Food Sci. Technol. Res., +- (+), /.ῌ0*, ,**1

Anti-Allergic from the Edible Brown Alga, Eisenia Arborea

+ - + + , , Yoshimasa SUGIURA , , Kohji MATSUDA , Yasuhiro YAMADA , Masashi NISHIKAWA , Kazufumi SHIOYA , , , -῍ Hirotaka KATSUZAKI , Kunio IMAI and Hideomi AMANO

+ Department of Technology, Kanehatsu Food Co., Ltd., -ῌ+3ῌ,. Yutaka, Minani-ku, Nagoya, Aichi ./1ῌ2///, Japan , Laboratory of Bioorganic Chemistry, Graduate School of Bioresources, Mie University, Kurimamachiya-cho +/11, Tsu, Mie /+.ῌ 2/*1, Japan - Laboratory of Marine Biochemistry, Graduate School of Bioresources, Mie University, Kurimamachiya-cho +/11, Tsu, Mie /+.ῌ2/*1, Japan

Received August ,+, ,**0; Accepted October ,/, ,**0

Eisenia arborea, an edible brown alga, is occasionally used as a folk medicine due to its anti-allergic e#ect. In the present study to identify the anti-allergic constituents in the alga, the extract of the alga was purified by partition between solvents and by reversed phase chromatography. Separation of the extract was guided by the inhibitory activity upon b-hexosaminidase release from the rat basophilic leukemia-,H- cells. HPLC purification a#orded six active compounds. Spectral analyses clarified their structures as , 0,0῎-bieckol, 0,2῎-bieckol, 2,2῎-bieckol, phlorofucofuroeckol-A, and phlorofucofuroeckol-B. Most of the phlorotannins exhibited activities similar to or greater than the typical inhibitor, epigallocatechin gallate. Phlorofucofuroeckol-B showed the greatest activity among the tested phlorotannins at ,.2 times greater than epigallocatechin gallate.

Keywords: Eisenia arborea, b-hexosaminidase, , RBL-,H- cell Abbreviations: DSCG: disodium cromoglycate, EGCg: epigallocatechin gallate, b-Hex: b-hexosaminidase, HR-FAB- MS: high resolution FAB-Ms, PFF: phlorofucofuroeckol, RBL: rat basophilic leukemia

Introduction finding that the ethanol extract of the alga showed a Brown algae are a popular health food in Japan. These similar e#ect on the BN rats. This finding encouraged us algae are known to contain many phlorotannins (Fuku- to identify the active constituents of the alga. To moni- yama et al., +32/, +323, +33*; Glombitza and Gerstberger, tor the anti-allergic activity of the purified samples, a +32/a; Glombitza and Vogels, +32/b). They contain vari- bioassay system using rat basophilic leukemia (RBL)-,H- ous biological activities, such as antioxidative activity cells was established, according to the procedure reported (Nakamura et al., +330), anti-tumor activity (Kobayashi et by Nishibe et al. (,**+). The RBL cells release histamine al., ,**+), anti-HIV activity (Ahn et al., ,**.), bactericidal which is responsible for the allergy, together with b- activity (Nagayama et al., ,**,), anti-inflammation activi- hexosaminidase (b-Hex) when they are stimulated by the ty (Shibata et al., ,**,, ,**-), and so on. However, no antigen-antibody reaction or a calcium ionophore (A reports have been found related to the inhibiting ac- ,-+21). The amount of the released histamine is known tivities of the phlorotannins regarding histamine release to be proportional to that of b-Hex (Schwartz et al., +32+). from cultured cells. By monitoring the inhibiting activity of the b-Hex release, Our preliminary investigation of the anti-allergic algae the M/C (mixture of methanol and chloroform) extract of found that the dried powder of the brown alga, E. arborea, the dried powder of E. arborea was purified and 0 active possessed a strong anti-allergic e#ect on Brown Norway compounds isolated. Their structures were identified by (BN) rats, an allergic model animal. The amount of hista- spectral analyses. They were eckol, 0,0῎-bieckol, 0,2῎- mine in the blood of BN rats fed a diet containing the bieckol, 2,2῎-bieckol, phlorofucofuroeckol-A (PFF-A), and powdered alga (/ῌ) was significantly lower (+...3ῌ0-.* phlorofucofuroeckol-B (PFF-B). Most of these phloro- nM, n῍/) than that of the animals fed a diet without the showed inhibiting activities similar to or greater alga (-++.2ῌ.,.* nM, n῍/). The details of these results than that of the typical inhibitor, epigallocatechin gallate will be discussed elsewhere in the near future. Although (EGCg), and PFF-B showed a ,.2 times greater activity it was postulated that the anti-allergic e#ect could be due than EGCg. This result indicated that the anti-allergic to the algal polysaccharides, the low molecular weight e#ect of E. arborea could be due to the histamine release- active principle was suggested in the alga, based on the inhibiting activity of these phlorotannins isolated from the alga. ῍ To whom correspondence should be addressed. This paper describes the extraction, purification, and E-mail: [email protected] identification of the anti-allergic constituents from E. ar- Anti-Allergic Phlorotannins from the Edible Brown Alga, Eisenia Arborea 55 borea and their inhibiting activity on histamine release *.+ mM nonessential amino acid (Invitrogen), and + mM from the stimulated RBL cells, which was estimated by sodium pyruvate (Cambrex Bio Science, Inc., Walkersville measuring the amount of the released b-Hex. MD, USA) at -1ΐC in a humidified atmosphere containing /ῌ CO,. The cells were passaged every . or / days by Materials and Methods trypsinization with one medium change between the pas- Material, Extraction and Partial Purification (Fig. +) sage until they attained confluence. The brown alga, E. arborea, was collected from the Mugi- Cell Stimulation and Determination of Released b- zaki coast in Mie prefecture, Japan. The alga was Hexosaminidase (b-Hex) The cells were stimulated and washed, dried in air, and powdered using an ultracen- the released b-Hex (equivalent to histamine) was deter- trifugal mill (ZM,**, Retsch Co., Ltd., Haan, Germany). mined according to a previously reported procedure The dried powder (/** g) was immersed in ,./ L of hexane (Nishibe et al., ,**+) with some modifications. for ,. h, and the hexane extract removed by filtration. The RBL cells were precultured for - days in EMEM The remaining powder was re-extracted with -.* LofM/ supplemented with ,*ῌ FBS and sensitized with anti- C (methanol: chloroformῒ+ : ,), after extraction with /** dinitrophenyl (DNP) IgE (final ,./ mg/mL, Sigma-Aldrich mL portions of a mixture of hexane and ethyl acetate (. : Co., St. Louis MO, USA) for +2 hr. The medium was then + and + : +). The M/C extract was partitioned with 1/* mL changed to Tyrode bu#er (pH 1.,) (Matsuo et al., +331), and of water, and then the aqueous methanol layer was extra- the test sample solution was added. After incubation for cted with 1/* mL of diethyl ether. The ether layer was +* min, the cells were treated with the antigen, DNP-BSA evaporated to dryness. The residue was re-dissolved in (final *./ mg/mL, LSL Co., Ltd., Tokyo, Japan), for -/ min. +* mL of methanol and the solution diluted with +** mL of To stimulate the RBL cells, the calcium ionophore A,-+21 water. The water solution was subjected to the reversed (Nacalai Tesque, Inc., Kyoto, Japan), was added to the phase HPLC analysis and purification. culture medium to make the final concentration of + mM, Reversed Phase HPLC A high performance liquid following which, the cells were incubated for -/ min with- chromatography (HPLC) system (0A, Shimadzu Co., Kyo- out sensitization by anti-DNP IgE and stimulation by to, Japan) was used in this study. An analytical De- DNP-BSA. The stimulation reaction was stopped by velosil ODS-/ column (..0 mm IDῑ,/* mm L, Nomura cooling the reaction vessel on ice for +/ min. The culture Chemical Co., Aichi, Japan, flow rate: *./ mL/min) and a medium was divided into two wells of a 30-well plate, and preparative Develosil ODS-/ column (+* mm IDῑ,/* mm the substrate solution (+ mM p-nitrophenyl-N-acetyl-b-D- L, flow rate: +.* mL/min) were used. For the analytical glucosaminide, Wako) was added to each well. After chromatography, a -*-min gradient program between incubation for 0* min at -1ΐC, +** mM sodium bicarbonate *.+ῌ TFA (A) and acetonitrile containing *.+ῌ TFA (B) was added to each solution to alkalize them and develop a was employed. For the preparative chromatography, yellow color. The color was measured at the optical the following three elution conditions were applied. density of .*/ nm to determine the amount of p-nitro- Condition +: gradient elution between A and B (*ῌ-*ῌ B phenol released from the substrate by the action of the for +,* min, maintain -*ῌ Bfor-* min and -*ῌ+**ῌ Bfor released enzyme. The inhibition ratio of the b-Hex was -* min); Condition ,: isocratic elution with +2ῌ B; and calculated using the following equation: Condition -: isocratic elution with .1ῌ methanol contain- Inhibition ratioῌῌ῍ῒ῎+ῐῌTῐNC῍ῌῌPCῐNC῍῏ῑ+** ing *.+ῌ TFA. The eluate was monitored by UV absorb- ance at ,2* nm. where NC is the OD.*/ of the blank cell medium without + +- Structure Analysis The H-NMR, C-NMR, HSQC, the test sample and the stimulations; PC is the OD.*/ of HMBC and ROESY spectra were measured in DMSO-d0 the cell medium without the test sample, but with stimu- using a JEOL NM-A-/** spectrometer (JEOL, Tokyo, Ja- lation; and T is the OD.*/ of the cell medium with both the pan). The mass analyses were performed using a Kom- test sample and stimulation. pact Discovery MALDI-TOF mass system (Shimadzu Co. Kyoto, Japan) with ,,/-dihydroxybenzoic acid as the Results and Discussion matrix. High resolution FAB-MS was measured by a MS Extraction and Isolation of Active Constituents The 1** HR-FAB-MS system (JEOL Co.) using glycerin as the flow diagram for the extraction and partial purification of matrix. The spectral data are presented in Tables + and the active constituents from the alga is shown in Figure + ,. (Folch et al., +3/1; Shibata et al., ,**,). The M/C extract Cell Culture RBL-,H- cells, (Bursumian et al., +32+) (yield: .-* mg) obtained by the extraction was re-dis- obtained from the Japanese Cancer Research Resource solved in water containing a small amount of methanol, Bank, (JCRB, Ibaraki, Osaka, Japan) were cultured in and the solution was subjected to further purification. Eagle’s Modified Essential Medium (EMEM, MP Bio- The solution was analyzed by RP-HPLC, and the medicals, Inc., Illkirch, France) supplemented with ,*ῌ chromatogram (Fig. ,) indicated that it contained 0 major fetal bovine serum (FBS, Lot. No.: A*0*1,-/**, Trace Sci- components together with several minor substances. To entific, Ltd., Melbourne, Australia), ,. mM sodium bicar- obtain su$cient amounts of the pure substances for the bonate (Wako Pure Chemical Co., Ltd., Tokyo, Japan), , bio-activity tests and for the structure analyses, the mM L-glutamine (Invitrogen Co., Carlsbad, CA, USA), 1/ k remaining solution was separated by preparative RP- unit/L penicillin (Wako), *.+/ mM streptomycin (Wako), HPLC (elution condition + in the experimental section). 56 Y. SUGIURA et al.

This chromatographic separation a#orded two active bieckol, -: 0,2῍-bieckol, and .: 2,2῍-bieckol were obtained. fractions which included active compounds +ῌ. (Fr. +)and To purify the active Fr. ,, elution Condition - was applied, active compounds / and 0 (Fr. ,), and they showed 0*.0῍ and two active compounds, /: phlorofucofuroeckol-B *.2ῌ and 12.-῍+/.0ῌ inhibitions on b-Hex release from (PFF-B) and 0: phlorofucofuroeckol-A (PFF-A) were pro- RBL cells at a concentration of *.+ mg/mL, respectively. duced. The yields of compounds +ῌ0 were +: ++.- mg, ,: The other fractions were inactive. These active fractions ..+ mg, -: -.- mg, .: ,,.- mg, /: -.- mg and 0: /.- mg, respec- were subjected to the second RP-HPLC purification proce- tively, from /** g of the dried alga. dure. To purify the active Fr. +, elution Condition , was Structure Analysis and Identification of the Isolated employed, and four active compounds, +: eckol, ,: 0,0῍- Phlorotannins The structures of the isolated compounds were analyzed by mass spectra, +H-NMR, +-C-NMR, +H-+H COSY, HSQC, HMBC and ROESY spectra. The spectral data are summarized in Tables + and ,, and the elucidated structures are shown in Fig. -. Compounds +, ,, .,and0 were identified as eckol, 0,0῍- bieckol, 2,2῍-bieckol, and phlorofucofuroeckol-A (PFF-A), respectively, by comparing their spectra with those re- ported in the references (Fukuyama et al., +32/, +323, +33*). Compound - showed its [MῌH]ῌ ion at M/Z῎1.- in the MALDI-TOF Ms analysis, and this result indicated its molecular weight of 1.,. The molecular weight was the same as those of the bieckols, and it indicated that com- pound - could be an isomer of them. Compound - showed ,2 carbon signals, ,, signals containing one carbon, / signals containing two carbons, and one signal containing four carbons in its +-C-NMR spectrum (Table +). These results suggested that compound - was an asymmetric molecule. Most of the +Hand+-C signals resembled those of eckol, except for the signals for the 0- and 2῍-protons and carbons. In compound -, the protons corresponding to H-0 and H-2῍ of two eckol moieties were lost, and the C-0 and C-2῍ signals shifted to 33./ and +*../ ppm from 3-.2 and 32./ ppm of the corresponding carbons of the eckols, respectively. These analyses, in addition to the +H-+H COSY, HSQC, HMBC analyses, clearly suggested that compound - was 0,2῍-bieckol. Although the exist- ence of 0,2῍-bieckol had already been pointed out by Glombitza et al. (+32/b), no spectral data for the intact Fig. +. Flow diagram for extraction and partial purifica- tion of the phlorotannins from E. arborea. phlorotannin were indicated. Only those of its per-

Fig. ,. Analytical RP-HPLC chromatogram of the partially purified phlorotannins from E. arborea. The elution conditions are listed in the “Materials and Methods” section. Anti-Allergic Phlorotannins from the Edible Brown Alga, Eisenia Arborea 57

Table +. Ms and NMR spectra of compound +ῌ..

Table ,. Ms and NMR spectra of compound / and 0. 58 Y. SUGIURA et al.

Fig. -. Structures of the phlorotannins isolated from E. arborea.

Table -. The inhibitory e#ects of the active compounds on b-Hex release from RBL cells.

acetate were reported. Accordingly, this paper is the shown in Fig. -. The details of the structure analyses first one that shows the exact spectral data for the intact will be reported elsewhere (Sugiura et al., ,**0). Com- 0,2ῌ-bieckol. pound / was a new structural isomer of PFF-A, and it was Compound / showed its [MῌH]ῌ ion at M/Z῍0*- in the called phlorofucofuroeckol-B (PFF-B). MALDI- TOF-Ms analysis, and its molecular weight was Inhibitory Activities on b-Hex Release from RBL-,H- estimated to be 0*,. Its molecular formula was deter- Cells by the Isolated Phlorotannins The degree of inhibi- mined to be C-*H+2O+. on the basis of its HR-FAB-MS tion of the b-Hex release by the test sample is summarized spectrum (positive, in glycerin, m/z῍0*-.*1+1, D ,.* mmu). in Table -, together with that of a positive control, epi- The molecular formula was the same as that of PFF-A, gallocathecin gallate (EGCg). All of the isolated phloro- and suggested compound / could be an isomer of PFF-A. tannins showed inhibitory e#ects to various degrees. Al- Most of the NMR signals of compound / resembled those though compound +, eckol, showed a lesser activity than of PFF-A except for C-/a, C-0,C-0a, C-2,C-+*,C-+,,C-+-,C- the positive control by several to ten times, the other +.,H-0 and H-+*. A comparison of their +Hand+-CNMR isolated phlorotannins showed inhibitory activities simi- spectra and analyses of the +H-+H COSY, HSQC, HMBC, lar to or greater than EGCg. The newly identified pholo- ROESY spectra of compound / revealed its structure as rofucofuroeckol-B (PFF-B) showed the strongest activity Anti-Allergic Phlorotannins from the Edible Brown Alga, Eisenia Arborea 59 among the tested samples, including EGCg at any dose. The results of the present study revealed one of the rea- The IC/* value of PFF-B was 1.2 mM. This result indicated sons why E. arborea is popular as a folk medicine or as a that PFF-B had a ,.2ῌ0.* times greater inhibitory activity health food in Japan. than those of ECGg (IC/*῍,,.* mM) or Tranilast (IC/*῍.0.0 mM) (Matsubara et al., ,**.), a clinically used anti-allergic References drug. Ahn, M.J., Yoon, K.D., Min, S.Y., Lee, J.S., Kim, J.H., Kim, T.G., Similar inhibition tendencies by the phlorotannins Kim, S.H., Kim, N.G., Hun, H. and Kim, J. (,**.). Inhibition of + were observed when the cells were stimulated by the HIV- reverse transcriptase and protease by phlorotannins from the brown alga Ecklonia cava. Biol. Pharm. Bull., ,1. /..ῌ ionophore, A,-+21, as shown in Table -. PFF-B again /.1. showed the greatest inhibitory activity among the tested Bursumian, E.L., Isersky, C., Petorino, M.G. and Siraganian, R.P. phlorotannins or ECGg. These results implied that the (+32+). IgE-induced histamine release from rat basophilic leuke- phlorotanins could inhibit not only cellular events tri- mia cell lines: isolation of releasing and nonreleasing clones. ggered by the antigen-antibody reaction before calcium Eur. J. Immunol., ++, -+1ῌ-,-. influx caused by A,-+21, but also cellular events later Folch, J., Lees, M. and Stanley, G.H.S. (+3/1). A simple method for than the calcium influx until degranulation of the RBL the isolation and purification of total lipids from animal tissues. J. Biol. Chem., ,,0, .31ῌ/*3. cells. Fujimura, Y., Tachibana, H., Maeda-Yamamoto, M., Miyase, T., The results showed that although eckol, with a lower Sano, M. and Yamada, K. (,**,). Anti-allergic tea catechin, (ῌ)- molecular weight, exhibited weaker activity, the other epigallocatechin---O-(--O-mathyl)-gallate, suppresses FceRI phlorotannins with higher molecular weights showed expression in human basophilic KU2+, cells. J. Agric. Food stronger activities comparable to EGCg, indicating that Chem., /*, /1,3ῌ/1-.. the molecular size or the number of phenol groups could Fukuyama, Y., Miura, I., Kinzyo, Z., Mori, H., Kido, M., Nakayama, +32/ be an important factor for expressing their activities. Y., Takahashi, M. and Ochi, M. ( ). Eckols, novel phloro- tannins with a dibenzo-p-dioxin skeleton possessing inhibitory The importance of the molecular size in exhibiting such e#ects on a,-macroglobulin from the brown alga Ecklonia an inhibition was reported for apple (Kanda kurome OKAMURA. Chem. Lett., +., 1-3ῌ1.,. et al., +332). The apple condensed (ACT) that had Fukuyama, Y., Kodama, M., Miura, I., Kinzyo, Z., Mori, H., Naka- a larger molecular size showed a greater histamine release yama, Y. and Takahashi M. (+323). Anti-plasmin inhibitor. V. inhibitory activity than the monomer of ACT, epica- Structures of novel dimeric eckols isolated from the brown thecin. Although the reason why PFF-B possessed a alga Ecklonia kurome OKAMURA. Chem. Pharm. Bull., -1, ,.-2ῌ ,..* greater activity than the others is not presently clear , its . Fukuyama, Y., Kodama, M., Miura, I., Kinzyo, Z., Mori, H., Naka- unique structure might be the reason for its strong activ- yama, Y. and Takahashi M. (+33*). Anti-plasmin inhibitor. VI. ity. Structures of phlorofucofuroeckol A, a novel phlorotannin This paper reported the first finding that the phlo- with both dibenzo-+,.-dioxin and elements, from rotannins, the major polyphenols of algae, possessed an Ecklonia kurome OKAMURA. Chem. Pharm. Bull., -2, +--ῌ+-/. inhibitory activity on b-Hex release from cultured cells. Glombitza, K.W. and Gerstberger, G. (+32/a). Phlorotannins with A similar inhibitory e#ect has been studied for the poly- dibenzodioxin structural elements from the brown alga Ei- ,. /.-ῌ//+ phenols of terrestrial plants, such as tea (Matsuo et al., senia arborea. Phytochemistry, , . Glombitza, K.W. and Vogels, H.P. (+32/b) Antibiotics from algae. +331), perilla (Ueda et al., ,**,), persimmon (Kotani et al., XXXV. Phlorotannins from Ecklonia maxima. Planta Med., /+, ,*** ), as well as on those of clinically used drugs: dis- -*2ῌ-+,. odium cromoglycate (DSCG) and Tranilast (Kakegawa et Kakegawa, H., Matsumoto, H. and Satoh, T. (+32/a). Activation of al., +33,). The inhibition mechanism was well inves- hyaluronidase by metallic salts and compound .2/2*, and in- tigated for the tea catechin, EGCg. EGCg inhibited the hibitory e#ect of anti-allergic agants on hyaluronidase. Chem. tyrosine phosphorylation of the protein kinase involved Pharm. Bull., --, 0.,ῌ0.0. in the RBL degranulation (Yamashita et al., ,***)and Kakegawa, H., Matsumoto, H., Endo, K., Satoh, T., Nonaka, G. and Nishioka, I. (+32/b). Inhibitory e#ects of tannins on hyalu- expression of the IgE receptor in the cultured human ronidase activation and on the degranulation from rat mesen- 2+, ,**, basophile leukemia cells, KU (Fujimura et al., ). tery mast cells. Chem. Pharm. Bull., --, /*13ῌ/*2,. In addition, the tea catechins inhibited hyalulonidase Kakegawa, H., Matsumoto, H. and Satoh, T. (+33,). Inhiibtory (Kakegawa et al., +32/b), which is closely related to the e#ects of some natural products on the activation of hyalu- inflammatory reaction. The same enzyme inhibition was ronidase and their anti-allergic actions. Chem. Pharm. Bull., .*, observed for the clinically used drugs, DSCG or Tranilast +.-3ῌ+..,. (Kakegawa et al., +32/a). The phlorotannins were also Kobayashi, A., Hashimoto, M., Nakano, T., Tamura, S. and Hidano, N. (,**+). Japan Kokai Tokkyo Koho, P,**+ῌ-+/2/A. reported to inhibit the enzyme (Shibata et al., ,**,). The Feb. 0. # similarities in the inhibitory e ects between the phlo- Kanda, T., Akiyama, H., Yanagida, A., Tanabe, M., Goda, Y., Toyo- rotannins and tea catechins or the clinical drugs indicated da, M., Teshima, R. and Saito, Y. (+332). Inhibitory e#ects of the possibility that similar mechanisms could be involved apple on induced histamine release from RBL-,H- in the expression of their anti-allergic e#ects. cells and rat mast cells. Biosci. Biotechnol. Biochem., 0,, +,2.ῌ Moreover, an improvement in the allergy symptoms of +,23. the model rats fed a diet including the alga, E. arborea, Kotani, M., Matsumoto, M., Fujita, A., Higa, S., Wang, W., Sue- mura, M., Kishimoto, T. and Tanaka, T. (,***). Persimmon leaf could be due to the inhibitory e#ects of the isolated phlo- extract and astragalin inhibit development of dermatitis and rotannins on both the enzyme and histamine release. 60 Y. SUGIURA et al.

IgE elevation in NC/Nga mice. J. Allergy Clin. Immunol., +*0, persed human lung mast cells. J. Immunol., +,0, +,3*ῌ+,3.. +/3ῌ+00. Shibata, T., Fujimoto, K., Nagayama, K., Yamaguchi, K. and Matsubara, M., Masaki, S., Ohmori, K., Karasawa, A. and Hase- Nakamura, T. (,**,). Inhibitory activity of brown algal phloro- gawa, K. (,**.). Di#erential regulation of IL-. expression and tannins against hyaluronidase. Int. J. Food Sci. Technol., -1, degranulation by anti-allergic olopatadine in rat basophilic 1*-ῌ1*3. leukemia (RBL-,H-) cells. Biochem. Pharmacol., 01, +-+/ῌ+-,0. Shibata, T., Nagayama, K., Tanaka, R., Yamaguchi, K. and Naka- Matsuo, N., Yamada, K., Shoji, K., Mori, M. and Sugano, M. (+331). mura, T. (,**-). Inhibitory e#ects of brown algal phlorotannins E#ects of tea polyphenols on histamine release from rat on secretory phospholipase A,s, lipoxygenases and cyclooxy- basophilic leukemia (RBL-,H-) cells: the structure-inhibitory genases. J. Appl. Phycol., +/, 0+ῌ00. activity relationship. Allergy, /,, /2ῌ0.. Sugiura, Y., Matsuda, K., Yamada, Y., Nishikawa, M., Shioya, K., Nagayama, K., Iwamura, Y., Shibata, T., Hirayama, I. and Naka- Katsuzaki, H., Imai, K. and Amano, H. (,**0). Isolation of a new mura, T. (,**,). Bactericidal activity of phlorotannins from the anti-allergic phlorotannin, Phlorofucofuroeckol-B, from an brown alga Ecklonia kurome. J. Antimicrob. Chemother., /*, 223ῌ edible brown alga, Eisenia arborea. Biosci. Biotechnol. Biochem., 23-. 1*, ,2*1ῌ,2++. Nakamura, T., Nagayama, K., Uchida, K. and Tanaka, R. (+330). Ueda, H., Yamazaki, C. and Yamazaki, M. (,**,). Luteolin as an Antioxidant activity of phlorotannins isolated from brown anti-inflammatory and anti-allergic constituent of Perilla fru- alga Eisenia bicyclis. Fisheries Sci., 0,, 3,-ῌ3,0. tescens. Biol. Pharm. Bull., ,/, ++31ῌ+,*,. Nishibe, S., Han, Y., Noguchi, Y., Ueda, H., Yamazaki, M., Mizutani, Yamashita, K., Suzuki, Y., Matsui, T., Yoshimaru, T., Yamaki, M., K., Kambara, T. and Kishida, N. (,**+). The inhibition e#ects of Suziki-Karasaki, M., Hayakawa, S. and Shimizu, K. (,***). compounds from oliveleaf on tumor necrosis factor production Epigallocatechin gallate inhibits histamine release from rat and on b-hexosaminidase release. Natural Medicines, //, ,*/ῌ basophilic leukemia (RBL-,H-) cells: Role of tyrosine phos- ,*2. pholylation pathway. Biochem. Biophys. Res. Commun., ,1., Schwartz, L.B., Lewis, R.A., Seldin, D. and Austen, K.F. (+32+). 0*-ῌ0*2. Acid hydrolases and tryptase from secretory granules of dis-