Fisheries Science 62(6), 923-926 (1996)

Antioxidant Activity of Phlorotannins Isolated

from the Brown Alga Eisenia bicyclis*l

Takashi Nakamura*2, Kohki Nagavama, Kenii Uchida. and Rvusuke Tanaka

Laboratory of Fisheries Technology, Faculty of Agriculture ,

Kyushu University, Hakozaki, Fukuoka 812, Japan

(Received October 18, 1995)

Five antioxidative substances in the brown alga Eisenia bicyclis were isolated by silicic acid column
chromatography and thin-layer chromatography. They were identified to be phloroglucinol and its poly
mers, namely eckol, phlorofucofuroeckol A, dieckol, and 8,8•Œ-bieckol on the basis of spectroscopic evi
dence (IR, FABMS, and 1H and 13C NMR). Their potent antioxidant effect extended the induction time
of autoxidation of methyl ƒ¿-linolenate.

Key words: antioxidant, phlorotannin, phloroglucinol, brown alga, Eisenia bicyclis, isolation,
identification

The literature on algal antioxidants is sparse. Fujimoto Yamatokagaku). The extracts were concentrated to ca.
and Kaneda1) investigated antioxidative substances of the 200ml, to which were added CHC13 (400 ml) and
brown alga Eisenia bicyclis and found phos deionized water (150ml). The upper layer, corresponding

phatidylethanolamine to be an antioxidant. Later, they to the non-lipid fraction,11) was extracted with ethyl ether
reported bromophenols, 5-bromo-3,4-dihydroxy benzalde (2 •~ 150 ml).
hyde as the main constituent and 5-bromo-benzylalcohols, The ether extract was fractionated by silicic acid chro
in the red alga Polysiphonia urceolate.2,3) Nishibori and matography on a column of Wakogel C-300 (10mm
Namiki4) studied antioxidative substances of a green alga i.d. •~ 30cm, Wako pure chemical Ind.) with CHC13
Enteromorpha sp., and identified pheophytin a, one of the - methanol-water (50:30:7, v/v) as eluent. The eluates were
Mg-free chlorophyls, as the main component. Cahyana et monitored by UV absorption at 310nm using a Hitachi L
al.5) found a related compound, pyropheophytin a, in the 400 UV detector. If necessary, preparative TLC was used
brown alga Eisenia bicyclis and showed its higher antiox under the conditions mentioned below.
idant activity than that of ƒ¿-tocopherol. Tocopherols,
which are popular antioxidants in terrestrial organisms, Structural Analysis of Antioxidative Substances
are also present in algae.6-8) Recently, we have reported an 1H and 13C nuclear magnetic resonance (NMR), and 1H
extremely high content of tocopherols in the brown alga -13 C long-range shift correlation spectra (COLOC) were
Ishige okamurae and their wide distribution in Phaeophy measured with a JEOL FX-400 spectrometer (JEOL Ltd.)
ta, Rhodophyta and Chlorophyta.9) with tetramethylsilane as an internal standard. Negative

These antioxidative substances described above are usu fast atom bombardment mass spectra (FABMS) were ob
ally isolated from the lipid fraction of algal extracts. tained on a JMS-DX300 (JEOL Ltd.) at an ion source ac
However, using a TLC-screening method,10) we detected celerating voltage of 10 kV, with glycerol as the matrix.

prominent antioxidative bands in the non-lipid fraction11) Electron impact mass spectra (EIMS) were recorded with a
of the brown alga Eisenia bicyclis. We describe here the iso GC-MS QP-1000 (Shimadzu Co.). Gas liquid chro
lation and identification of the antioxidative substances in matography (GLC) was carried out using a Shimadzu GC
the alga and their potent antioxidant activity. - 14A gas chromatograph on a capillary column (DBP-1, 30
m •~ 0.25 mm i.d. Shimadzu Co). IR spectra were mea

Materials and Methods sured in KBr tablets with a Hitachi EPI-G21 spectrometer.
Trimethylsilyl ether derivative of phloroglucinol was pre

Materials pared by treatment with N,O-bis(trimethylsilyl) trifluoro

The brown alga Eisenia bicyclis collected from the coast acetoamide in pyridine. 12)

of Tsuzumi island, Fukuoka Prefecture in July 1992 was

washed with tap water, air-dried, and pulverized. The al Antioxidant Assay
gal powder was stocked at -40•Ž until use. Thin-layer chromatography (TLC) plates (Silica Gel 60
F254, 0.25mm, Merck Co.), which had been activated at

Extraction and Separation of Antioxidative Substances 110•Ž for 1 h before use, were developed with CHC13
- methanol-water-acetic acid (50:25:4:3, v/v). One plate
The algal powder (100g) was extracted with methanol
was sprayed with 50% sulfuric acid and charred on a hot
(800ml) in an iced bath using an Ultra disperser (LK-21,

*1 Presented at the General Meeting of JSSF held in Tokyo University of Fisheries, Tokyo, in April, 1993.
*2 To
whom correspondence should be addressed.
924 Nakamura et al.

plate. Another plate was sprayed with a paprika pigment by column chromatography, while T-2 and T-3 were
solution (10mg/ml benzene) and exposed for several finally purified by preparative TLC. About 20 mg of T-1
minutes to UV light (254 nm) using a UV illuminator and 100 to 150mg for each of T-2 - 5 were obtained from
(7000ƒÊW, model 20-TC, Atto Co.) after removal of the 100g of the dry alga.
solvent.10) Antioxidative substances appeared as colored IR spectra (KBr) of the purified T-2?5 were similar to
spots. A paprika pigment, the main component of which is each other, showing bands at 3400 (OH), 1650-1450
capsanthin, was donated by Takeda pharmaceutical Ind. (phenyl), 1200 and 1050 (ether), and 820 cm-1 (1,3,5-sub
Antioxidant activity was measured by the weighing stituted phenyl). These results suggested that they were
method developed by Olcott and Einset.l3) Mixtures of phloroglucinol derivatives. 14)
methyl a-linolenate (200 mg) and aliquot amounts of T-1 was identified as phloroglucinol by comparison with
phlorotannins in Petri dishes (i.d. 36mm) were left in an GC-MS data of the authentic sample.
incubator at 40•Ž. Increasing weight was measured at ap T-2 showed an [M-H] ion peak at m/z 371 (rel. int.

propriate intervals. 100%) in negative FABMS, matching a formula of

Phloroglucinol was purchased from Wako pure chem. C18H1109, along with fragment ions m/z 355 (M-17,3.9),
Ind., while methyl ƒ¿-linolenate was from Tokyo kasei 263 (M-109,12.3), 247 (M-125, 5.3), and 139 (M-233, 6.5).
Ind. All reagents used in this experiment were of analytical The 13C NMR spectrum measured in CD3OD exhibited sig

grade. nals at ƒÂ 95.4 (C6), 95.8 (C2•Œ, C6•Œ), 97.8 (C4•Œ), 99.5 (C3),
99.9 (C8), 124.6 (C4a), 124.9 (C9a), 125.6 (C1), 138.6
Results (ClOa), 143.4 (C4), 144.3 (C5a), 147.1 (C2), 147.3 (C9),
154.6 (C7), 160.2 (C3•Œ, C5•Œ), and 161.9 (C1•Œ). In addition,
Isolation and Characterization NMR data obtained in DMSO-d6 were in good agreement
Antioxidative substances in the ether extract, 0.90 g with those of eckol, T-2 in Fig. 2. Thus, T-2 was identified
from 100 g of the dry alga, were separated on TLC plates. with eckol reported by Fukuyama et al.15,16)
As shown in Fig. 1, five antioxidative bands (T-1?5) were T-3 exhibited an [M-H] ion peak at m/z 601 (rel. int.
detected with little contaminants other than antioxidative 100%), matching a formula of C30H17014, and fragment

substances. An upper band of T-1 is a pigment since the ions m/z 585 (M-17, 8.0), 492 (M-109, 21.3), 476 (M-125,
color was apparent before spraying the paprika pigment so 7.1), 369 (M-233, 10.6), 353 (M-249, 6.6), and 339 (M

lution. These five antioxidative substances were isolated by 263, 6.5) in negative FABMS. The 13C NMR spectrum

column chromatography on a Wakogel C-300 and prepara measured in CD3OD contained signals at 6 95.5 (C2•Œ,

tive TLC. T-1, T-4, and T-5 were successfully isolated only C6•Œ, C2••, C6••), 96.2 (C13), 97.7 (C4•Œ), 97.8 (C4••), 99.5

(C3), 100.0 (C9), 105.3 (C6, C7), 122.4 (Cl1), 124.7 (Cl),
125.0 (C4a), 128.1 (C14a), 135.4 (C5a), 138.4 (C15a),
144.0 (C4), 146.0 (C14), 148.3 (C2, C8), 151.2 (Clla),

Fig. 1. Separation and detection of antioxidative substances in the

brown alga Eisenia bicyclis by thin-layer chromatography.
Ether extract was applied onto a pair of plates (Silica gel 60 F254,
0.25mm, Merck Co.) and developed with CHC13-methanol-water
acetic acid (50:25:4:3, v/v). After removal of the solvent, one plate
(A) was sprayed with a paprika pigment solution followed by ex
posure to UV-light, while the other plate (B) was sprayed with 50%
H2SO4 and charred on a hot plate. Fig. 2. Structure of phlorotannins found in Eisenia bicyclis.
 Antioxidant Activity of Phlorotannins 925

151.7 (CIO), 153.2 (C12a), 160.1 (C3•Œ, C5•Œ, C3••), 160.2

(CS••), 161.8 (C1••), and 161.9 (C1). NMR data obtained in Discussion
DMSO-d6 were in good agreement with those of phloro
fucofuroeckol A.") Thus, T-3 was phlorofucofuroeckol It has long been known that brown algae contain
A. phlorotannins, phloroglucinol and its polymers, in subcel
T-4 revealed an [M-H] ion peak at m/z 741 (rel. int. lular bodies called physodes.19,20) In the present study,
4.6%), matching a formula of C36H21018, and fragment phloroglucinol and four phlorotannins: eckol,
ions m/z 725 (M-17, 0.7), 633 (M-109, 0.6), 617 (M-125, phlorofucofuroeckol A, dieckol, 8,8•Œ-bieckol were iden
0.4), 495 (M-247, 0.7), 479 (M-263, 0.3), 387 (0.7), 371 tified from Eisenia bicyclis. These compounds were firstly

(1.2), and 355 (0.5) in negative FABMS. The 13C NMR detected in Ecklonia kurome OKAMURA as anti-plasmin
recorded in CD3OD showed signals at S 95.4 (C6), 95.8 inhibitors .15-18,21) Studying the inhibitory spectra of eckol

(C2•Œ), 95.9 (C6•Œ, C6••), 96.2 (C2 ? , C6?), 97.7 (C4•Œ), 99.4 derivatives, Nakayama et al.21) suggested that the dibenzo

(C3••), 99.5 (C3), 99.8 (C8), 99.9 (C8••), 124.6 (C4a), 124.7 1,4-dioxane skeleton was necessary for the inhibition of a

(C4a••), 124.9 (C9a••), 125.6 (C1••), 125.7 (C1), 126.2 (C9a), plasmin inhibitor. Geiselman and McConnel22) and
126.5 (C4?), 138.5 (ClOa••), 138.7 (ClOa), 143.3 (C4), Taniguchi et al.23-25) reported that phlorotannins of vari

143.4 (C4••), 144.2 (C5a), 144.3 (C5a••), 146.9 (C9), 147.1 ous molecular weights played important roles in chemical

(C9••), 147.3 (C2), 147.4 (C2••), 152.4 (C3?, C5?), 154.5 defenses against marine herbivores. However, with the ex

(C7••), 156.0 (C7), 157.8 (C1?), 160.1 (C3•Œ, CS•Œ), and ception of phloroglucinol, the same compounds described
161.8 (C1•Œ). NMR data obtained in DMSO-d6 were in in the present paper were not reported. Kita et a1.26) iden

good agreement with those of dieckol.18) COLOC data also tified eckol and its dimeric compounds (not identified) as
suggested the dimeric structure of eckol. Therefore, T-4 oral deodorants in Eisenia bicyclis. However, there is no
was identified as dieckol. documented research on the antioxidant activities of poly
T-5 gave an [M-H] ion peak at m/z 741 (rel. int. phloroglucinols of isolated compound.
1.8%), matching a formula of C36H21018, and fragment In this paper, we described the antioxidant activities of
ions at m/z 725 (M-17, 0.3), 633 (M-109, 0.3), 617 (M- polyphloroglucinols and their possible usefulness as an
125, 0.3), 509 (M-233, 0.2), 493 (M-249, 0.3), and 477 (M- tioxidants. The method of preparation used in our experi
265, 0.2). The 13C NMR spectrum measured in CD3OD re ment is simple and convenient for preparing phlorotannins
vealed signals at .5 95.4 (C2•Œ, C6•Œ), 95.9 (C6), 97.8 (C4•Œ), since most of the contaminant lipids were removed before
99.4 (C3), 105.4 (C8), 124.6 (C4a), 125.2 (C9a), 125.6 (C 1), the chromatographic purification.
138.4 (ClOa), 143.4 (C5a), 143.5 (C4), 145.4 (C9), 147.2
Acknowledgments This work was part of an investigation supported by
(C2), 152.8 (C7), 160.2 (C3•Œ, C5•Œ), and 161.8 (CI). NMR
data obtained in DMSO-d6 were in good agreement with a grant from the NS Science Promoting Foundation. We thank Dr. G-I.
Nonaka for his helpful discussion on the structural determination, and M.
those for 8,8•Œ-bieckol.17) COLOC data suggested the sym
Ohara, who provided useful comments on the manuscript.
metrical structure. Thus, T-5 was 8,8•Œ-bieckol.

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