Reactive oxygen species (ROS) play an important role in the pathogenesis of many human degenerative diseases such as cancer, aging, arteriosclerosis, and rheumatism. Much attention has been focused on the development of safe and effective antioxidants. To discover sources of antioxidative activity in marine algae, extracts from 17 kinds of seaweed were screened for their inhibitory effect on total ROS generation in kidney homogenate using 2',7'-dichlorofluorescein diacetate (DCFH-DA). ROS inhibition was seen in three species: Ulva pertusa, Symphyocladia latiuscula, and Ecklonia stolonifera. At a final concentration of 25 microg/mL, U. pertusa inhibited 85.65+/-20.28% of total ROS generation, S. latiscula caused 50.63+/-0.09% inhibitory, and the Ecklonia species was 44.30+/-7.33% inhibition. E. stolonifera Okamura (Laminariaceae), which belongs to the brown algae, has been further investigated because it is commonly used as a foodstuff in Korea. Five compounds, phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5), isolated from the ethyl acetate soluble fraction of the methanolic extract of E. stolonifera inhibited total ROS generation.

Extracts from seventeen seaweeds were determined for tyrosinase inhibitory activity using mushroom tyrosinase with L-tyrosine as a substrate. Only one of them, Ecklonia stolonifera OKAMURA (Laminariaceae) belonging to brown algae, showed high tyrosinase inhibitory activity. Bioassay-guided fractionation of the active ethyl acetate (EtOAc) soluble fraction from the methanolic extract of E. stolonifera, led us to the isolation of phloroglucinol derivatives [phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5)]. Compounds 1 approximately 5 were found to inhibit the oxidation of L-tyrosine catalyzed by mushroom tyrosinase with IC50 values of 92.8, 126, 33.2, 177, and 2.16 microg/mL, respectively. It was compared with those of kojic acid and arbutin, well-known tyrosinase inhibitors, with IC50 values of 6.32 and 112 microg/ mL, respectively. The inhibitory kinetics analyzed from Lineweaver-Burk plots, showed compounds 1 and 2 to be competitive inhibitors with Ki of 2.3x10(-4) and 3.1x10(-4) M, and compounds 3 approximately 5 to be noncompetitive inhibitors with Ki of 1.9x10(-5), 1.4x10(-3) and 1.5x10(-5) M, respectively. This work showed that phloroglucinol derivatives, natural compounds found in brown algae, could be involved in the control of pigmentation in plants and other organisms through inhibition of tyrosinase activity using L-tyrosine as a substrate.

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Ecklonia stolonifera OKAMURA is a member of the family of Laminariaceae, belonging to the order Laminariales as a perennial brown alga. The previous phytochemical investigations performed on this species resulted in the isolation of phloroglucinol, 1) phlorotannins 2) and ecklonialactones. 3,4) In the course of a continuous study on the active principles of this alga, we isolated a new phlorotannin with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, along with three known ones, of the methanolic extract of E. stolonifera. Column chromatography of the EtOAc soluble part from the methanolic extract of this alga yielded four phlorotannins, compounds 1-4 in the order of increasing polarity. The structures of 2, 3, and 4 were identified by comparison with published spectral data as eckol, phlorofucofuroeckol A, and dieckol, respectively (Fig. 1). [5][6][7][8] Compound 1 was obtained as off-white amorphous powder. The molecular formula of 1 was determined as C 18 . The carbon-13 nuclear magnetic resonance ( 13 C-NMR) spectrum of 1 indicated the presence of five non-substituted and thirteen O-bearing aromatic carbons, whereas the proton nuclear magnetic resonance ( 1 H-NMR) spectrum contained signals characteristic of five aromatic protons, i.e. two AB systems at d 6.04 (1H, Jϭ2.7 Hz) and 5.82 (1H, Jϭ2.7 Hz), and d 6.01 (1H, Jϭ2.7 Hz) and 5.84 (1H, Jϭ2.7 Hz), and a singlet at 6.10 (1H) as well as five singlets indicating phenolic hydroxy protons at d 9.77, 9.64, 9.60, 9.27, and 9.26. These NMR spectral features are very similar to those of eckol (2) isolated from Eisenia bicyclis and Ecklonia kurome, 5,6) indicating that 1 is composed of three phloroglucinol units. The only difference between the 1 H-NMR spectra of 1 and 2 is that the former lacks the signals for one phenolic hydroxyl proton and one aromatic proton, suggesting that 1 has an additional aryl-ether linkage. This was supported by the presence of a new oxygen-bearing carbon signal (d 122.7), which is characteristic of an aromatic carbon with two oxygenated neighbors, and also by the formation of a pentaacetate (1a) on usual acetylation. Analysis of HMQC, HMBC and NOESY spectra of 1 allowed an unambiguous assignment of all the proton and carbon signals (Table 1, Fig. 2). In the HMBC spectrum, each cross peak between d 9.77 and C-1 (d 146.1), C-2 (d 98.8), and C-14a , and C-10 (d 98.8) designated the existence of the hydroxyl groups at C-11 and C-9, respectively. Each cross peak between d 9.60 and C-5a (d 125.9), C-6 (d 140.1), and C-7 (d 97.6), established the presence of the hydroxyl group at C-6. The stereostructure of compound 1 was deduced to be planar and achiral by its specific rotation and a loss of additional anisotropic effect for the aromatic protons. It was found that specific rotation showed zero value and the similar chemical shifts not only between H-2 and H-10, but also between H-4 and H-12. Consequently, the structure of 1 was established as 5,8,13,14-tetraoxa-pentaphene-1,3,6,9,11-pentaol, named eckstolonol. Compounds 1-...

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