/acetic acid (70:30:1, v/v/v) and visualized after fumigating with iodine steam. The baseline bands corresponding to fucoxanthinol and fucoxanthin were scraped off the plates and extracted four times with 5 mL methanol. Lipid components were separated from fucoxanthinol or fucoxanthin and pure fucoxanthinol and fucoxanthin were obtained. The purity of fucoxanthinol and fucoxanthin were 8

confirmed by TLC. They were subjected to TLC plates (10  20 cm) coated with silica gel 60 (Merck, USA). Solvents used were hexane/diethyl ether/acetic acid (70:30:1, v/v/v) for neutral lipids and chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5, v/v/v/v/v) for polar lipids. Bands corresponding to fucoxanthinol and fucoxanthin were identified by co-chromatography with authentic standards, and the visualization of lipids was by copper sulfate. 2.7 Identification and Quantification of Fucoxanthinol and Fucoxanthin 2.7.1 UPLC-PDA-TWIMS-QTOF-MS Analysis of Fucoxanthinol and Fucoxanthin UPLC separation was performed on an ACQUITY UPLC (Waters, Milford, USA) using a UPLC HSS T3 C18 column (100 × 2.1 mm, 1.7 μm) (Waters, Milford, USA). A PDA detector (Waters, Milford, USA) was used for online acquisition of UV/Vis spectra from 200 to 800 nm and detected at 450 nm (carotenoids). The mobile phases were composed of (A) methanol: isopropanol=68:32 (0.1% formic acid) and (B) acetonitrile: methanol: water=84:2:14 (0.1% formic acid). The gradient elution program was from 0% A at 0 min to 100% A at 8 min, 100% A at 16 min. The flow rate was 0.4 mL /min, injection volume was 1L. The UPLC-UV/Vis system was coupled in line with a SYNAPT G2-Si (Waters, Wilmslow, UK) operating in the positive electrospray ionization mode. The capillary and cone voltage were 1.5 kV and 30 V, respectively. The source and desolvation temperature were 100 and 500 °C, respectively, and the desolvation gas flow was 800 L/h. Leucine enkephalin (2 ng/ μL) was used as lock mass (m/z 556.2771). Data were acquired in high definition mass spectrometer elevated experiment mode (HDMSE) from m/z 100 to 1200 with an acquisition speed of 1.5 scan/s, creating two discrete and independent interleaved acquisition functions. Argon served as collision gas, and the collision energy in the trap cell was 4 eV (Function 1); in the transfer cell it was ramped from 30 to 40 eV (Function 2). 9

Data analysis was performed with UNIFI 1.8.2 (Waters, Milford, MA, USA). Data were processed with the results containing individually elucidated parameters including retention time, exact masses of pseudo molecular ions, adducts and fragments, and CCS. The identification criteria were as follows: precision of the mass error  5ppm for compounds and isotopes, retention time shift  0.1 min, and additionally a CCS delta  2%. 2.7.2 UPLC-PDA Quantification of Fucoxanthinol and Fucoxanthin The determination of fucoxanthinol and fucoxanthin was carried out according to the absorbance at 450 nm. The calibration curves (10-200 μg. mL-1) using authentic fucoxanthinol and fucoxanthin were established for quantification of the two compounds. The amounts of them were calculated from the peak areas by the standard curves. The concentrations were expressed as milligram fucoxanthinol or fucoxanthin per gram cell dry weight (mg. g-1 DW). 2.8 Analysis of Fucoxanthin and Fucoxanthinol by NMR Purified fucoxanthinol (20 mg) and fucoxanthin (10 mg) was dissolved in 0.25 mL of CDCl3 and used for NMR spectroscopy. The 1H and 13C NMR experiments were performed on a Bruker 500 MHz NMR system (1H with 500 MHz, 13C with 125 MHz). Two-dimensional (2D) NMR (COSY) and 13C- DEPT 135 NMR spectra were recorded with a Bruker 800 MHz spectrometer. Resonance assignments were based on chemical shifts (δ). The chemical shifts (δ) relative to the solvent signals are expressed in ppm whereas coupling constant (J) are reported in Hertz (Hz). Data were processed using the MestReNova program (Mestrelab Research, Santiago de Compostela, Spain).

3 Results and Discussion 3.1 Identification and Quantification of Fucoxanthinol and Fucoxanthin in the Diatom N. 10

laevis under Continuous Illumination The marine diatom N. laevis was selected in this study due to its high growth rate and abundant high-value products, in particular eicosapentaenoic acid (EPA) (Chen et al., 2007), fucoxanthinol and fucoxanthin (Sun, 2018). It has been reported that mixotrophic culture had the highest specific growth rate and maximum cell density among the three growth modes of photoautotrophic, mixotrophic and heterotrophic conditions (Wen & Chen, 2001). In the present study, a continuous illumination under mixotrophic condition of growth was used. 3.1.1 Identification of Fucoxanthinol and Fucoxanthin by UPLC-PDA-TWIMS-QTOF-MS To identify fucoxanthinol and fucoxanthin in the pigmented extracts (carotenoids and chlorophyll species), an UPLC-PDA-TWIMS-QTOF-MS in full scan HDMSE mode was used. This strategy provides simultaneous UV detection, accurate mass measurements, MS/MS spectra for structural elucidation, and CCS measurements. Identification and confirmation of fucoxanthinol and fucoxanthin were performed by comparison with reference standards. Hence, mass measurements, fragmentation information, and retention times against the databases based on the reference standards were accurately searched. Fucoxanthinol is a deacetylated derivative of fucoxanthin and the presence of hydroxyl instead of an acetyl gives fucoxanthinol a more polar characteristic. Therefore, it is less retained and yielded similar fragmentation pattern with fucoxanthin. UPLC-PDA yielded to a first set of information for the identification of the two compounds (Fig. 1b). Peaks at 1.86 min and 3.14 min were initially identified as fucoxanthinol and fucoxanthin, respectively, based on the comparison of their retention time and UV absorption spectra with reference standards. Furthermore, analyses of fragmentation patterns by MS/MS provided unambiguous identification. In previous studies (Repeta & Gagosian, 1982; Sun, 2018), 11

m/z 411.2693 and m/z 109.1023 were the characteristic fragments of fucoxanthinol and fucoxanthin. Protonated ion peaks of fucoxanthinol and fucoxanthin were at m/z 617.4256 and 659.4300, respectively. We observed two peaks on the extracted ion chromatogram (XIC) at m/z 617.4206 and 659.4312 in low-energy mode (Fig. 1c) and these peaks overlapped with the ones obtained from XIC in the high energy mode at m/z 411.2693 and 109.1023 (Fig. 1d). In addition to the mass spectral data, the CCS value is a unique physicochemical property of a compound that can act as identification indicator and aid in compound identification (Paglia et al., 2015; Paglia et al., 2014). HDMSE measurement obtained from ion mobility separation of the fragments can be specifically assigned to the parent molecules via their corresponding drift times. The features identified fucoxanthinol and fucoxanthin with specific CCS values as shown in table 1. These data suggest that the peaks at 1.86 min and 3.14 min were identified as fucoxanthinol and fucoxanthin. 3.1.2 Quantification of Fucoxanthinol and Fucoxanthin To evaluate the potential of fucoxanthinol and fucoxanthin production in N. laevis, growth experiments were conducted in bubble column photobioreactors. The pigment composition of crude extracts was analyzed by UPLC-PDA (Fig. 2a) and the results showed that N. laevis contained three major carotenoids (Fig. 2b). Quantification of fucoxanthinol and fucoxanthin was achieved by the use of their corresponding pure standards (Table 2). Under experimental conditions, the concentration of fucoxanthinol was as high as 4.64 mg·g-1 DW whereas that of fucoxanthin was 1.68 mg·g-1 DW. There has been no published information on the carotenoids of N. laevis, thus, this is the first report on the carotenoids of this alga, in particular for fucoxanthinol and fucoxanthin. The results indicated that the microalga N. laevis was a potential source for 12

production of fucoxanthinol and fucoxanthin. 3.2 Isolation and Purification of Fucoxanthinol and Fucoxanthin by SPE and TLC In light of the result of pigment composition (Fig. 2), non-polar and polar solvents (n-hexane and Et2O) were applied to isolate fucoxanthinol and fucoxanthin from the pigment extracts. In previously reported protocols on carotenoids purification (Kuczynska & Jemiola-Rzeminska, 2017), hexane and Et2O were used to remove lipids from pigment extracts. Since the microalga N. laevis can also accumulate polyunsaturated fatty acids (Ethier et al., 2011; Wen & Chen, 2001), a two-step purification process combining SPE and TLC was performed. 3.2.1 Purification of Fucoxanthinol and Fucoxanthin by SPE Fractions 1 and 2 were eluted with n-hexane/Et2O (50:50, v/v) and Et2O, respectively, and then collected and analyzed by UPLC-PDA. The results showed that fractions 1 and 2 mainly contain fucoxanthin and fucoxanthinol, respectively, according to the retention time of the standards. The purity of both fucoxanthinol and fucoxanthin was estimated to be of > 95%, as shown by the chromatographic spectra (Fig. 2). The concentrations of purified fucoxanthin and fucoxanthinol obtained from 0.1 g methanol extracts (0.27 g biomass) after SPE were 1.18 and 3.96 mg. g-1 DW according to the external standard curves, respectively (Table 2). The recovery rates of fucoxanthinol and fucoxanthin reached  85% and 70%, respectively. Hence, the application of gradient elution with hexane and diethyl ether efficiently separated fucoxanthinol and fucoxanthin from other pigments and also from each other. Additionally, as this solvent system was often used to isolate lipids (He et al., 2017), our method might simultaneously attain the separation of functional lipids from the extracts. 3.2.2 TLC Purification of Fucoxanthinol and Fucoxanthin 13

In this study, algal cells were harvested at the stationary phase. Thus, less lipids could be extracted in the crude pigment extracts, especially neutral lipids that account for a majority of the total lipids in N. laevis. Total lipids could be separated into neutral lipids (NLs), glycolipids (GLs) and phospholipids (PLs) using SPE (Chen et al., 2007). For polar lipids, methanol and acetone were usually used for eluting phospholipid and glycolipid, respectively (Chen et al., 2007). Nevertheless, hexane was a good solvent for the extraction of NLs (Yao, et al, 2015). Therefore, the main co-effluents were NLs instead of polar lipids in the SPE fractions. For the above reasons, further purification was carried out by preparative TLC to obtain pure fucoxanthinol and fucoxanthin. Analytical TLC images of preparative TLC purified fucoxanthinol and fucoxanthin were shown in Fig. 3. As observed by analytical TLC analysis (Fig. 3), lipids of the methanol extracts in N. laevis are consisted mainly of NLs, along with a small amount of polar lipids which were not quantified in this study. After SPE isolation and purification, there were a few NLs contaminants, but not polar lipids, in fractions containing fucoxanthinol and fucoxanthin. On the other hand, no NLs and polar lipids contaminants were found after prep-TLC purification. Collectively, pure fucoxanthinol and fucoxanthin production were obtained by two-step purification. Natural pigments are among the most influential compounds in determining the overall revenue from the microalgal biomass. As noted by Ruiz et al. (Ruiz et al., 2016), the market volume for natural pigments is limited. For commercially feasible cases of pigment production from microalgae, their current revenue averaged 30.4 €·kg-1 of dry biomass. The integration of high cell density cultivation and high-value products such as fucoxanthinol and fucoxanthin in N. laevis could potentially allow its commercialization into a new industrial algae strain. 14

3.3 NMR Analysis of Fucoxanthinol and Fucoxanthin To unambiguously confirm the identity of purified fucoxanthinol and fucoxanthin, NMR spectroscopy was utilized as it was considered the gold standard for assignment of the structures of compounds. The complete assignments and structural elucidation data of 1H and 13C NMR spectra were listed in Table 3. 1H and 13C spectra elucidated signals were assignable to conjugated ketone and polyene with or without acetyl group (fucoxanthin or fucoxanthinol), quaternary geminal dimethyls and methyls of oxygen, and olefinic methyls. 1H and 13C spectral data of the purified compounds were identical with those of authentic fucoxanthinol and fucoxanthin standards, which confirmed their identity as fucoxanthinol and fucoxanthin. 4 Conclusion Microalgae show an enormous potential as sustainable feedstocks for numerous bioproducts. The current study establishes a new and simple method for simultaneous isolation and purification of fucoxanthinol and fucoxanthin from the microalga N. laevis by SPE coupled with TLC techniques. The solvent elution system of n-hexane/Et2O based on SPE is efficient and achieves high purity. Taken together, our study represents a pioneering work of obtaining the high-value production of fucoxanthinol and fucoxanthin from microalgae and underscores N. laevis as a promising production strain. Moreover, it enables the purification of them to the preparative scale by using common large-scale silica gel column chromatography. In addition, due to the great potential of the two compounds in food and pharmaceutical industry, present results may aid the extensive development of this method for commercial-scale production.

Conflicts of interest 15

The authors declare no conflict of interest. Acknowledgments This work was funded by National Key R&D Program of China (2016YFD0400204), and partly supported by Public Science and Technology Research Funds Projects of Ocean (Project No.: 201505032).

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FIGURE CAPTIONS

Fig. 1 Isolating pigment signatures of N. laevis. Retention time information for carotenoids absorbing at 450 nm was used as a filter to obtain the UV-Vis spectra (b). Low-energy XIC of m/z 617.4256 and 659.4300 (c). High-energy XIC of m/z 411.2693 and 109.1023 (d).

Fig. 2 The chromatographic spectra and structures of fucoxanthinol (a) and fucoxanthin (b) by SPE.

Fig. 3 The visualization of fucoxanthinol and fucoxanthin by TLC. The developing solvents of the plates were used for separation of neutral lipids (a) and polar lipids (b). The samples and standards on the two plates were the same. Lanes: 1, TLC purified fucoxanthinol; 2, TLC purified fucoxanthin; 3, the standard fucoxanthinol; 4, the standard fucoxanthin; 5, the pigment extracts; 6, Et2O fraction; 7, hexane/Et2O (50:50) fraction.

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Fig. 1

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Fig. 2

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Fig. 3

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TABLES Table 1 Tentative identification of fucoxanthinol and fucoxanthin by UPLC-PDA-TWIMS-MSE pigments formula RT(min) adduct measured (m/z) CCS (Å2) fucoxanthinol C40H56O5 1.86 [M+H]+ 617.4256 250 + fucoxanthin C42H58O6 3.14 [M+H] 659.4300 279

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Table 2 The contents of fucoxanthinol and fucoxanthin in crude extracts and SPE fractions. Fucoxanthinol(mg·g-1 DW) Fucoxanthin(mg·g-1 DW) Crude extracts of 4.640.01 1.680.01 N. laevis Fractions isolated 3.960.01 1.180.01 by SPE Values are expressed as the mean ± SD from three replicates.

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Table 3 NMR data of fucoxanthinol and fucoxanthin in CDCl3 Positi on 1 2 3 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’ 10’ 11’

Fucoxanthinol 13 H (multiplicity, C J=Hz, 500 MHz) (125 MHz) 1

1.37 (1H, m) 1.50 (1H, m) 3.82 (1H, m) 1.79 (1H, J=14.0, 9.3 Hz, dd) 2.33 (1H, m)

2.61 (1H, J=18.3Hz, d) 3.66 (1H, J=18.3Hz, d)

7.16 (1H, J=11.8Hz, d) 6.58 (1H, J=11.4, 15.4 Hz, dd) 6.68 (1H, J=14.7 Hz, d) 6.42 (1H, J=11.6Hz, d) 6.64 (1H, J=11.8, 15.4 Hz, dd) 1.04 (3H, s) 0.97 (3H, s) 1.23 (3H, s) 1.95 (3H, s) 2.00 (3H, s) 1.35 (1H, m) 1.97 (1H, m) 4.32 (1H, m) 1.42 (1H, m) 2.27 (1H, m)

6.04 (1H, s) 6.13 (1H, J=11.3 Hz, d) 6.61 (1H, J=11.8, 15.4 Hz, dd)

35.1 47.0 64.3 41.6

66.1 67.1 40.8 197.8 134.5 139.1 123.3 145.0 138.1 136.6 129.3 25.0 28.1 21.1 11.8 12.7 35.8 49.4 64.3 48.9 73.0 117.8 202.4 103.2 132.7 128.3 125.7

Fucoxanthin H (multiplicity, J=Hz, 500 MHz) 1

1.28 (1H, m) 1.50 (1H, m) 3.69 (1H, m) 1.77 (1H, J=13.9, 9.2 Hz, dd) 2.26 (1H, m)

2.59 (1H, J=18.2Hz, d) 3.64 (1H, J=18.2Hz, d)

6.39 (1H, J=11.9 Hz, d) 6.75 (1H, J=11.9 Hz, d) 6.65 (1H, J=14.8 Hz, d) 6.39 (1H, J=11.6Hz, d) 6.60 (1H, J=14.8 17.7 Hz, dd) 1.02 (3H, s) 0.95 (3H, s) 1.22 (3H, s) 1.98 (3H, s) 2.05 (3H, s) 1.41 (1H, m) 1.99 (1H, m) 5.40 (1H, m) 1.45 (1H, m) 2.27 (1H, m)

6.04 (1H, s) 6.11 (1H, J=11.4 Hz, d) 6.72 (1H, J=11.9 Hz, d) 28

13

C (125 MHz) 35.1 47.0 64.2 41.6

66.1 67.1 40.8 170.4 134.5 139.1 123.4 145.0 138.0 136.6 129.3 25.0 28.1 21.1 11.8 12.7 35.7 45.4 68.0 45.2 72.6 117.8 202.3 103.3 132.5 128.8 125.6

12’ 13’ 14’ 15’ 16’ 17’ 18’ 19’ 20’ 21’ 22’

6.35 (1H, J=15.1 Hz, d) 6.27 (1H, J=11.7 Hz, d) 6.76 (1H, J=12.1, 14.2 Hz, dd) 1.34 (3H, s) 1.08 (3H, s) 1.36 (3H, s) 1.82 (3H, s) 2.00 (3H, s)

137.0 135.3 132.1 132.5

6.33 (1H, J=15.0 Hz, d)

29.3 32.1 31.9 14.1 12.8

1.34 (3H, s) 1.30 (3H, s) 1.42 (3H, s) 1.80 (3H, s) 2.05 (3H, s)

6.25 (1H, J=11.6 Hz, d) 6.91 (1H, J=8.24 Hz, t)

2.02 (3H, s)

29

137.1 135.3 132.2 132.4 29.3 31.9 31.4 14.0 12.8 197.7 21.1

Highlights Nitzschia laevis was a promising resource to develop fucoxanthinol and fucoxanthin production A simple two-step method was mediated with SPE and TLC techniques This exploited approach could effectively purified fucoxanthinol and fucoxanthin The purities of the two purified compounds were over 95%

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