J Sci Food Agric 1998, 76, 56È62

Possible Immunodulating Activities in an Extractof Edible Brown Alga, Hijikia fusiforme (Hijiki)

Yasuji Okai,1* Kiyoka Higashi-Okai,1 Shigeaki Ishizaka,2 Kimiko Ohtani,3Isao Matsui-Yuasa3 and Uki Yamashita4

1 Department of Human Life Science, Osaka Kun-Ei Womens College, Showjaku, Sets City, Osaka 566,Japan2 Department of Parasitology, Nara Medical University, Shijho-cho, Kashihara, Nara 634, Japan3 Department of Food and Nutrition, Faculty of Human Life science, Osaka City University,Sugimoto-cho, Sumiyoshi-Ku, Osaka 558, Japan4 Department of Immunology, University of Occupational and Environmental Health, Iseigaoka,Yahata Nishi-Ku, Kitakyushu 807, Japan

(Received 31 May 1996 ; revised version received 25 April 1997 ; accepted 13 June 1997)

Abstract : A signiÐcant immunomodulating activity was found in the hot-water-soluble extract of an edible brown seaweed, Hijikia fusiforme (Hijiki in Japanese)which showed an enhancing activity for the proliferative response of spleen cellsin endotoxin-nonresponder C3H/HeJ mice. This activity was separated intopolysaccharide and nonpolysaccharide fractions. The former fraction exhibited amuch higher activity than that of the latter fraction. The enhancing e†ect of thepolysaccharide fraction on the proliferative response of spleen cells was associ-ated with the response of the B cell population, but not with that of the T cellpopulation judged by experiments using nylon wool column-puriÐed T cells andantisera against B cell- or T-cell-speciÐc antigens. The active component of thepolysaccharide fraction was further fractionated using DEAE cellulose columnchromatography which also caused enhancing e†ects on polyclonal antibody(IgM and IgG) production and the release of interleukin-1a or tumour necrosisfactor-a from peritoneal macrophages of C3H/HeJ mice. In addition, theseimmunomodulating activities of the polysaccharide fraction were associated withthe polysaccharides themselves, but not with the artiÐcial activities induced bycontaminated endotoxins. The biochemical and physicochemical properties ofthe immunoenhancing polysaccharides were partially characterised and the sig-niÐcance of the present Ðnding is discussed from the viewpoint of the immuno-potentiating activity of edible seaweeds against carcinogenesis. 1998 SCI.(

J Sci Food Agric 76, 56È62 (1998)

Key words : immunodulating activity ; edible alga ; polysaccharidesHijiki ;

INTRODUCTION

Epidemiological studies have indicated that the major,but not all causes of important diseases such as cancerand coronary heart disease are associated with environ-mental factors including foods and the mode of lifestyle

* To whom correspondence should be addressed.

(Willet 1994). For example, in Japan, various kinds ofseaweed have been traditionally used as an additive orseasoning in cooking and it has been purported to havebeneÐcial properties, for example, in curing various dis-eases and maintaining the healthy state of individuals(Carper 1989). Other investigators reported that oraladministration of the powder of edible seaweeds causeda signiÐcant decrease in the incidence of chemically

561998 SCI. J Sci Food Agric 0022-5142/98/$17.50. Printed in Great Britain(

Immunodulating activities of edible brown alga 57

induced tumours in in vivo animal experiments(Yamamoto and Maruyama 1985). However, a detailedmechanism for the antitumour e†ects by seaweeds havenot been analysed. Recently, signiÐcant antigenotoxicactivities were observed in hot-water-soluble extractsfrom edible brown algae such as L aminaria japonica,Undaria pinnatiÐda and Hijikia fusiforme, which showedsuppressive e†ects on umu C gene expression in the SOSresponse of Salmonella typhimurium (TA 1535/pSK1002) induced by genotoxic substances (Okai et al 1993 ;Okai and Higashi-Okai 1994). The authors also foundthat methanol-soluble extracts of some edible seaweedscaused suppressive e†ects on tumour promotor-inducedbiochemical activation in in vitro cell culture experi-ments (Okai et al 1994). As another possible mechanismfor the antitumour e†ects by seaweeds, their immuno-potentiating activities can be considered. However, thedetails of the immunomodulating activities derived fromedible seaweeds have been poorly elucidated to date. Arecent report has showed that the polysaccharide frac-tions from an edible marine alga (Porphyra yezoensis)stimulated some functions of mouse macrophages in invitro and in vivo experiments (Yoshizawa et al 1993).

In the present paper, immunomodulating activities ofhot-water-soluble extract of an edible seaweed, Hijikiafusiforme were analysed using in vitro experiments.

MATERIALS AND METHODS

Preparation of seaweed extract and fractionation of thepolysaccharides

Hijikia fusiforme was harvested in the Ise district inJapan and dried fronds of the seaweed were mincedbrieÑy by an electric cutter apparatus (IFM-100,Iwatani Co, Tokyo, Japan) and added to a 20-foldvolume of hot water (80È90¡C). After cooling to roomtemperature for 30 min, the extract of seaweeds wasrecovered by centrifugation at 1500] g for 15 min andthen stored at [ 20¡C. The preparation of the poly-saccharides from the seaweeds was carried out by a pre-viously described method (Okai and Higashi-Okai1994). BrieÑy, after thawing the extract was mixed withthree volumes of cold ethanol in the presence of 0É3 M

NaCl, kept overnight at [ 20¡C, then centrifuged at5000 ] g for 15 min. The precipitate was dissolved withdistilled water and dialysed by a dialysis membranewith a cut-o† size of 13 kDa (Spectrum MedicalIndustries Inc, Los Angeles, CA, USA) against 1 litre ofdistilled water overnight at 4¡C. The supernatant(nonpolysaccharide fraction) was concentrated using arotary evaporator at 45¡C and adjusted to equal thesame volume of the polysaccharide fraction with dis-tilled water. The polysaccharide fraction was applied toa DEAE cellulose (DE 52) column (1É8 ] 24 cm,

Whatman Co, Maidstone, Kent, UK) which waswashed with distilled water and eluted with 25 mM

phosphate bu†er (pH 7É4) containing 0É5, 1É0 and 2É0 M

NaCl in a stepwise fashion. All fractions were stored at[ 20¡C. The sugar content in each fraction was mea-sured by the phenolÈsulphuric acid method (Dubois etal 1956).

Assay for the proliferative response of mouse spleen cells

The proliferative response of spleen cells were assayedby a slight modiÐcation of our previous method (Okaiet al 1985). Spleen cells (5] 105) from C3H/HeJ mice(8È10 weeks old, Seiwa Experimental Animal Co, Ohita,Japan) were suspended in 200 ll of RPMI 1640 medium(Nissui Seiyaku Co, Tokyo, Japan) supplemented with10% faetal bovine serum (FBS, Gibco Co, New York,NY, USA) and then 25 ll of test solution was added.The cells were cultured at 37¡C in 5% and 95%CO2humidiÐed air for 3 days and labelled with 0É5 lCi of[3H]thymidine (6 Ci mmol~1, Amersham, Buck-inghamshire, UK) for the last 18 h. The cells were har-vested using a semiautomatic cell harvester (AbeKagaku Co, Chiba, Japan) and the radioactivity incorp-orated into the cells was counted using a BeckmanLS-6500 scintillation counter.

Assay for the proliferative response of T cells puriÐedfrom mouse spleen cells

PuriÐcation of T cells from spleen cells was performedby the previously described by Yamashita andHamaoka (1979). BrieÑy, approximately 109 spleen cellssuspended in 10 ml RPMI 1640 medium-5% FBS wereapplied to a nylon wool column. After incubation onthe column for 1 h at 37¡C, nonadherent cells wereeluted in a dropwise fashion using RPMI-1640 medium-5% FBS. The proliferative response of puriÐed T cellswas assayed using [3H]thymidine as described abovefor the proliferative response of spleen cells.

Assay for the proliferative response of mouse spleen cellstreated with antiserum against B cell- or T cell-speciÐcantigen

Treatment of spleen cells with antiserum and com-plement was carried out by the method of Yamashitaand Hamaoka (1979). Spleen cells were treated with60-fold diluted anti-Thy 1 serum (Olac, Blackthorn, UK)or serum (Cederlane, Ontario, Canada) atanti-IgG14¡C for 25 min, then incubated at 37¡C for 25 min.After washing with RPMI 1640 medium, the spleen cellswere resuspended with 30-fold diluted rabbit com-plement, then incubated at 37¡C for 50 min. After

58 Y Okai et al

TABLE 1E†ects of the extract of H fusiforme on the proliferative

response of spleen cells from C3H/HeJ micea

T est sample [3H]T dR incorporation(CPM)

Control (PBS) 462 ^ 25Dilution ratio

1 : 20 15 820^ 13561 : 50 5104 ^ 3921 : 150 2072 ^ 179

a The hot water-soluble extract of H fusiforme was prepared asdescribed. The extract was successively diluted withphosphate-bu†ered saline (PBS) and 20 ll of each dilutionwas added to the cell culture at a Ðnal volume of 200 ll whichcorresponded to 10 ll, 4 ll or 1É33 ll of the extract. Thevalues shown represent the mean ^ SD of triplicate assays.

washing the cells three times with RPMI 1640 medium,the proliferative response of these cells was assayedusing [3H]-thymidine as described above for the pro-liferative response of spleen cells.

Assay for antibody production by mouse spleen cells

Assay for antibody production was carried out by apreviously described method (Okai and Ishizaka 1989).Spleen cells of C3H/HeJ mice were washed twice withHankÏs balanced salt solution (HBSS, Nissui SeiyakuCo, Tokyo, Japan) and the cells (2] 106) were sus-pended in 100 ll of RPMI 1640 medium-10% FBS,

TABLE 2E†ects of the polysaccharide and nonpolysaccharide fractionsof the extract of H fusiforme on the proliferative response of

C3H/HeJ mouse spleen cellsa

T est sample [3H]T dR incorporation(CPM)

Control (PBS) 539 ^ 28Polysaccharide fraction

Dilution ratio1 : 20 9054 ^ 5111 : 50 4633 ^ 420

Nonpolysaccharide fractionDilution ratio

1 : 20 3172 ^ 2791 : 50 2293 ^ 182

E coli lipopolysaccharide 848^ 17(25 lg ml~1)

a Dilutions were made by the same method as described inTable 1. The values shown represent the mean^ SD of tripli-cate assays.

then cultured with 25 ll of test solution supplementedwith 75 ll of RPMI 1640 medium-10% FBS in 5% CO2and humidiÐed 95% air at 37¡C for 4 days. The numberof antibody-forming cells was determined by protein A-plaque forming cell assay as follows : 25 ll of 10-folddiluted protein A-coupled SRBC suspension (FunakoshiCo, Tokyo, Japan), 25 ll splenocyte suspension, 25 ll ofa 100-fold diluted anti-mouse IgM and IgG serum(Litton Bionetics, Kensington, USA) and 25 ll of10-fold diluted guinea pig complement were mixed with200 ll of 1% melted agar (Difco, Detroit, MI, USA)containing 0É05% DEAE dextran (Pharmacia, Uppsala,Sweden). The mixture was placed on Petri dishes,covered with a coverglass, and incubated at 37¡C for4 h. The number of plaque-forming cells (PFC) werethen counted. The results were expressed as themean^ SD of triplicate assays.

Preparation of mouse peritoneal macrophages

Mouse macrophages were prepared by our previousmethod (Okai and Ishizaka 1994). Saline was injectedinto the abdominal cavity of C3H/HeJ mouse and per-itoneal cells were recovered by suction with a Pasteurpipette. The recovered cells were washed with saline,suspended with RPMI 1640 medium and 5% FBS, andoverlaid on a plastic plate to stand for 1 h at 37¡C.After removing nonadherent cells by washing with thesame medium, the adherent cells were treated with 500-fold diluted monoclonal anti-Thyl.2 (Olac Co, Black-thorn, UK) supplemented with guinea pig complementand incubated for 1 h at 37¡C in 5% CO. The adherentcells were recovered by gentle sweeping with a rubberpoliceman, then suspended with RPMI 1640 mediumand 5% FBS. The purity of phagocytes was about 98%as judged by an ingestion test of latex bead particles.

Assay for the content of interleukin-1a (IL-1a) andtumour necrosis factor-a (TNF-a) released from mousemacrophages

Mouse macrophages (5 ] 105) were suspended in 1 mlof RPMI 1640 medium-1% FBS and appropriate dosesof polysaccharide from H fusiforme were added to thecell culture medium. After incubation at 37¡C for 24 hin 5% the culture medium was recovered, andCO2 ,frozen at [80¡C. The amount of IL-1a or TNF-a in theculture medium was measured by commercial cytokineassay kit (Genzyme Co, Cambridge, MA, USA) accord-ing to the manufactureÏs instructions. Serial dilutions ofthe test samples were compared with the colour devel-opment of standard puriÐed IL-1a or TNF-a using aspectrophotometer for EIA (Intermed, Tokyo, Japan).

Immunodulating activities of edible brown alga 59

Fig 1. Fractionation of the polysaccharides from H fusiforme by DEAE cellulose column chromatography. The crude poly-saccharide fraction from H fusiforme was dialysed extensively against distilled water and applied on a DE 52 column as describedin the Materials and Methods section. The arrows in the Ðgure show the positions of the elution with distilled water : 25 mM

phosphate bu†er (pH 7É4) containing 0É5 M, 1É0 M or 2É0 M NaCl from the left to the right. Open and closed circles show the[3H]thymidine incorporating activities of splenocytes and the sugar content in each fraction, respectively.

RESULTS

As shown in Table 1, a dose-dependent enhancing activ-ity for the proliferative response of spleen cells fromendotoxin-nonresponder C3H/HeJ mice was observedin a hot-water-soluble extract of H fusiforme. Theextract was further separated into polysaccharide andnonpolysaccharide fractions. As indicated in Table 2,although both fractions showed signiÐcant enhancinge†ects on the proliferative response of spleen cells, thepolysaccharide fraction caused a much higher activity.

In addition, included as a control, a relatively high con-centration of E coli lipopolysaccharide (25 lg ml~1)showed a very weak activity compared with that of thepolysaccharide fraction (Table 2).

Further elucidation of the polysaccharide fraction byDEAE cellulose column chromatography detected thedominant activity in the bound fractions of the chroma-tography (Fig 1). The major activity was eluted withbu†er (pH 7É4) containing 0É5 NaCl. The active frac-tions of the DEAE cellulose column chromatography(nos 13È18 in Fig 1) were collected, concentrated and

TABLE 3The polysaccharide fraction-induced proliferative response is associ-

ated with the response of B cells but not with T cellsa

(3H)T dR incorporationExperimental condition (CPM)

Experiment 1Spleen cells] polysaccharide fraction 17090 ^ 302T cells] polysaccharide fraction 1292^ 302

Experiment 2Spleen cells] polysaccharide fraction 14696 ^ 139Anti-Thy 1 serum ] complement

] polysaccharide fraction 13121 ^ 365[ polysaccharide fractions 1747 ^ 161

Anti-IgG1 serum ] complement] polysaccharide fraction 5721 ^ 191[ polysaccharide fraction 2346 ^ 125

a The concentration of polysaccharides in the cell culture mediumwas 100 lg ml~1. The values shown represent the mean^ SD oftriplicate assays.

60 Y Okai et al

analysed for their e†ects on various functions of immuno-competent cells of C3H/HeJ mice using in vitro cellculture experiments. IdentiÐcation of the cell populationassociated with the proliferative response of spleen cellsinduced by the polysaccharide fraction was then under-taken. PuriÐed T cells did not respond to the poly-saccharide fraction at a high concentration(100 lg ml~1) (did not show signiÐcant incorporation of[3H]thymidine) (Table 3). When spleen cells weretreated with anti-Thy 1 serum, they exhibited a remark-able incorporation of [3H]thymidine induced by thepolysaccharide fraction indicating that the antiserumagainst T cell-speciÐc antigen did not cause signiÐcantsuppression of the polysaccharide fraction-induced pro-liferative response (Table 3).

In contrast, the treatment with serumanti-IgG1,caused a considerable reduction of [3H]thymidineincorporation (Table 3).

These results indicate that the polysaccharidefraction-induced proliferative response of spleen cells is

associated with the response of the B cell population,but not with that of T cell population.

As shown in Table 4, the polysaccharide fractioncaused stimulatory e†ects on the production of anti-body (IgM and IgG) in a dose-dependent manner.These results suggest that the polysaccharide fractionfrom H fusiforme has an adjuvant activity for antibodyproduction of B lymphocytes in C3H/HeJ mice.

The e†ect of the polysaccharide fraction on therelease of IL-1a into the culture medium from phago-cytic cells of C3H/HeJ mice was also investigated. Asshown in Table 5, when the cells were treated with10 lg ml~1 polysaccharide, a signiÐcant increase inIL-1a concentration was observed in the cell culturemedium compared with that of the control experiment.Furthermore, the addition of higher concentrations ofpolysaccharide (25È50 lg ml~1) caused much morestimulation of release of IL-1a from the cells. However,E coli lipopolysaccharide (50 lg ml~1) did not showsigniÐcant stimulation for the release of IL-1a.

TABLE 4E†ects of DEAE-fractionated polysaccharides from H fusiforme on poly-

clonal antibody production in mouse spleen cellsa

Antibody production(PFC per 106 cells)

T est sample IgM IgG

Control (PBS) 185 ^ 23 228 ^ 19] Polysaccharide

10 lg ml~1 386 ^ 27 562 ^ 4225 lg ml~1 770 ^ 61 1012 ^ 54100 lg ml~1 1078 ^ 125 1315 ^ 188

E coli lipopolysaccharide (25 lg ml~1) 205^ 31 286 ^ 35

a The active fractions (nos 13È18) of DE 52 column chromatographypuriÐed polysaccharide fraction (see Fig 1) were added to the assaysystem in di†ering amounts. The value shown represent the mean^ SDof triplicate assays.

TABLE 5E†ects of DEAE-fractionated polysaccharides from H fusiforme

on the release of interleukin-1a from mouse macrophagesa

IL 1a concentrationT est sample (pg ml~1)

Control (PBS) 7 ^ 0É3] Polysaccharide

10 lg ml~1 75 ^ 1425 lg ml~1 191 ^ 28100 lg ml~1 364 ^ 40

E coli lipopolysaccharide (50 lg ml~1) 12^ 1

a The active fractions (nos 13È18) of DE 52 column chromatog-raphy puriÐed polysaccharide fraction were added to the culturesystem of mouse macrophages in di†ering amounts. The valuesare expressed as the mean ^ SD of triplicate assays.

Immunodulating activities of edible brown alga 61

The e†ect of the polysaccharide on the release ofTNF-a was also examined. As indicated in Table 6, thepolysaccharide-treated cells released much more TNF-athan the control. These results indicate that poly-saccharide from H fusiforme has a possible enhancingactivity for the release of cytokines such as IL-1a andTNF-a from mouse macrophages.

DISCUSSION

The results of this study indicate that the hot-water-soluble extract of an edible brown alga, H fusiforme,contains immunomodulating polysaccharides whichinduced proliferative responses of spleen cells fromC3H/HeJ mice. This activity was associated with theresponse of the B cell population, but not with that ofthe T cell population. Enhancing e†ects were alsoobserved on the polyclonal antibody production of Blymphocytes. Furthermore, macrophages were stimu-lated to release typical cytokines such as IL-1a andTNF-a. The immunomodulating activities by the poly-saccharide fraction are associated with the poly-saccharides themselves, but not with contaminatingendotoxins for the following reasons. First of all, toexclude the artiÐcial activity by contaminating endo-toxins from the true immunological activity, the presentstudy was carried out in an endotoxin-nonresponderstrain of mice. As shown in Table 2, the polysaccharidefraction from H fusiforme caused a marked proliferativeresponse of spleen cells, but a similar concentration of Ecoli lipopolysaccharide exhibited a very weak response.

Furthermore, the experimental system for the anti-body production was designed to remove theendotoxin-induced antibody production according to aprevious report by Ishizaka (1983) which showed thatthe spleen cells of endotoxin-nonresponder mice arecompletely nonresponsive to endotoxins under high celldensity conditions. In the present study, the poly-

saccharide fraction from H fusiforme stimulated poly-clonal antibody production of spleen cells, but a similarconcentration of lipopolysaccharide did not cause sig-niÐcant antibody production (Table 4). In addition, thepolysaccharide fraction exhibited a stimulatory e†ect onthe release of cytokines from macrophages, but a rela-tively high concentration of lipopolysaccharide did notcause signiÐcant stimulation (Tables 5 and 6). ToconÐrm that the immunomodulating activities wereassociated with the polysaccharides themselves, thepolysaccharide fraction was treated with sodium perio-date which destroys the structure of polysaccharides.This treatment caused a drastic decrease in stimulatorye†ects such as proliferative response of spleen cells andthe release of cytokines from macrophages (data notshown). To analyse the possibility of the association ofthe proteins with the immunomodulating activities, theprotein content of the polysaccharide fraction was mea-sured. However, the protein content was too low todetect its amount which indicated that the proteincontent was less than 0É02% in total by mass. Further-more, treatment with proteases such as trypsin,chymotrypsin and papain did not a†ect theimmunomodulating activities of the polysaccharidefraction from H fusiforme (data not shown). Theseresults suggest that the immunomodulating activities inthe polysaccharide fraction from H fusiforme are associ-ated with the polysaccharides themselves, but not withthe contaminating endotoxins or proteins. Previously,several polysaccharide or polysaccharide-rich fractionsfrom extracts of brown algae with antitumor activityhave been reported. To compare these polysaccharideswith polysaccharides in the present paper, we pre-liminarily characterised the physicochemical propertiesof the polysaccharides from H fusiforme. Fujihara et al(1984) reported the sodium alginate from Sargassum ful-vellum and it contained as a major component, uronicacid (about 85% of total mass) which is composed ofmannuronic acid and gluronic acid. However, the

TABLE 6E†ects of DE 52-puriÐed polysaccharide fraction from H fusiforme

on TNF-a released from mouse phagocytic cells

T est sample T NF-a concentration(pg ml~1)

Control (PBS) 9 ^ 1] Polysaccharide

10 lg ml~1 38 ^ 525 lg ml~1 105 ^ 18100 lg ml~1 231 ^ 12

E coli lipopolysaccharide (50 lg ml~1) 14 ^ 2

a The active fractions (nos 13È18) of DE 52 column chromatog-raphy puriÐed polysaccharide fraction were added to the phago-cytic cell culture. The values are expressed as the mean^ SD oftriplicate assays.

62 Y Okai et al

content of uronic acid in the polysaccharides of H fusi-forme was much lower than that of the previous report(less than 30% of total mass) and a signiÐcant amountof mannuronic acid and gluronic acid could not bedetected. The same study group has shown that sul-phinated galactofucans from Sargassam kjellmanianumcontain a considerable amount of neutral sugars com-posed of the major sugar monomer, galactose andfucose (20È39% and 48È77% in total neutral sugars,respectively) (Nagumo et al 1988). The polysaccharidesfrom H fusiforme are composed of the major neutralsugars, mannose and galactose. Recently Yoshizawa etal (1993) reported macrophage-stimulating activities ofporphyran-rich fractions from a red alga, Porphyrayezoensis which include dominantly a basic disacchariderepeating unit of 3-O-(3,6-anhydro-L-galactopyranosyl)-b-D-galactopranose. Although the detailed physico-chemical analysis of the polysaccharides from H fusi-forme has not been carried out to date, the preliminaryanalysis suggests that the polysaccharides from H fusi-forme might have di†erent structures compared withalgal polysaccharides reported previously.

The results seem to o†er a possible explanation forthe anti-tumour e†ects by edible seaweeds, but immuno-modulating activity by seawood may be insufficientto explain the anti-tumour e†ect completely. Otheractivities derived from seaweed besides immuno-modulating activity may be associated with anti-tumoure†ects. Recently, signiÐcant antigenotoxic activities weredetected in the hot-water-soluble extracts from ediblebrown algae which were divided into the polysaccharideand nonpolysaccharide fractions (Okai et al 1993 ; Okaiand Higashi-Okai 1994). The authors also found thatmethanol-soluble extract of edible seaweeds caused sup-pressive e†ects on tumour promotor-induced biochemi-cal activation in in vitro experiments (Okai et al 1994).Although it cannot be determined at this stage whichactivity plays a dominant role in protection against car-cinogenesis, the additive or synergistic e†ects of theseactivities in seaweed extracts seem to contribute to pro-tection.

REFERENCES

Carper J 1989 Seaweed or Kelp. In : T he Food PharmacyBantam Books, New York, pp 264È268.

Dubois M, Gillis K A, Hamilton J K, Rebers P A, Smith F1956 Collorimetric method for determination of sugars andrelated substances. Anal Chem 28 350È356

Fujihara M, Iijima N, Yamamoto I, Nagumo T 1984 PuriÐ-cation and chemical and physical characterization of ananti-tumor polysaccharide from the brown seaweed, Sar-gassum fevellum Carbohydr Res 125 97È106.

Ishizaka S 1983 Modulation of lipopolysaccharide non-responder cells in low cell density culture. Immunol L ett 6343È350.

Nagumo T, Iizima-Mizui N, Fujihara M, Himeno J,Komiyama K, Umezawa I 1988 Separation of sulfated,fucose-containing polysaccharides from brown seaweed,Sargassum kjellmanianum and their heterogeneity andantitumor activity. Kitasato Arch of Exp Med 61 59È67.

Okai Y, Gotoh S, Yamashita U 1985 Thymocyte-activatingfactors from SV40-transformed human embryo Ðbroblasts.Immunol L ett 9 153È159.

Okai Y, Higashi-Okai K, Nakamura S 1993 IdentiÐcation ofheterogenous antimutagenic activities in the extract ofedible brown seaweeds, L aminaria japonica (Makonbu) andUndaria pinnatiÐda (Wakame) by umu gene expressionsystem in Salmonella typhimurium (TA 1535/pSK 1002).Mutation Res 302 63È70.

Okai Y, Higashi-Okai K 1994 IdentiÐcation of antimutagenicactivities in the extract of an edible brown alga, Hijikia fusi-forme (Hijiki) by umu gene expression system in Salmonellatyphimurium (TA 1535/pSK 1002), J Sci Food Agric 66 103È110.

Okai Y, Higashi-Okai K, Nakamura S, Yano Y, Otani S 1994Suppressive e†ects of the extracts of Japanese edible sea-weeds on mutagen-induced umu C gene expression in Sal-monella typhimurium (TA 1535/pSK 1002) and tumorpromotor-dependent ornithine decarboxylase induction inBALB/c 3T3 Ðbroblast cells. Cancer L ett 87 25È32.

Okai Y, Ishizaka S 1989 Spontaneous release of B lympho-cytes enhancing factors for growth and di†erentiation byYH-l cells in serum-free culture media-IdentiÐcation ofInterleukin l activities. Zool Science 6 523È532.

Okai Y, Ishizaka S 1994 A possible immunomodulating activ-ity of Arbekacin (ABK), A newly synthesized antibioticagainst methicillin-resistant Staphylococcus aureus (MRSA).Int J Immunopharmacol 16 321È327.

Willet W C 1994 Diet and Health : What should we eat?Science 264 532È537.

Yamamoto I, Maruyama H 1985 E†ect of dietary seaweedpreparation on 1,2-dimethylhydrazine-induced intestinalcarcinogenesis in rats. Cancer L ett 26 241È251.

Yamashita U, Hamaoka T 1979 The requirement of Ia-positive accessary cells for the induction of hapten-reactivecytotoxic T lymphocytes in vitro. J Immunol 123 2637È2643.

Yoshizawa Y, Enomoto A, Todho H, Ametani A, Kamino-gawa S 1993 Activation of murine macrophages by poly-saccharide fractions from marine algae (Porphyra yezoensis).Biosci Biotech Biochem 57 1862È1866.