Fucoxanthin and Its Metabolites in Edible Brown Algae Cultivated in Deep Seawater

Three metabolites of fucoxanthin were isolated from a brown alga, Scytosiphon lomentaria, and the structure of a new compound was determined by NMR. The content of fucoxanthin, a biologically active carotenoid, in four edible brown algae, cultivated in deep seawater, was studied.


Introduction
Deep seawater (DSW) is attracting biological and industrial interest because of the following advantages: (1) abundance of mineral nutrients, (2) cleanness (pathogen-and pollution-free), and Mar. Drugs 2004, 2

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(3) low temperature convenient for aquaculture. Kochi Prefectural DSW Laboratory is supplying about 1000 tons of DSW per day, and the water has been industrialized as a material for cosmetics, beverages, and foods such as miso (fermented soybean paste), tofu (bean curd), and dried fish.
Recently, the laboratory has started a new project that aims to use DSW as a cultivation medium for edible seaweeds based on the above-mentioned merits. Actually, it turned out that a green alga, Enteromorpha plolifera (Müller) J. Agardh, which is a valuable seafood in Japan, grew rapidly in the tank supplied DSW throughout the year. The average daily growth rate was 40% [1]. Mass production of this alga (3 ton dry weight per year) is in progress at the East Muroto Fishery Cooperative in Kochi Prefecture.
During the course of our study to obtain pharmacologically active compounds from the algae that are cultivated in DSW, we focused our attention on fucoxanthin (FUCOX) (1) [2], a carotenoid commonly distributed in brown algae. FUCOX is a potent drug candidate and can be utilized as an excellent supplement like astaxanthin [3], since it acts as an antioxidant [4] and inhibits GOTO cells of neuroblastoma and colon cancer cells [5]. Recently, the apoptosis activity against HL-60 and Caco-2 cells has been reported for FUCOX [6]. This paper deals with the content of FUCOX in four brown algae cultivated in DSW in addition to the structure determination of a new compound (2) isolated from Scytosiphon lomentaria (Lingbye) Link.

Results and Discussion
At first, a brown alga, Scytosiphon lomentaria, cultivated in DSW, was selected as a model for analyzing the content of FUCOX (1). In order to obtain pure 1, the dichloromethane extract was subjected to flash column chromatography followed by preparative TLC and HPLC. By these procedures, pure 1 was obtained in 0.0081% yield from the crude extract together with compounds 2, 3, and 4. The identity of FUCOX was established by comparing its 1 H-and 13 C-NMR properties with those reported in the literature [2]. The structures of compounds 2, 3, and 4 will be

a) Content of FUCOX in the brown algae cultivated in DSW
The isolation yield of FUCOX (1) described above does not reflect the actual content of 1 in S. lomentaria because of the loss sustained during the prolonged isolation procedure. For the purpose of determining the exact content of 1, the methanol (MeOH) extract of S. lomentaria was subjected to HPLC and UV analyses. The HPLC (Mightsil ® RP-18, 5 µm, 250 x 20 mm; 450 nm; MeOH-H 2 O= 95:5) showed many peaks, a few of which were overlapped with the signal due to 1.
The UV spectrum showed a maximum at 450 nm ascribable to 1. This band, however, was obscured by those of concomitant chlorophylls. Therefore, we devised a quick and simple pretreatment method for separating 1 in the following manner.
The algal body of S. lomentaria was cut with a razor into pieces, which were stored in MeOH (30 mL/1 g wet body) in the dark for two days in a stoppered 50 mL Erlenmeyer flask (Extraction for 4 and 5 days gave the same result.). A 1.0 mL aliquot of the supernatant was taken out and diluted with distilled water (1.0 mL), and the mixture was charged on the top of Sep-Pak ® Vac C18 (Waters, 1 cc), which had been washed with MeOH (2 mL) and then MeOH-H 2 O (1:1) (2 mL).
The HPLC (Mightsil ® RP-18 GP, 3 µm, 250 x 4.6 mm; 450 nm; MeOH) also showed a single peak due to 1. The eluate (fraction 3) was diluted to 5.0 mL with MeOH-H 2 O (9:1), and the solution was analyzed by UV spectroscopy. The E value (1%, 1 cm) of 1197 at 451 nm determined for authentic FUCOX in MeOH-H 2 O (9:1) was used to calculate the amount of 1. The same experiment was performed three times, and the mean value was expressed as the weight of 1 [mg/g (wet algal body)]. In the case of S. lomentaria, the content of 1 was analyzed to be 0.241±0.005 mg/g. This value did not change significantly when other parts of the algal body were selected.  It can be seen that the content of FUCOX in the young thallus (5-10 cm long) algae (S1, L1, P1, and U1 in Fig. 1) is in the range 0.24 -0.43 mg/g and is not so different among the species. It is interesting that the content found in commercial dried U. pinnatifida (U2) is the same as that in the raw material (U1). This indicates that FUCOX is quite stable when mixed with the organic ingredients and survives the drying process and storage at ambient temperature, while pure FUCOX is susceptible to air oxidation. It is remarkable that the younger stages of the algae contain a higher content of FUCOX, S2/S1 = 2. Compounds 3 and 4 were identified as apo-9'-fucoxanthinone [7] and apo-13'-fucoxanthinone [8], respectively, by comparing their 1 H-and 13 C-NMR properties with those reported in the literature. The deacetyl derivative of compound 3 was first isolated as an allelopathic substance from a grasshopper [9] and is reported to exhibit cytotoxicity against murine lymphoma L-1210 and human epidoemoid carcinoma KB cells [7]. Compound 4 has been obtained from marine diatoms, and it shows a feeding deterrent effect in a copepod [8].  8 Hz,. From the triplet pattern of the methine signal at δ 3.81 (tt, J=4. 8,9.1 Hz,, it was deduced that the oxymethine proton was axial, and, the orientation of the protons at 1.36 and 1.69 is axial, and that at UV-VIS spectra were measured on a Beckman DU-650 spectrophotometer.

Mass culture and harvest of Scytosiphon lomentaria
Scytosiphon lomentaria was collected at Aikappu in Akkeshi, Hokkaido on 1 July 1989 by Dr.
Kogame, Hokkaido University. The prostrate-branched filaments were kept as unialgal culture at the laboratory of Kochi Prefectural Deep Seawater Laboratory. In order to induce the erect thalli, the filaments were cultured as free-floating form in glass vessels containing 500 mL of PES medium with continuous aeration at 10 °C in short day length (10:14 h Light:Dark cycle) with white fluorescent light of 40-60 µmol m -2 s -1 . Several erect thallus germlings occurred in a filament and became 'germling clusters', each of which contained 10-100 germlings. When the germling clusters grew to more than 5 mm diameter, they were transplanted as free-floating forms to an outdoor tank and cultured with continuous aeration and deep seawater supply at a rate of 3 volume exchanges per day.
Other brown algae were cultivated essentially in the same manner.
The whole plant was immediately soaked in MeOH and extracted at rt for 4 d. The MeOH extract was concentrated under a reduced pressure to give a residue. The residue was treated with H 2 O.