Carotenoid Profiling of a Red Seaweed Pyropia Yezoensis

Home , Antheraxanthin, Apocarotenal, Pyropia

marine drugs

Article Carotenoid Proﬁling of a Red Seaweed Pyropia yezoensis: Insights into Biosynthetic Pathways in the Order Bangiales

Jiro Koizumi 1, Naoki Takatani 1, Noritoki Kobayashi 1, Koji Mikami 2, Kazuo Miyashita 2, Yumiko Yamano 3, Akimori Wada 3 , Takashi Maoka 4 and Masashi Hosokawa 2,*

1 Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan; [email protected] (J.K.); [email protected] (N.T.); [email protected] (N.K.) 2 Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan; komikami@fish.hokudai.ac.jp (K.M.); kmiya@fish.hokudai.ac.jp (K.M.) 3 Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University, Kobe 658-8558, Japan; [email protected] (Y.Y.); [email protected] (A.W.) 4 Research Institute for Production Development, 15 Shimogamo, Morimoto Cho, Sakyoku, Kyoto 606-0805, Japan; [email protected] * Correspondence: hoso@fish.hokudai.ac.jp; Tel.: +81-138-40-5530

Received: 17 October 2018; Accepted: 30 October 2018; Published: 1 November 2018

Abstract: Carotenoids are natural pigments that contribute to light harvesting and photo-protection in photosynthetic organisms. In this study, we analyzed the carotenoid profiles, including mono-hydroxy and epoxy-carotenoids, in the economically valuable red seaweed Pyropia yezoensis, to clarify the detailed biosynthetic and metabolic pathways in the order Bangiales. P. yezoensis contained lutein, zeaxanthin, α-carotene, and β-carotene, as major carotenoids in both the thallus and conchocelis stages. Monohydroxy intermediate carotenoids for the synthesis of lutein with an ε-ring from α-carotene, α-cryptoxanthin (β,ε-caroten-3’-ol), and zeinoxanthin (β,ε-caroten-3-ol) were identified. In addition, β-cryptoxanthin, an intermediate in zeaxanthin synthesis from β-carotene, was also detected. We also identified lutein-5,6-epoxide and antheraxanthin, which are metabolic products of epoxy conversion from lutein and zeaxanthin, respectively, by LC-MS and 1H-NMR. This is the first report of monohydroxy-carotenoids with an ε-ring and 5,6-epoxy-carotenoids in Bangiales. These results provide new insights into the biosynthetic and metabolic pathways of carotenoids in red seaweeds.

Keywords: Pyropia yezoensis; α-cryptoxanthin; zeinoxanthin; lutein-5,6-epoxide; antheraxanthin; carotenoid synthesis pathway; red seaweed

1. Introduction Carotenoids are yellow-orange tetraterpenoid pigments generated from C4 isoprenoid units. To date, more than 700 carotenoids have been identiﬁed in nature. Carotenoids are synthesized in plants, algae (unicellular type), seaweeds (macrophytic type), bacteria, and fungi, but not animals. In photosynthetic organisms, carotenoids contribute to photosynthesis in light-harvesting antenna complexes in the thylakoid membrane of chloroplasts. They also play an important role in photo-protection from damage by reactive oxygen species under excess light energy [1]. In addition, carotenoids have various health beneﬁts in humans [2]. For example, β-carotene, α-carotene, and β-cryptoxanthin have pro-vitamin A activity [3]. Lutein and zeaxanthin decrease the risk of age-related macular degeneration [4,5].

Mar. Drugs 2018, 16, 426; doi:10.3390/md16110426 www.mdpi.com/journal/marinedrugs Mar. Drugs 2018, 16, 426 2 of 14 Mar. Drugs 2018, 16, x 2 of 14

Pyropia yezoensis,, belongingbelonging toto BangialesBangiales (Rhodophyta),(Rhodophyta), isis oneone ofof the most economically valuable marine foods in EastEast Asia.Asia. In Japan, approximately 300,000–350,000 tons (wet weight) of thalli are produced everyevery yearyear [6[6].]. Moreover,Moreover,P. P. yezoensis yezoensisis is valuable valuable for for studies studies of of seaweed seaweed biology biology owing owing to itsto heteromorphicits heteromorphic life life cycle, cycle, characterized characterized by independentby independent macroscopic macroscopic leafy leafy gametophyte gametophyte (thallus) (thallus) and microscopic,and microscopic, ﬁlamentous filamentous sporophyte sporophyte (conchocelis) (conch stagesocelis) [7 ].stages The genome[7]. The sequence genome of sequenceP. yezoensis ofhas P. beenyezoensis determined has been [ 8determined] and it is readily [8] and cultivated it is readily at acultivated laboratory at scale; a laboratory accordingly, scale; it accordingly, is a useful model it is a organismuseful model for studiesorganism of redfor studies seaweeds of red [9]. seaweeds [9]. In most most red red seaweeds, seaweeds, major major carotenoids carotenoids include include α-carotene,α-carotene, β- carotene,β-carotene, lutein, and lutein, zeaxanthin and zeaxanthin[10–12]. Lutein [10– consists12]. Lutein of two consists hydroxyl of two groups hydroxyl with groups α-carotene with composedα-carotene of composed a β-ring and of a βε-ring,-ring and εthus,-ring, has and an thus, asymmetrical has an asymmetrical structure. In structure. terrestrial In plants, terrestrial lutein plants, is produced lutein isby produced two biosynthetic by two biosyntheticpathways via pathways α-cryptoxanthin via α-cryptoxanthin (β,ε-caroten-3’-ol) (β,ε and-caroten-3’-ol) zeinoxanthin and (β, zeinoxanthinε-caroten-3-ol) (β from,ε-caroten-3-ol) α-carotene fromby caroteneα-carotene hydroxylases, by carotene such hydroxylases, as CYP97A such, CYP97C, as CYP97A, and CYP97C,BHY [13]. and In BHYthe genome [13]. In theof Porphyra genome ofumbilicalisPorphyra, which umbilicalis belongs, which to Bangiales, belongs to CYP97A Bangiales,, CYP97CCYP97A, and, CYP97C BHY genes, and BHYhave genesnot been have found not been[14]. found [14]. PuCHY belonging to the CYP97B subfamily shows β-hydroxylation activity in the PuCHY1 belonging to1 the CYP97B subfamily shows β-hydroxylation activity in the synthesis of synthesiszeaxanthin of from zeaxanthin β-carotene from inβ -carotenePo. umbilicalis in Po. [15]. umbilicalis Moreover,[15 ].intermediate Moreover, intermediate monohydroxy monohydroxy carotenoids carotenoidsin lutein synthesis in lutein from synthesis α-carotene from withα-carotene an ε-ring with are not an εwell-ring known are not (Figure well known 1). These (Figure results1). suggest These resultsthat Bangiales suggest thathave Bangiales characteristic havecharacteristic biosynthetic biosyntheticpathways different pathways from different those from of thoseland ofplants. land plants.Therefore, Therefore, it is interesting it is interesting to investigate to investigate the thelutein lutein synthesis synthesis pathway pathway in inP.P. yezoensis, yezoensis, including intermediateintermediate carotenoids.carotenoids.

Figure 1. Occurrence of carotenoids in Pyropia yezoensisyezoensis..

We recentlyrecently foundfound aa genegene candidatecandidate forfor zeaxanthinzeaxanthin epoxidaseepoxidase (ZEP),(ZEP), whichwhich catalyzescatalyzes the conversion fromfrom zeaxanthin zeaxanthin to violaxanthin,to violaxanthin, in P. yezoensis in P. [yezoensis16]. Abscisic [16]. acid Abscisic (ABA), aacid phytohormone, (ABA), a hasphytohormone, also been detected has also in beenP. yezoensis detected[17 in]. P. Genes yezoensis involved [17]. inGenes ABA involved biosynthesis in ABA have biosynthesis also been found have inalso the been genomes found ofin Bangiales.the genomes ABA of Bangiales. attenuates ABA oxidative attenuates stress oxidative under desiccation stress under in seaweedsdesiccation [18 in]. Theseseaweeds findings [18]. suggestThese findings that intermediates suggest that in intermed the biosyntheticiates in pathwaythe biosynthetic of ABA frompathway zeaxanthin of ABA might from bezeaxanthin found in might Bangiales. be found However, in Bangiales. owing toHowever, the lack owing of information to the lack about of information epoxy-carotenoids, about epoxy- their identificationcarotenoids, their is necessary identification to clarify is necessary the ABA to synthetic clarify the pathway ABA synthetic in this order. pathway in this order. In the the present present study, study, we weanalyzed analyzed intermediate intermediate carotenoids carotenoids to clarify to th clarifye carotenoid the carotenoid synthetic syntheticpathways pathwaysin P. yezoensis in P.. yezoensis Based on. Based the identification on the identification of monohydroxy-carotenoids of monohydroxy-carotenoids and epoxy- and epoxy-carotenoids,carotenoids, we proposed we proposed novel carotenoid novel carotenoid metabolic metabolic pathways pathways in P. yezoensis in P. yezoensis. .

2. Results

2.1. Lutein, Zeaxanthin,Zeaxanthin, and A/B-CaroteneΑ/Β-Carotene Contents in P. yezoensisyezoensis Total lipidslipids werewere extracted from P. yezoensisyezoensis atat thethe conchocelis stage, and major carotenoids were analyzed byby high-performancehigh-performance liquid liquid chromatography chromatography (HPLC) (HPLC) at 450at 450 nm (Figurenm (Figure2). Carotenoids 2). Carotenoids were identiﬁedwere identified by comparing by comparing the HPLC the retentionHPLC retention times and times absorption and absorption spectra with spectra those with of their those authentic of their α β standards.authentic standards. Lutein, zeaxanthin, Lutein, zeaxanthin,-carotene, α and-carotene,-carotene and wereβ-carotene detected were as major detected carotenoids as major in ± thecarotenoids total lipids in the from totalP. yezoensis lipids from. Lutein P. yezoensis was the. mostLutein abundant was the most carotenoid abundant at 3.46 carotenoid0.57 mg/g at 3.46 dry ± ± weight0.57 mg/g and dry 1.44 weight0.03 mg/gand dry1.44 weight± 0.03 inmg/g the cultureddry weight conchocelis in the andcultured the aquacultured conchocelis thallusand the of aquacultured thallus of P. yezoensis, respectively. The contents of these carotenoids were greater in the conchocelis than in the thallus of P. yezoensis (Figure 3).

Mar. Drugs 2018, 16, 426 3 of 14

Mar.P. yezoensis Drugs 2018, respectively., 16, x The contents of these carotenoids were greater in the conchocelis than3 in of the 14 thallusMar. Drugs of 2018P. yezoensis, 16, x (Figure3). 3 of 14

Figure 2. Chromatogram of total lipids extracted from the conchocelis of Pyropia yezoensis . Figure 2. Chromatogram of total lipids extracted from the conchocelis of Pyropia yezoensis. Carotenoids CarotenoidsFigure 2. Chromatogram were measured of usin totalg alipids high-performance extracted from liquid the chconchocelisromatography of Pyropia(HPLC) yezoensissystem . were measured using a high-performance liquid chromatography (HPLC) system equipped with a equippedCarotenoids with were a diode measured array detector using aat high-performance450 nm. Two Develosil liquid C30-UG-5 chromatography columns were(HPLC) connected system diode array detector at 450 nm. Two Develosil C30-UG-5 columns were connected for the analysis. The forequipped the analysis. with Thea diode mobile array phase detector was methanolat 450 nm. fo Twor 20 min,Develosil followed C30-UG-5 by dichloromethane, columns were increasedconnected mobile phase was methanol for 20 min, followed by dichloromethane, increased linearly from 0% to linearlyfor the analysis.from 0% Theto 50% mobile over phase 20 min, was and methanol methanol: for 20Dichloromethane min, followed by (1:1, dichloromethane, v/v) for an additional increased 20 50% over 20 min, and methanol: Dichloromethane (1:1, v/v) for an additional 20 min. The ﬂow rate min.linearly The from flow 0% rate to was50% over1.0 mL/min, 20 min, andand methanol:the sample Dichloromethane injection volume (1:1, was v/v )50 for μ anL. additionala, lutein; b,20 was 1.0 mL/min, and the sample injection volume was 50 µL. a, lutein; b, zeaxanthin; c, α-carotene; zeaxanthin;min. The flow c, α-carotene; rate was d,1.0 β -carotene.mL/min, and the sample injection volume was 50 μL. a, lutein; b, d, β-carotene. zeaxanthin; c, α-carotene; d, β-carotene.

Figure 3. Carotenoid contents in the thallus and conchocelis of Pyropia yezoensis. Values are the Figure 3. Carotenoid contents in the thallus and conchocelis of Pyropia yezoensis. Values are the means means ± SD (n = 3). Asterisks show signiﬁcance by the Student’s t-test (* p < 0.05). ±Figure SD (n =3. 3). Carotenoid Asterisks contentsshow significance in the thallus by the and Student’s conchocelis t-test of (*Pyropia p < 0.05). yezoensis . Values are the means 2.2. Isolation± SD (n = and 3). Asterisks Identiﬁcation show of significance Monohydroxy-Carotenoids by the Student’s int-test P. yezoensis(* p < 0.05). 2.2. Isolation and Identification of Monohydroxy-Carotenoids in P. yezoensis 2.2. IsolationWe observed and Identification a yellow band of Mo (Fractionnohydroxy-Carotenoids A in Figure4a) between in P. yezoensisα/β-carotene and lutein/zeaxanthin on theWe silica observed gel by thina layeryellow chromatography band (Fraction (TLC) A usingin Figure total lipids 4a) frombetween the P. yezoensisα/β-caroteneconchocelis. and lutein/zeaxanthinTo removeWe observed chlorophyll on thea yellow insilica Fraction gel band by Athin eluted(Fraction layer fromchromatography A the in silicaFigure gel (TLC) with4a) usingbetween acetone, total silica αlipids/β-carotene gel from TLC the wasand P. yezoensisperformedlutein/zeaxanthin conchocelis. using ethylon theToacetate: removesilica gel n-hexanechlorophyll by thin layer (6:4, in chromatographyvFraction/v) (Figure A eluted4b). (TLC) Peak from 1,using the peak silica total 2, andgel lipids with peak from acetone, 3 werethe P. silicaseparatedyezoensis gel TLC conchocelis. from was Fraction performed To A remove by using silica chlorophyll gel-HPLCethyl acetate: within Fractionn-hexane n-hexane: A (6:4, eluted Acetone v/v) from(Figure (9:1, thev 4b)./ silicav) (FigurePeak gel 1, with4 peakc). acetone, 2, and peaksilica 3 gel were TLC separated was performed from Fraction using ethylA by acetate:silica gel-HPLC n-hexane with (6:4, n-hexane: v/v) (Figure Acetone 4b). Peak (9:1, 1, v peak/v) (Figure 2, and 4c).peak 3 were separated from Fraction A by silica gel-HPLC with n-hexane: Acetone (9:1, v/v) (Figure 4c).

Mar. Drugs 2018, 16, 426 4 of 14 Mar. Drugs 2018, 16, x 4 of 14

FigureFigure 4. SchemeScheme for for the the separation separation of of monohydroxyl monohydroxyl ca carotenoidsrotenoids in in total total lipids lipids extracted extracted from from the the conchocelisconchocelis of of PyropiaPyropia yezoensis. yezoensis .((a):a )Silica Silica gel gel TLC TLC of of total total lipids lipids extracted extracted from from the the conchocelis conchocelis of P. of yezoensisP. yezoensis; (b;(): bSilica) Silica gel gelTLC TLC of Fraction of Fraction A to A remove to remove the chlorophyll the chlorophyll fraction; fraction; (c): Fractionation (c) Fractionation of peak of 1,peak peak 1, 2, peak and 2,peak and 3 peakin Fraction 3 in Fraction A by an A HPLC by an system HPLC systemequipped equipped with a Mightysil with a Mightysil silica gel silica column gel (250column × 4.6 (250 mm),× 4.6 with mm), a flow with rate a ﬂow of 1.0 rate mL/min of 1.0 mL/min with n-hexane: with n-hexane: acetone acetone (9:1, v/v (9:1,) andv/ detectionv) and detection at 450 nm.at 450 α-Car/ nm. βα-Car:-Car/ αβ-carotene-Car: α-carotene and β-carotene; and β-carotene; Lu/Zx: lu Lu/Zx:tein and lutein zeaxanthin. and zeaxanthin.

PeakPeak 3 3 was was further separated into into peak peak 3-1 3-1 an andd peak 3-2 using the LC-MS system (Shimadzu TripleTriple Quadrupole Mass Spectrometer LCMS8040) LCMS8040) with with an an ODS ODS column column and and methanol methanol as as a a solvent (Figure(Figure 55a).a). TheThe retentionretention times times of of peak peak 3-1 3-1 and and peak peak 3-2 3-2 detected detected at at 445 445 nm nm were were the the same same as as those those of ofthe the zeinoxanthin zeinoxanthin and andβ-cryptoxanthin β-cryptoxanthin standards, standards, respectively respectively (Figure (Figure5a–c). 5a–c). Mass Mass chromatography chromatography at atm/z m/z552.40 552.40 [M] [M]+ also+ also indicated indicated the the same same patterns patternsas asthose those ofof thethe zeinoxanthinzeinoxanthin and β-cryptoxanthin + standards,standards, respectively respectively (Figure 55d–f)).d–f)). Furthermore,Furthermore, productproduct ionsions atat m/z 535.40535.40 [MH-H[MH-H22O]+ werewere not not detecteddetected in in peak peak 3-1 and peak 3-2, consistent with zeinoxanthin and β-cryptoxanthin-cryptoxanthin with with an an OH OH groupgroup on on the β-ring,-ring, but but not not on on the ε-ring-ring.. These These data data indicated indicated that that peak peak 3-1 3-1 and and peak peak 3-2 3-2 were were zeinoxanthinzeinoxanthin and and ββ-cryptoxanthin,-cryptoxanthin, respectively. respectively. Peak 1 (Figure4c) corresponded to a minor carotenoid compared to peak 3, which included zeinoxanthin and β-cryptoxanthin. Therefore, we analyzed the peak by TOF-MS (Waters Acquity LC Xevo G2-S Q TOF Mass Spectrometer) with high sensitivity. An LC-MS analysis of peak 1 showed ions + + at m/z 552.4337 [M] (C40H56O, calc. for 552.4331) and m/z 535.4299 [MH-H2O] as products (Figure6). + UV-VIS wavelengths were 420, 444, and 471 nm (methanol, Table1). In particular, [MH-H 2O] product ions showed higher intensities than those of [M]+ molecular ions. OH group binding at the 30 position of the ε-ring more easily produces –H2O product ions compared with binding to the hydroxylated β-ring in zeinoxanthin and β-cryptoxanthin. Furthermore, 1H-NMR signals indicate that peak 1 is α-cryptoxanthin with an OH group at the allylic position on the ε-end group [19] (Table1).

Mar. Drugs 2018, 16, 426 5 of 14 Mar. Drugs 2018, 16, x 5 of 14

FigureFigure 5. LC-MSLC-MSanalysis analysis of of peak peak 3 separated 3 separated from from the conchocelis the conchocelis of Pyropia of yezoensisPyropia .yezoensis Peak-3 separated. Peak-3 separatedfrom Fraction from A Fraction was evaluated A was byevaluated LC-MS (Shimadzuby LC-MS (Shimadzu LCMS8040) LCMS8040) with an ODS-UG-3 with an columnODS-UG-3 and columnmethanol and as methanol a solvent atas0.1 a solvent mL/min. at Detection0.1 mL/min. at 445Detection nm of (ata) 445 peak nm 3 separated of (a) peak from 3 separated the conchocelis from β theof Pyropia conchocelis yezoensis of ,(Pyropiab) zeinoxanthin yezoensis, standard,(b) zeinoxanthin (c) β-cryptoxanthin standard, ( standard.c) -cryptoxanthin MS chromatogram standard. at MSm/z chromatogram552.40 (black) andat m/zm/z 552.40535.40 (black) (red) and of (d m/z) peak 535.40 3 separated (red) of (d from) peak the 3 conchocelisseparated from of Pyropia the conchocelis yezoensis , β of(e )Pyropia zeinoxanthin yezoensis standard,, (e) zeinoxanthin (f) β-cryptoxanthin standard, ( standard.f) -cryptoxanthin standard.

Peak 1 (Figure 4c) corresponded to a minor carotenoid compared to peak 3, which included zeinoxanthin and β-cryptoxanthin. Therefore, we analyzed the peak by TOF-MS (Waters Acquity LC Xevo G2-S Q TOF Mass Spectrometer) with high sensitivity. An LC-MS analysis of peak 1 showed ions at m/z 552.4337 [M]+ (C40H56O, calc. for 552.4331) and m/z 535.4299 [MH-H2O]+ as products (Figure 6). UV-VIS wavelengths were 420, 444, and 471 nm (methanol, Table 1). In particular, [MH-H2O]+ product ions showed higher intensities than those of [M]+ molecular ions. OH group binding at the 3′ position of the ε-ring more easily produces –H2O product ions compared with binding to the hydroxylated β-ring in zeinoxanthin and β-cryptoxanthin. Furthermore, 1H-NMR signals indicate that peak 1 is α-cryptoxanthin with an OH group at the allylic position on the ε-end group [19] (Table 1).

Mar. Drugs 2018, 16, 426 6 of 14

Mar. Drugs 2018, 16, x 6 of 14

FigureFigure 6. 6.Mass Mass spectrum spectrum ofof peakpeak 1 separated from from the the conchocelis conchocelis of of PyropiaPyropia yezoensis. yezoensis .

We triedTable to identify 1. Spectrum peak data 2 in ofthe peak total 1 isolatedlipids isolated from the from conchocelis P. yezoensis of Pyropia. However, yezoensis it was. difficult to determine the structure owing to the low content of the compound. UV-VIS 420, 444, 471 nm Methanol These results showed that P. yezoensis contains three monohydroxy-carotenoids, i.e., α- 1 cryptoxanthin, β-cryptoxanthin,H-NMR and zeinoxanthin, as intermediateδ ppm carotenoids in the biosynthetic pathways from α-carotene 16/17to lutein or β-carotene to zeaxanthin. 1.03 18 1.73 Table 1. Spectrum19 data of peak 1 isolated from the 1.97 conchocelis of Pyropia yezoensis. 20 1.97 4’UV-VIS 420, 444, 471 nm 5.55 Methanol 17’H-NMR δ ppm 5.43 17’16/17 1.03 0.85 18’18 1.73 1.63 19’19 1.97 1.91 20’20 1.97 1.97 4’ 5.55 7’ 5.43 We tried to identify peak 2 in the total lipids isolated from P. yezoensis. However, it was difficult to 17’ 0.85 determine the structure owing to18’ the low content of the1.63 compound. These results showed that19’ P. yezoensis contains1.91 three monohydroxy-carotenoids, i.e., α-cryptoxanthin, β-cryptoxanthin,20’ and zeinoxanthin, as1.97 intermediate carotenoids in the biosynthetic pathways from α-carotene to lutein or β-carotene to zeaxanthin. 2.3. Isolation and Identification of Epoxy-Carotenoids in P. yezoensis 2.3. Isolation and Identification of Epoxy-Carotenoids in P. yezoensis We evaluated epoxy-carotenoids to reveal novel carotenoid metabolic pathways in P. yezoensis. WeWe extracted evaluated total epoxy-carotenoids lipids from the aquacultured to reveal novel thallus carotenoid of P. yezoensis, metabolic and the pathways concentrated in P. yezoensis polar . Wecarotenoid extracted fraction total lipids (Fraction from B) the was aquacultured expected to thalluscontain ofepoxy-carotenoidsP. yezoensis, and according the concentrated to silica gel polar carotenoidopen column fraction chromatography. (Fraction B) was Furthermore, expected to by contain HPLC epoxy-carotenoids separation using a according C30 column to silica (Develosil gel open columnC30-UG-5, chromatography. two columns Furthermore, were connected by HPLCto enhance separation separation), using apeak C30 4 column and peak (Develosil 5 were isolated C30-UG-5, twofrom columns Fraction were B (Figure connected 7). to enhance separation), peak 4 and peak 5 were isolated from Fraction B (Figure7). Peak 4 had the same HPLC retention time (19.80 min) and maximal absorption wavelength as those of the lutein-5,6-epoxide standard (Figure8). An LC-MS (Shimadzu LCMS8040) analysis of peak 4 showed that the [MH]+ ion and product ion [MH-18]+ at m/z 585.40 and m/z 567.40 were identical to those of the standard. 1H-NMR data indicated that peak 4 is lutein-5,6-epoxide (Table2). The HPLC retention time (22.18 min), maximal absorption wavelength, LC-MS, and 1H-NMR data showed that peak 5 is antheraxanthin (5,6epoxide derived from zeaxanthin) (Figure9, Table2). This is the first identification of lutein-5,6-epoxide and antheraxanthin in Bangiales.

Mar. Drugs 2018, 16, x 7 of 14

Mar. Drugs 2018, 16, 426 7 of 14 Mar. Drugs 2018, 16, x 7 of 14

Figure 7. Purification of peak 4 and peak 5 in Fraction B separated from the thallus of Pyropia yezoensis by the HPLC system. Peak 4 and Peak 5 were isolated by the HPLC system equipped with a diode array detector at 450 nm. Two Develosil C30-UG-5 columns were connected and used for carotenoid isolation. The mobile phase was methanol. The flow rate was 1.0 mL/min.

Peak 4 had the same HPLC retention time (19.80 min) and maximal absorption wavelength as those of the lutein-5,6-epoxide standard (Figure 8). An LC-MS (Shimadzu LCMS8040) analysis of peak 4 showed that the [MH]+ ion and product ion [MH-18]+ at m/z 585.40 and m/z 567.40 were 1 identicalFigure to 7. those PurificationPuriﬁcation of the ofstandard. peak 4 and H-NMR peak 5 indata Fr Fractionaction indicated B separated that peak from 4 the is lutein-5,6-epoxidethallus of Pyropia yezoensis (Table 2). 1 The byHPLC the HPLC retention system. time Peak (22.18 4 and min), Peak maximal 5 were isolated absorption by the wavelength, HPLC system LC-MS, equipped and with H-NMR a diode data showedarray that detector peak at 5 450is antheraxanthin nm. Two Develosil (5,6epoxide C30-UG-5 de columnsrived from were zeaxanthin) connected and (Figure used for 9, carotenoidTable 2). This is theisolation. first identification The mobile phaseof lutein-5,6-epoxide was methanol.methanol. The and ﬂowflow antheraxanthin rate was 1.0 mL/min. in Bangiales.

FigureFigure 8.8. HPLCHPLC andand LC-MSLC-MS analyses of peakpeak4 4 and and lutein-5,6-epoxide lutein-5,6-epoxide..

Figure 8. HPLC and LC-MS analyses of peak4 and lutein-5,6-epoxide.

Mar. Drugs 2018, 16, 426 8 of 14 Mar. Drugs 2018, 16, x 8 of 14

Figure 9. HPLCHPLC and and LC-MS analyses of peak 5 and antheraxanthin antheraxanthin..

1 Table 2. 1H-NMRH-NMR spectral spectral data data of of peak peak 4 and pe peakak 5 isolated from the thallus of Pyropia yezoensis. yezoensis.

Peak 4Peak 4 Peak Peak 5 5 PositionPosition dd mult. mult. J (Hz) J (Hz) d d mult. mult. J (Hz) J(Hz) 22 1.251.25 dd dd (12, (12, 7) 7) 1.25 1.25 dd (12, dd 7) (12, 7) 22 1.631.63 ddd ddd (12, (12, 3, 1.5)3, 1.5) 1.63 1.63 ddd ddd(12, 3, (12, 1.5) 3, 1.5) 33 3.913.91 mm 3.91 3.91 m m 44 1.631.63 dd dd (14, (14, 9) 9) 1.63 1.63 dd (14, dd 9) (14, 9) 44 2.392.39 ddd ddd (14, (14, 5, 1.5)5, 1.5) 2.39 2.39 ddd ddd(14, 5, (14, 1.5) 5, 1.5) 77 5.885.88 d (16)d (16) 5.88 5.88 d (16) d (16) 88 6.296.29 d (16)d (16) 6.29 6.29 d (16) d (16) 1010 6.206.20 d (11)d (11) 6.20 6.20 d (11) d (11) 11 6.61 dd (15, 11) 6.61 dd (15, 11) 11 6.61 dd (15, 11) 6.61 dd (15, 11) 12 6.38 d (15) 6.38 d (15) 12 6.38 d (15) 6.38 d (15) 14 6.25 m 6.25 m 1514 6.636.25 mm 6.25 6.63 m m 1615 0.986.63 sm 6.63 0.98 m s 1716 1.150.98 ss 0.98 1.15 s s 1817 1.191.15 ss 1.15 1.19 s s 1918 1.931.19 ss 1.19 1.93 s s 2019 1.971.93 ss 1.93 1.97 s s 2’ 1.37 dd (13, 7) 1.48 0 20 1.97 s 1.97 s 2 2’ 1.851.37 dd dd (13, (13, 6) 7) 1.48 1.77 30 4.25 m 4.00 m 2′ 1.85 dd (13, 6) 1.77 40 5.55 br. S 2.05 dd (14, 9) 3′ 4.25 m 4.00 m 40 2.39 ddd (14, 5.5, 1.5) 60 4′ 2.405.55 d (9)br. S 2.05 dd (14, 9) 4′ 2.39 ddd (14, 5.5, 1.5) 6′ 2.40 d (9) 7′ 5.43 dd (15.5, 9) 6.10 d (16) 8′ 6.14 d (16) 6.16 d (16) 10′ 6.14 d (10) 6.16 d (11) 11′ 6.62 dd (15, 11) 6.60 dd (15, 11) 12′ 6.36 d (15) 6.35 d (15)

Mar. Drugs 2018, 16, 426 9 of 14

Table 2. Cont.

Peak 4 Peak 5 Position d mult. J (Hz) d mult. J (Hz) 70 5.43 dd (15.5, 9) 6.10 d (16) 80 6.14 d (16) 6.16 d (16) 100 6.14 d (10) 6.16 d (11) 110 6.62 dd (15, 11) 6.60 dd (15, 11) 120 6.36 d (15) 6.35 d (15) 140 6.26 m 6.25 d (11) 150 6.63 m 6.63 m 160 1.00 s 1.08 s 170 0.85 s 1.08 s 180 1.63 s 1.74 s 190 1.91 s 1.93 s 200 1.97 s 1.97 s

3. Discussion Bangiales contains not only β-carotene and its derivatives, but also α-carotene and lutein with an ε-ring [11,15], and these carotenoids have important biological and nutraceutical functions in red seaweeds [20,21]. In the present study, we analyzed the carotenoid profile of one of the most popular edible red seaweeds, P. yezoensis. Lutein was the predominant carotenoid in the conchocelis and thallus of P. yezoensis, similar to other red seaweeds (Figure3). Lutein and zeaxanthin have established health benefits, such as protection against aged-related macular degeneration [4,5]. In addition, the improvement of cognitive function by dietary lutein and zeaxanthin has been reported in a clinical trial [22]. Thus, P. yezoensis containing lutein and zeaxanthin is a highly valuable food with respect to human health. Minor intermediate carotenoids, which are important components of carotenoid biosynthetic pathways, have not been comprehensively identified [11,12]. We identified intermediate carotenoids in P. yezoensis and found that both α-cryptoxanthin and zeinoxanthin, which are monohydroxy-carotenoids, are produced as intermediates in the synthesis of lutein from α-carotene in P. yezoensis (Figure 10). α-Cryptoxanthin has been detected in the red seaweeds Antithamnion plumula (Ceramiales) and Jania rubens (Corallinales) [23,24]. However, α-cryptoxanthin has not been detected in Bangiales to date. To the best of our knowledge, this is the first report of both zeinoxanthin and α-cryptoxanthin in P. yezoensis of Bangiales, as evidenced by LC-MS and 1H-NMR analyses. In higher plants, lutein is synthesized via α-cryptoxanthin and zeinoxanthin from α-carotene [25]. The hydroxylation of α-carotene is catalyzed by nonheme diiron β-hydroxylase (BHY) and heme-containing cytochrome P450-type carotene hydroxylase (CYP97A and C) [26–28]. CYP97C1 (the Lut1 locus) catalyzes the ε-ring hydroxylation of both α-carotene and zeinoxanthin [28], and CYP97A3 catalyzes β-ring hydroxylation in Arabidopsis thaliana [13]. In the liverwort Marchantia polymorpha, CYP97C catalyzes the ε-ring hydroxylation of zeinoxanthin but not α-carotene, and BHY catalyzes β-ring hydroxylation [29]. The differences in lutein biosynthesis among taxa are likely to be associated with differences in the specificity of carotene hydroxylases for β- and ε-rings in the α-carotene molecules. Recently, PuCHY1, which belongs to the CYP97B subfamily, was functionally characterized as a carotene hydroxylase in zeaxanthin synthesis from β-carotene in Po. umbilicalis [15]. It is noteworthy that there are no homologues of CYP97A, CYP97C, and BHY genes in the genomes of Po. umbilicalis [14] and P. yezoensis [unpublished observation]. Therefore, the identification of α-cryptoxanthin and zeinoxanthin together with genome information provides insight into the lutein biosynthetic pathway with unique enzymes in Bangiales. Mar. Drugs 2018, 16, 426 10 of 14 Mar. Drugs 2018, 16, x 10 of 14

Figure 10.10. SummarySummary of of proposed proposed pathways pathways of carotenoid of carotenoid synthesis synthesis and metabolism and metabolism in Pyropia in yezoensis Pyropia. yezoensis. Antheraxanthin and violaxanthin are converted from zeaxanthin in land plants [30]. The reversible conversionRecently, among PuCHY1, these carotenoidswhich belongs in responseto the CYP97B to light subfamily, stress plays was a pivotal functionally role in characterized photoprotection as aand carotene photoabsorption, hydroxylase and in zeaxanthin is referred synthesis to as the xanthophyll from β-carotene cycle in [31 Po.]. umbilicalis The xanthophyll [15]. It is cycle noteworthy requires thattwo there enzymes, are no i.e., homologues zeaxanthin of epoxidaseCYP97A, CYP97C (ZEP),, whichand BHY converts genes in zeaxanthin the genomes to violaxanthinof Po. umbilicalis via [14]antheraxanthin, and P. yezoensis and [unpublished violaxanthin observation]. de-epoxidase, Therefore, which converts the identification violaxanthin of α-cryptoxanthin to zeaxanthin [and30]. Antheraxanthinzeinoxanthin together has been with detected genome in information red seaweeds provides in the ordersinsight Corallinales,into the lutein Ceramiales, biosynthetic and pathwayGracilariales, with within unique the enzymes class Florideophyceae in Bangiales. [11]. However, it has not been detected in Bangiales [11]. In theAntheraxanthin present study, weand evaluatedviolaxanthin epoxy-carotenoids are converted fromfrom zeaxanthinzeaxanthin ininP. land yezoensis plants, because [30]. The we reversiblepreviously conversion found a ZEP-related among these sequence carotenoids in the inP. response yezoensis togenome, light stress as well plays as ABA,a pivotal which role is in a photoprotectiondown-stream product and ofphotoabsorption, violaxanthin [17 ].and Since is it re wasferred difficult to as to detectthe xanthophyll epoxy-carotenoids cycle directly[31]. The in xanthophylltotal lipids extracted cycle requires from P. two yezoensis enzymes,, the polari.e., zeaxan lipid fractionthin epoxidase (Fraction (ZEP), B) containing which converts epoxy-carotenoids zeaxanthin wasto violaxanthin first separated via antheraxanthin, from the total lipids and violaxanth by silica gelin de-epoxidase, open column chromatography.which converts violaxanthin Then, peak to 4 zeaxanthinand peak 5 (Figure[30]. Antheraxanthin7) were isolated has from been the polardetected lipid in fraction red seaweeds by HPLC in and the their orders structures Corallinales, were Ceramiales,determined byand LC-MS Gracilariales, and 1H-NMR within analyses. the class The analyticalFlorideophyceae data demonstrated [11]. However, that peakit has 4 andnot peak been 5 detectedin P. yezoensis in Bangialeslipids were [11]. lutein-5,6-epoxide In the present study, and we antheraxanthin evaluated epoxy-carotenoids with an epoxide from group, zeaxanthin respectively. in P.These yezoensis, results because provide we the previously first evidence found of a theseZEP-related 5,6-epoxy sequence carotenoids in the inP. yezoensisP. yezoensis genome,, belonging as well to asBangiales, ABA, which and provideis a down-stream insight into product the epoxidation of violaxanthin pathways [17]. forSince lutein it was and difficult zeaxanthin to detect (Figure epoxy- 10), carotenoidsalthough its directly activity isinweak total aslipids indicated extracted by the from low P. contents yezoensis of, epoxy-carotenoids.the polar lipid fraction (Fraction B) containingA ZEP-related epoxy-carotenoids sequence haswas been first detected separated in thefrom genomes the total of lipids Bangiales by silica [17]. gel Dautermann open column and chromatography.Lohr [32] reported Then, ZEP enzyme peak 4 activityand peak in 5Madagascaria (Figure 7) were erythrocladioides isolated from(order the Erythropeltidales),polar lipid fraction and by HPLCthe accumulation and their structures of antheraxanthin were determined as a predominant by LC-MS carotenoid and 1H-NMR in a ZEP-deficientanalyses. The tobaccoanalytical mutant. data Ademonstrated distant relationship that peak between 4 and peak the ZEP 5 in proteinP. yezoensis candidates lipids were from lutein-5,6-epoxideP. yezoensis and M. and erythrocladioides antheraxanthinwas withobserved an epoxide in an in silicogroup, analysis. respectively. In addition, These theresult ZEPs proteinprovidecandidate the first evidence in P. yezoensis of theseis not 5,6-epoxy involved carotenoidsin carotenoid in metabolism P. yezoensis, basedbelonging on its to lack Bangiales, of a transit and peptideprovide andinsight the into lack the of epoxy-carotenoidsepoxidation pathways [32]. forHowever, lutein and in the zeaxanthin present study, (Figure we clearly10), although detected its antheraxanthin activity is weak and as lutein-5,6-epoxide indicated by the low in P. contents yezoensis of(Figures epoxy-carotenoids.8 and9). Since antheraxanthin and lutein-5,6-epoxide are derived from zeaxanthin and lutein by ZEP,A ZEP-related our findings sequence provide has evidence been detected for ZEP activityin the genomes in P. yezoensis of Bangiales. Moreover, [17]. theDautermann production and of Lohr [32] reported ZEP enzyme activity in Madagascaria erythrocladioides (order Erythropeltidales), and the accumulation of antheraxanthin as a predominant carotenoid in a ZEP-deficient tobacco

Mar. Drugs 2018, 16, 426 11 of 14 antheraxanthin may be related to violaxanthin synthesis in P. yezoensis (Figure 10). The present results contribute to the elucidation of the biosynthesis of epoxy-carotenoids in the order Bangiales.

4. Materials and Methods

4.1. Chemicals α-Carotene, β-carotene, β-cryptoxanthin, lutein, zeaxanthin, antheraxanthin, and lutein-5,6-epoxide were purchased from Carote Nature GmbH (Münsingen, Switzerland). Zeinoxanthin used for standard was synthesized by Wittig condensation of C25-apocarotenal derived from commercially available α-ionone, with previously prepared C15-phosphonium salt possessing 3-hyxroxy-β-end group [33]. 1H NMR data of synthetic zeinoxanthin were identical with those reported in Reference [34].

4.2. Seaweed Materials Thalli and conchocelis filaments of P. yezoensis strain U-51 were used in the present study. Thalli maricultured at Shichigahama (Miyagi, Japan) were frozen and kept under −20 ◦C until extraction of total lipid. Conchocelis materials were prepared by laboratory culture in ESL medium [35] at 15 ◦C, under irradiation of 40 µmol photons m−2 s−1 provided by cool white fluorescent lamps with a photoperiod of 10 h light/14 h dark. The medium was bubbled continuously with filter-sterilized air and changed weekly. We used approximately 40–80 mg of conchocelis stage of P. yezoensis during cultivation for total lipid extraction.

4.3. Extraction of Total Lipids from P. yezoensis Total lipid was extracted from conchocelis and thallus of P. yezoensis with 20-fold methanol (v/w) for 24 h at room temperature under shading conditions. The extraction with methanol was conducted twice. Then, methanol was evaporated, and the extracts was solved in chloroform-methanol-water (10:5:3, v/v/v) to remove water soluble components. The total lipid fraction containing carotenoids was obtained from chloroform layer.

4.4. Lutein, Zeaxanthin, A/B-Carotene Contents in P. yezoensis Carotenoids content in P. yezoensis were measured by a high-performance liquid chromatography (HPLC) system (Hitachi, Tokyo, Japan) equipped with a diode array detector (Hitachi L2455, Tokyo, Japan). Two Develosil C30-UG-5 (Nomura Chemical Co., Aichi, Japan) columns were connected and used for carotenoid analysis. Detection was set at 450 nm. Mobile phase was methanol until 20 min, and thereafter, dichloromethane content increased linearly from 0% to 50% in 20 min, then was held at methanol:dichloromethane (1:1, v/v) for additional 20 min. The ﬂow rate was maintained at 1.0 mL/min, and sample injection volume was 50 µL. α-Carotene, β-carotene, lutein, and zeaxanthin were identiﬁed by comparison of retention time and absorption spectra of HPLC analysis with their authentic standards. The contents of α-carotene, β-carotene, lutein, and zeaxanthin in P. yezoensis were calculated using a calibration curve prepared by each authentic standard.

4.5. Isolation of Monohydroxy Carotenoids The total lipid of P. yezoensis was developed on a silica gel TLC plate (Merk KGaA, Darnstadt, Germany) with petroleum ether:acetone (7:3, v/v) or n-hexane:acetone (7:3, v/v). Three pigment fractions with yellow-orange color were observed on the TLC plate. The yellow pigment fraction with relative front (Rf) value 0.61 between strong two yellow-orange fractions with Rf value 0.92 (α/β-carotene) and 0.42 (lutein/zeaxanthin) was scrapped off from the TLC plate developed with petroleum ether:acetone (7:3, v/v), and extracted with acetone. Separation of the yellow pigment fraction (Fraction A) was conducted by silica gel TLC plate with ethyl acetate:n-hexane (6:4, v/v) to remove remaining chlorophyll. Then, peak 1, peak 2, and peak 3 were separated from Fraction A by Mar. Drugs 2018, 16, 426 12 of 14

HPLC system (Hitachi, Tokyo, Japan) equipped with Mightysil silica gel column (Kanto Chemical Co., Tokyo, Japan, 250 × 4.6 mm), ﬂow rate: 1.0 mL/min with n-hexane:acetone (9:1, v/v), detection at 450 nm. Peak 3 was further fractionated and analyzed using an LC-MS system (Shimadzu Triple Quadrupole mass spectrometer LCMS8040, Shimadzu, Kyoto, Japan), equipped with ODS column (Develosil ODS-UG-3 150 × 2.0 mm, Nomura Chemical Co., Aichi, Japan). Methanol was used as a mobile phase at a ﬂow rate of 0.1 mL/min.

4.6. Isolation of Epoxy-Carotenoids The total lipid extracted from aquacultured thallus of P. yezoensis was separated silica gel column by n-hexane:acetone (7:3, v/v). Four fractions [strong yellow layer (α/β-carotene fraction), yellow-green layer (α/β-cryptoxanthin, zeinoxanthin and chrolophyll faction), strong orange layer (lutein/zeaxanthin fraction), and light-yellow layer (Fraction B)] were eluted in order. Then, peak 4 and peak 5 were isolated from Fraction B (light yellow layer) using an HPLC system (Hitachi, Tokyo, Japan) equipped with a Develosil C30-UG-5 column (250 × 4.6 mm, two columns were connected), ﬂow rate: 1.0 mL/min with methanol, detection at 450 nm.

4.7. Identiﬁcation of Monohydroxy- and Epoxy-Carotenoids in P. yezoensis The chemical structures of peaks 1–5 were determined by LC-MS and 1H-NMR analyses. LC-MS analysis was carried out using a Shimadzu Triple Quadrupole mass spectrometer LCMS8040, as described in 2.6 or a Waters Xevo G2S Q TOF mass spectrometer (Waters Corporation, Milford, CT, USA), equipped with an Acquity UPLC system with Acquity 1.7 µm BEH UPLC C18 column (100 × 2.1 mm) (Waters Corporation, Milford, CT, USA), and methanol as a mobile phase at a ﬂow rate 1 of 0.4 mL/min. H-NMR (500 MHz) spectra in CDCl3 were measured with an UNITY INOVA-500 system (Varian Corporation, Palo Alto, CA, USA).

4.8. Statistical Analysis Analytical data of carotenoid content (Figure3) were expressed as the mean ± standard deviation (SD). Statistical significance was determined between the two groups using the Student’s t-test. A significant difference was defined at p < 0.05.

5. Conclusions The results of the present study demonstrated that P. yezoensis synthesizes zeinoxanthin and α-cryptoxanthin, as well as β-cryptoxanthin, as intermediate monohydroxy-carotenoids. These results indicate that P. yezoensis has two lutein biosynthetic pathways via zeinoxanthin and α-cryptoxanthin from α-carotene (Figure 10). Furthermore, antheraxanthin and lutein-5,6-epoxide were found for the ﬁrst time in Bangiales (Figure 10). In particular, antheraxanthin is considered an intermediate carotenoid in the conversion from zeaxanthin to violaxanthin. The carotenoid proﬁle of P. yezoensis provides new insight into the biosynthetic and metabolic pathways of carotenoids in red seaweeds.

Author Contributions: M.H. conceived and designed the experiments and wrote the paper; K.M. (Koji Mikami) designed the experiments and reviewed the manuscript. J.K., N.T., and N.K. performed the experiments; T.M. analyzed NMR data for isolated carotenoids; Y.Y. and A.W. synthesized standard carotenoids and evaluated the data for carotenoid identification; K.M. (Kazuo Miyashita) reviewed the manuscript. Funding: This research was funded by The Towa Foundation for Food Science & Research. Acknowledgments: We thank the Marine Resources Research Center of Aichi Fisheries Research Institute (Gamagori-shi, Aichi, Japan) for providing the P. yezoensis strain U-51. We are grateful to the Shichigahama branch office of the Miyagi Prefecture Fisheries Cooperative Association for kindly providing raw maricultured P. yezoensis thalli. Conflicts of Interest: The authors declare no conflict of interest. Mar. Drugs 2018, 16, 426 13 of 14

References

1. Nisar, N.; Li, L.; Lu, S.; Khin, N.C.; Pogson, B.J. Carotenoid metabolism in plants. Mol. Plant 2015, 8, 68–82. [CrossRef][PubMed] 2. Eggersdorfer, M.; Wyss, A. Carotenoids in human nutrition and health. Arch. Biochem. Biophys. 2018, 652, 18–26. [CrossRef][PubMed] 3. Castenmiller, J.J.; West, C.E. Bioavailability and bioconversion of carotenoids. Annu. Rev. Nutr. 1998, 18, 19–38. [CrossRef][PubMed] 4. Johnson, E.J. The role of carotenoids in human health. Nutr. Clin. Care 2002, 5, 56–65. [CrossRef][PubMed] 5. Fernández-Sevilla, J.M.; Acién Fernández, F.G.; Molina Grima, E. Biotechnological production of lutein and its applications. Appl. Microbiol. Biotechnol. 2010, 86, 27–40. [CrossRef][PubMed] 6. E-Stat, Portal Site of Official Statistics of Japan. Available online: https://www.e-stat.go.jp/ (accessed on 1 November 2018). 7. Drew, K.M. Life-history of Porphyra. Nature 1954, 173, 1243–1244. [CrossRef] 8. Nakamura, Y.; Sasaki, N.; Kobayashi, M.; Ojima, N.; Yasuike, M.; Shigenobu, Y.; Satomi, M.; Fukuma, Y.; Shiwaku, K.; Tsujimoto, A.; et al. The first symbiont-free genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis). PLoS ONE 2013, 8, e57122. [CrossRef][PubMed] 9. Sahoo, D.; Tang, X.R.; Yarish, C. Porphyra—The economic seaweed as a new experimental system. Curr. Sci. 2002, 83, 1313–1316. 10. Takaichi, S. Carotenoids in algae: Distributions, biosyntheses and functions. Mar. Drugs 2011, 9, 1101–1118. [CrossRef][PubMed] 11. Takaichi, S.; Yokoyama, A.; Mochimaru, M.; Uchida, H.; Murakami, A. Carotenogenesis diversification in phylogenetic lineages of Rhodophyta. J. Phycol. 2016, 52, 329–338. [CrossRef][PubMed] 12. Schubert, N.; Garcia-Mendoza, E.; Pacheco-Ruiz, I. Carotenoid composition of marine red algae. J. Phycol. 2006, 42, 1208–1216. [CrossRef] 13. Kim, J.; Smith, J.J.; Tian, L.; DellaPenna, D. The evolution and function of carotenoid hydroxylases in Arabidopsis. Plant Cell Physiol. 2009, 50, 463–479. [CrossRef][PubMed] 14. Brawley, S.H.; Blouin, N.A.; Ficko-Blean, E.; Wheeler, G.L.; Lohr, M.; Goodson, H.V.; Jenkins, J.W.; Blaby-Haas, C.E.; Helliwell, K.E.; Chan, C.X.; et al. Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta). Proc. Natl. Acad. Sci. USA 2017, 114, E6361–E6370. [CrossRef][PubMed] 15. Yang, L.E.; Huang, X.Q.; Hang, Y.; Deng, Y.Y.; Lu, Q.Q.; Lu, S. The P450-type carotene hydroxylase PuCHY1 from Porphyra suggests the evolution of carotenoid metabolism in red algae. J. Integr. Plant Biol. 2014, 56, 902–915. [CrossRef][PubMed] 16. Mikami, K.; Hosokawa, M. Biosynthetic pathway and health benefits of fucoxanthin, an algae-specific xanthophyll in brown seaweeds. Int. J. Mol. Sci. 2013, 14, 13763–13781. [CrossRef][PubMed] 17. Mikami, K.; Mori, I.C.; Matsuura, T.; Ikeda, Y.; Kojima, M.; Sakakibara, H.; Hirayama, T. Comprehensive quantification and genome survey reveal the presence of novel phytohormone action modes in red seaweeds. J. Appl. Phycol. 2016, 28, 2539–2548. [CrossRef] 18. Guajardo, E.; Correa, J.A.; Contreras-Porcia, L. Role of abscisic acid (ABA) in activating antioxidant tolerance responses to desiccation stress in intertidal seaweed species. Planta 2016, 243, 767–781. [CrossRef][PubMed] 19. Englert, G. NMR spectroscopy. In Carotenoids; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser: Basel, Switzerland, 1995; Volume 1B, pp. 147–260. 20. Chan, C.X.; Blouin, N.A.; Zhuang, Y.; Zäuner, S.; Prochnik, S.E.; Lindquist, E.; Lin, S.; Benning, C.; Lohr, M.; Yarish, C.; et al. Porphyra (Bangiophyceae) transcriptomes provide insights into red algal development and metabolism. J. Phycol. 2012, 48, 1328–1342. [CrossRef][PubMed] 21. Christaki, E.; Bonos, E.; Giannenas, I.; Florou-Paneri, P. Functional properties of carotenoids originating from algae. J. Sci. Food Agric. 2013, 93, 5–11. [CrossRef][PubMed] 22. Hammond, B.R., Jr.; Miller, L.S.; Bello, M.O.; Lindbergh, C.A.; Mewborn, C.; Renzi-Hammond, L.M. Effects of lutein/zeaxanthin supplementation on the cognitive function of community dwelling older adults: A randomized, double-masked, placebo-controlled trial. Front. Aging Neurosci. 2017, 9, 254. [CrossRef] [PubMed] Mar. Drugs 2018, 16, 426 14 of 14

23. Bjørnland, T.; Borch, G.; Liaaen-Jensen, S. Configurational studies on red algae carotenoids. Phytochemistry 1984, 23, 1711–1715. [CrossRef] 24. Hegazi, M.M.; Pérez-Ruzafa, A.; Almela, L.; Candela, M.E. Separation and identification of chlorophylls and carotenoids from Caulerpa prolifera, Jania rubens and Padina pavonica by reversed-phase high-performance liquid chromatography. J. Chromatogr. 1998, 829, 153–159. [CrossRef] 25. Quinlan, R.F.; Shumskaya, M.; Bradbury, L.M.; Beltrán, J.; Ma, C.; Kennelly, E.J.; Wurtzel, E.T. Synergistic interactions between carotene ring hydroxylases drive lutein formation in plant carotenoid biosynthesis. Plant Physiol. 2012, 160, 204–214. [CrossRef][PubMed] 26. Quinlan, R.F.; Jaradat, T.T.; Wurtzel, E.T. Escherichia coli as a platform for functional expression of plant P450 carotene hydroxylases. Arch. Biochem. Biophys. 2007, 458, 146–157. [CrossRef][PubMed] 27. Li, Q.; Farre, G.; Naqvi, S.; Breitenbach, J.; Sanahuja, G.; Bai, C.; Sandmann, G.; Capell, T.; Christou, P.; Zhu, C. Cloning and functional characterization of the maize carotenoid isomerase and β-carotene hydroxylase genes and their regulation during endosperm maturation. Transgenic Res. 2010, 19, 1053–1568. [CrossRef] [PubMed] 28. Tian, L.; Musetti, V.; Kim, J.; Magallanes-Lundback, M.; DellaPenna, D. The Arabidopsis LUT1 locus encodes a member of the cytochrome p450 family that is required for carotenoid epsilon-ring hydroxylation activity. Proc. Natl. Acad. Sci. USA 2004, 101, 402–407. [CrossRef][PubMed] 29. Takemura, M.; Maoka, T.; Misawa, N. Biosynthetic routes of hydroxylated carotenoids (xanthophylls) in Marchantia polymorpha, and production of novel and rare xanthophylls through pathway engineering in Escherichia coli. Planta 2015, 241, 699–710. [CrossRef][PubMed] 30. Hieber, A.D.; Bugos, R.C.; Yamamoto, H.Y. Plant lipocalins: Violaxanthin de-epoxidase and zeaxanthin epoxidase. Biochim. Biophys. Acta 2000, 1482, 84–91. [CrossRef] 31. Niyogi, K.K.; Grossman, A.R.; Björkman, O. Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. Plant Cell 1988, 10, 1121–1134. [CrossRef] 32. Dautermann, O.; Lohr, M. A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light-harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes. Plant J. 2017, 92, 879–891. [CrossRef][PubMed] 33. Yamano, Y.; Ito, M. Total synthesis of capsanthin and capsorubin using Lewis acid-promoted regio- and stereoselective rearrangement of tetrasubsutituted epoxides. Org. Biomol. Chem. 2007, 5, 3207–3212. [CrossRef][PubMed] 34. Khachik, F.; Chan, A.-N.; Gana, A.; Mazzola, E. Partial synthesis of (3R,60R)-α-cryptoxanthin and (3R)-β-cryptoxanthin from (3R,30R,60R)-lutein. J. Nat. Prod. 2007, 70, 220–226. [CrossRef][PubMed] 35. Kitade, Y.; Fukuda, S.; Nakajima, M.; Watanabe, T.; Saga, N. Isolation of a cDNA encoding a homologue of actin from Porphyra yezoensis (Rhodophyta). J. Appl. Phycol. 2002, 14, 135–141. [CrossRef]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).