Mar. Drugs 2016, 14, 93
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mytiloxanthin was due to the presence of the long conjugated polyene system described above.
Mytiloxanthin has an enolic -diketone group. Along with the 3-hydroxy-4-keto- -end group in
astaxanthin, this enolic β-diketone group may contribute to anti-oxidative activity.
Many crustaceans oxidatively convert dietary -carotene to astaxanthin [ ]. The scallop and sea
angel also oxidatively convert dietary diatoxanthin to pectenolone [ 16]. By these oxidative metabolic
β
β
β
,
conversions, the anti-oxidative activities of dietary carotenoids are increased. Therefore, these marine
animals metabolize dietary carotenoids to a more active anti-oxidative form and accumulate them in
their bodies and gonads.
Similarly, shellfish and tunicates accumulate fucoxanthin from dietary algae and convert it to
mytiloxanthin. By this conversion, the carotenoid changes color from orange to red and shows
increased anti-oxidative activities, as described above. Mytiloxanthin in shellfish and tunicates may
contribute to protection against oxidative stress and promote reproduction, similarly to astaxanthin in
crustaceans and pectenolonein in the scallop and sea angel.
3. Experimental Section
3.1. Reagents
Hematoporphyrin, riboflavin, and hydrogen peroxide were purchased from Wako Pure Chemicals
(Osaka, Japan); 2,2,6,6-Tetramethyl-4-piperidone (TMPD) was purchased from Aldrich (Milwaukee,
WI, USA); 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from Labotec (Tokyo, Japan);
2,21-Azobis(2,4-dimethylvaleronitrile) (AMVN) as purchased from Wako Pure Chemicals (Osaka,
Japan). Methyl linolate, β-carotene, and astaxanthin were purchased from Aldrich (Milwaukee, WI,
USA). Fucoxanthin was prepared from brown algae. Mytiloxanthin was synthesized as described
below. Figure 1 shows structures of carotenoids used in this study.
3.2. ESR Spin-Trapping Analysis
ESR spectra were recorded at room temperature on a JEOL JES-FR30 spectrometer (JEOL, Tokyo,
Japan) using an aqueous quartz flat cell (Labotec, Tokyo, Japan). TMPD was used as a singlet
oxygen-trapping agent, and DMPO was used as superoxide anion and hydroxyl radicals, respectively.
Superoxide anion radical (O2´) and OH were generated by addition of both the 100
riboflavin, or 100 L of 8 mM H2O2 solution and 10 L of 250 mM DMPO to 100
carotenoid CH3CN solution by UV-A irradiation. In a similar manner described above, O2 was
generated by the addition of both the 100 L of 0.25 mM hematoporphyrin and 10 L of 500 mM
TMPD to 100 L of 8.8 g/mL of carotenoid CH3CN solution by UV-A irradiation. ESR spectra
µL of 25
µM of
µ
µ
µ
L of 8.8
µg/mL
1
µ
µ
µ
µ
were started simultaneously to measure after UV-A irradiation. The all spin-trapped ESR spectra
were monitored between the third and fourth signals from the low magnetic field due to the external
standard, Mn(II)-doped MnO.
3.3. Inhibition of Lipid Peroxidation
Carotenoids were dissolved in EtOH at a concentration of 2 mM (final concentration of 167 µM
in the reaction mixture). The sample solution, 100
µL, was added to 1 mL of 100 mM methyl
linolate solution [n-hexane/2-propanol (1:1, v/v)], and the solution was incubated at 37 ˝C for 5 min.
As a control, EtOH alone was used instead of the sample solution. The oxidation reaction was
then performed by adding 100 µL of 100 mM n-hexane solution of AMVN and the mixture was
incubated with air at 37˝C. At regular intervals, the oxidation reaction products, methyl linolate
hydroperoxides, were quantified by high performance liquid chromatography (HPLC). HPLC was
performed with a Hitachi L-6000 intelligent pump and an L-4250 UV-VIS detector. The following
HPLC conditions were employed for the quantitative analysis of methyl linolate hydroperoxides:
column Lichrosorb Si 100 (5
system: 2-propanol/n-hexane (1:99, v/v); flow rate: 1 mL/min; and detection: 235 nm.
µ
m particle size) (4.6
ˆ
250 mm) (Merck, Damstraat, Germany); solvent
Maoka, Takashi
