J Sci Food Agric 1998, 76, 56È62 Possible Immunodulating Activities in an Extract of Edible Brown Alga, Hijikia fusiforme (Hijiki) Yasuji Okai,1* Kiyoka Higashi-Okai,1 Shigeaki Ishizaka,2 Kimiko Ohtani,3 Isao Matsui-Yuasa3 and Uki Yamashita4 1 Department of Human Life Science, Osaka Kun-Ei Womens College, Showjaku, Sets City, Osaka 566, Japan 2 Department of Parasitology, Nara Medical University, Shijho-cho, Kashihara, Nara 634, Japan 3 Department of Food and Nutrition, Faculty of Human Life science, Osaka City University, Sugimoto-cho, Sumiyoshi-Ku, Osaka 558, Japan 4 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-watersoluble extract of an edible brown seaweed, Hijikia fusiforme (Hijiki in Japanese) which showed an enhancing activity for the proliferative response of spleen cells in endotoxin-nonresponder C3H/HeJ mice. This activity was separated into polysaccharide and nonpolysaccharide fractions. The former fraction exhibited a much higher activity than that of the latter fraction. The enhancing e†ect of the polysaccharide fraction on the proliferative response of spleen cells was associated with the response of the B cell population, but not with that of the T cell population judged by experiments using nylon wool column-puriÐed T cells and antisera against B cell- or T-cell-speciÐc antigens. The active component of the polysaccharide fraction was further fractionated using DEAE cellulose column chromatography which also caused enhancing e†ects on polyclonal antibody (IgM and IgG) production and the release of interleukin-1a or tumour necrosis factor-a from peritoneal macrophages of C3H/HeJ mice. In addition, these immunomodulating activities of the polysaccharide fraction were associated with the polysaccharides themselves, but not with the artiÐcial activities induced by contaminated endotoxins. The biochemical and physicochemical properties of the immunoenhancing polysaccharides were partially characterised and the signiÐcance of the present Ðnding is discussed from the viewpoint of the immunopotentiating activity of edible seaweeds against carcinogenesis. ( 1998 SCI. J Sci Food Agric 76, 56È62 (1998) Key words : immunodulating activity ; edible alga ; Hijiki ; polysaccharides INTRODUCTION (Willet 1994). For example, in Japan, various kinds of seaweed have been traditionally used as an additive or seasoning in cooking and it has been purported to have beneÐcial properties, for example, in curing various diseases and maintaining the healthy state of individuals (Carper 1989). Other investigators reported that oral administration of the powder of edible seaweeds caused a signiÐcant decrease in the incidence of chemically Epidemiological studies have indicated that the major, but not all causes of important diseases such as cancer and coronary heart disease are associated with environmental factors including foods and the mode of lifestyle * To whom correspondence should be addressed. 56 ( 1998 SCI. J Sci Food Agric 0022-5142/98/$17.50. Printed in Great Britain Immunodulating activities of edible brown alga induced tumours in in vivo animal experiments (Yamamoto and Maruyama 1985). However, a detailed mechanism for the antitumour e†ects by seaweeds have not been analysed. Recently, signiÐcant antigenotoxic activities were observed in hot-water-soluble extracts from edible brown algae such as L aminaria japonica, Undaria pinnatiÐda and Hijikia fusiforme, which showed suppressive e†ects on umu C gene expression in the SOS response of Salmonella typhimurium (TA 1535/pSK 1002) induced by genotoxic substances (Okai et al 1993 ; Okai and Higashi-Okai 1994). The authors also found that methanol-soluble extracts of some edible seaweeds caused suppressive e†ects on tumour promotor-induced biochemical activation in in vitro cell culture experiments (Okai et al 1994). As another possible mechanism for the antitumour e†ects by seaweeds, their immunopotentiating activities can be considered. However, the details of the immunomodulating activities derived from edible seaweeds have been poorly elucidated to date. A recent report has showed that the polysaccharide fractions from an edible marine alga (Porphyra yezoensis) stimulated some functions of mouse macrophages in in vitro and in vivo experiments (Yoshizawa et al 1993). In the present paper, immunomodulating activities of hot-water-soluble extract of an edible seaweed, Hijikia fusiforme were analysed using in vitro experiments. MATERIALS AND METHODS Preparation of seaweed extract and fractionation of the polysaccharides Hijikia fusiforme was harvested in the Ise district in Japan and dried fronds of the seaweed were minced brieÑy by an electric cutter apparatus (IFM-100, Iwatani Co, Tokyo, Japan) and added to a 20-fold volume of hot water (80È90¡C). After cooling to room temperature for 30 min, the extract of seaweeds was recovered by centrifugation at 1500 ] g for 15 min and then stored at [ 20¡C. The preparation of the polysaccharides from the seaweeds was carried out by a previously described method (Okai and Higashi-Okai 1994). BrieÑy, after thawing the extract was mixed with three volumes of cold ethanol in the presence of 0É3 M NaCl, kept overnight at [ 20¡C, then centrifuged at 5000 ] g for 15 min. The precipitate was dissolved with distilled water and dialysed by a dialysis membrane with a cut-o† size of 13 kDa (Spectrum Medical Industries Inc, Los Angeles, CA, USA) against 1 litre of distilled water overnight at 4¡C. The supernatant (nonpolysaccharide fraction) was concentrated using a rotary evaporator at 45¡C and adjusted to equal the same volume of the polysaccharide fraction with distilled water. The polysaccharide fraction was applied to a DEAE cellulose (DE 52) column (1É8 ] 24 cm, 57 Whatman Co, Maidstone, Kent, UK) which was washed 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 measured by the phenolÈsulphuric acid method (Dubois et al 1956). Assay for the proliferative response of mouse spleen cells The proliferative response of spleen cells were assayed by a slight modiÐcation of our previous method (Okai et 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 with 10% 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% CO and 95% 2 humidiÐed air for 3 days and labelled with 0É5 lCi of [3H]thymidine (6 Ci mmol~1, Amersham, Buckinghamshire, UK) for the last 18 h. The cells were harvested using a semiautomatic cell harvester (Abe Kagaku Co, Chiba, Japan) and the radioactivity incorporated into the cells was counted using a Beckman LS-6500 scintillation counter. Assay for the proliferative response of T cells puriÐed from mouse spleen cells PuriÐcation of T cells from spleen cells was performed by the previously described by Yamashita and Hamaoka (1979). BrieÑy, approximately 109 spleen cells suspended in 10 ml RPMI 1640 medium-5% FBS were applied to a nylon wool column. After incubation on the column for 1 h at 37¡C, nonadherent cells were eluted in a dropwise fashion using RPMI-1640 medium5% FBS. The proliferative response of puriÐed T cells was assayed using [3H]thymidine as described above for the proliferative response of spleen cells. Assay for the proliferative response of mouse spleen cells treated with antiserum against B cell- or T cell-speciÐc antigen Treatment of spleen cells with antiserum and complement was carried out by the method of Yamashita and Hamaoka (1979). Spleen cells were treated with 60-fold diluted anti-Thy 1 serum (Olac, Blackthorn, UK) or anti-IgG serum (Cederlane, Ontario, Canada) at 1 4¡C for 25 min, then incubated at 37¡C for 25 min. After washing with RPMI 1640 medium, the spleen cells were resuspended with 30-fold diluted rabbit complement, then incubated at 37¡C for 50 min. After Y Okai et al 58 TABLE 1 E†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) Dilution ratio 1 : 20 1 : 50 1 : 150 462 ^ 25 15 820 ^ 1356 5104 ^ 392 2072 ^ 179 a The hot water-soluble extract of H fusiforme was prepared as described. The extract was successively diluted with phosphate-bu†ered saline (PBS) and 20 ll of each dilution was added to the cell culture at a Ðnal volume of 200 ll which corresponded to 10 ll, 4 ll or 1É33 ll of the extract. The values shown represent the mean ^ SD of triplicate assays. washing the cells three times with RPMI 1640 medium, the proliferative response of these cells was assayed using [3H]-thymidine as described above for the proliferative response of spleen cells. Assay for antibody production by mouse spleen cells Assay for antibody production was carried out by a previously described method (Okai and Ishizaka 1989). Spleen cells of C3H/HeJ mice were washed twice with HankÏs balanced salt solution (HBSS, Nissui Seiyaku Co, Tokyo, Japan) and the cells (2 ] 106) were suspended in 100 ll of RPMI 1640 medium-10% FBS, TABLE 2 E†ects of the polysaccharide and nonpolysaccharide fractions of 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) Polysaccharide fraction Dilution ratio 1 : 20 1 : 50 Nonpolysaccharide fraction Dilution ratio 1 : 20 1 : 50 E coli lipopolysaccharide (25 lg ml~1) 539 ^ 28 9054 ^ 511 4633 ^ 420 3172 ^ 279 2293 ^ 182 848 ^ 17 a Dilutions were made by the same method as described in Table 1. The values shown represent the mean ^ SD of triplicate assays. then cultured with 25 ll of test solution supplemented with 75 ll of RPMI 1640 medium-10% FBS in 5% CO 2 and humidiÐed 95% air at 37¡C for 4 days. The number of antibody-forming cells was determined by protein Aplaque forming cell assay as follows : 25 ll of 10-fold diluted protein A-coupled SRBC suspension (Funakoshi Co, Tokyo, Japan), 25 ll splenocyte suspension, 25 ll of a 100-fold diluted anti-mouse IgM and IgG serum (Litton Bionetics, Kensington, USA) and 25 ll of 10-fold diluted guinea pig complement were mixed with 200 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 for 4 h. The number of plaque-forming cells (PFC) were then counted. The results were expressed as the mean ^ SD of triplicate assays. Preparation of mouse peritoneal macrophages Mouse macrophages were prepared by our previous method (Okai and Ishizaka 1994). Saline was injected into the abdominal cavity of C3H/HeJ mouse and peritoneal cells were recovered by suction with a Pasteur pipette. The recovered cells were washed with saline, suspended with RPMI 1640 medium and 5% FBS, and overlaid on a plastic plate to stand for 1 h at 37¡C. After removing nonadherent cells by washing with the same medium, the adherent cells were treated with 500fold diluted monoclonal anti-Thyl.2 (Olac Co, Blackthorn, UK) supplemented with guinea pig complement and incubated for 1 h at 37¡C in 5% CO. The adherent cells were recovered by gentle sweeping with a rubber policeman, then suspended with RPMI 1640 medium and 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) and tumour necrosis factor-a (TNF-a) released from mouse macrophages Mouse macrophages (5 ] 105) were suspended in 1 ml of RPMI 1640 medium-1% FBS and appropriate doses of polysaccharide from H fusiforme were added to the cell culture medium. After incubation at 37¡C for 24 h in 5% CO , the culture medium was recovered, and 2 frozen at [80¡C. The amount of IL-1a or TNF-a in the culture medium was measured by commercial cytokine assay kit (Genzyme Co, Cambridge, MA, USA) according to the manufactureÏs instructions. Serial dilutions of the test samples were compared with the colour development of standard puriÐed IL-1a or TNF-a using a spectrophotometer 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 polysaccharide fraction from H fusiforme was dialysed extensively against distilled water and applied on a DE 52 column as described in 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 activity for the proliferative response of spleen cells from endotoxin-nonresponder C3H/HeJ mice was observed in a hot-water-soluble extract of H fusiforme. The extract was further separated into polysaccharide and nonpolysaccharide fractions. As indicated in Table 2, although both fractions showed signiÐcant enhancing e†ects on the proliferative response of spleen cells, the polysaccharide fraction caused a much higher activity. In addition, included as a control, a relatively high concentration of E coli lipopolysaccharide (25 lg ml~1) showed a very weak activity compared with that of the polysaccharide fraction (Table 2). Further elucidation of the polysaccharide fraction by DEAE cellulose column chromatography detected the dominant activity in the bound fractions of the chromatography (Fig 1). The major activity was eluted with bu†er (pH 7É4) containing 0É5 NaCl. The active fractions of the DEAE cellulose column chromatography (nos 13È18 in Fig 1) were collected, concentrated and TABLE 3 The polysaccharide fraction-induced proliferative response is associated with the response of B cells but not with T cellsa Experimental condition Experiment 1 Spleen cells ] polysaccharide fraction T cells ] polysaccharide fraction Experiment 2 Spleen cells ] polysaccharide fraction Anti-Thy 1 serum ] complement ] polysaccharide fraction [ polysaccharide fractions Anti-IgG serum ] complement 1 ] polysaccharide fraction [ polysaccharide fraction (3H)T dR incorporation (CPM) 17090 ^ 302 1292 ^ 302 14696 ^ 139 13121 ^ 365 1747 ^ 161 5721 ^ 191 2346 ^ 125 a The concentration of polysaccharides in the cell culture medium was 100 lg ml~1. The values shown represent the mean ^ SD of triplicate assays. Y Okai et al 60 analysed for their e†ects on various functions of immunocompetent cells of C3H/HeJ mice using in vitro cell culture experiments. IdentiÐcation of the cell population associated with the proliferative response of spleen cells induced by the polysaccharide fraction was then undertaken. PuriÐed T cells did not respond to the polysaccharide fraction at a high concentration (100 lg ml~1) (did not show signiÐcant incorporation of [3H]thymidine) (Table 3). When spleen cells were treated with anti-Thy 1 serum, they exhibited a remarkable incorporation of [3H]thymidine induced by the polysaccharide fraction indicating that the antiserum against T cell-speciÐc antigen did not cause signiÐcant suppression of the polysaccharide fraction-induced proliferative response (Table 3). In contrast, the treatment with anti-IgG , serum 1 caused a considerable reduction of [3H]thymidine incorporation (Table 3). These results indicate that the polysaccharide fraction-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 fraction caused stimulatory e†ects on the production of antibody (IgM and IgG) in a dose-dependent manner. These results suggest that the polysaccharide fraction from H fusiforme has an adjuvant activity for antibody production of B lymphocytes in C3H/HeJ mice. The e†ect of the polysaccharide fraction on the release of IL-1a into the culture medium from phagocytic cells of C3H/HeJ mice was also investigated. As shown in Table 5, when the cells were treated with 10 lg ml~1 polysaccharide, a signiÐcant increase in IL-1a concentration was observed in the cell culture medium compared with that of the control experiment. Furthermore, the addition of higher concentrations of polysaccharide (25È50 lg ml~1) caused much more stimulation of release of IL-1a from the cells. However, E coli lipopolysaccharide (50 lg ml~1) did not show signiÐcant stimulation for the release of IL-1a. TABLE 4 E†ects of DEAE-fractionated polysaccharides from H fusiforme on polyclonal antibody production in mouse spleen cellsa Antibody production (PFC per 106 cells) T est sample IgM IgG Control (PBS) ] Polysaccharide 10 lg ml~1 25 lg ml~1 100 lg ml~1 E coli lipopolysaccharide (25 lg ml~1) 185 ^ 23 228 ^ 19 386 ^ 27 770 ^ 61 1078 ^ 125 205 ^ 31 562 ^ 42 1012 ^ 54 1315 ^ 188 286 ^ 35 a The active fractions (nos 13È18) of DE 52 column chromatography puriÐed polysaccharide fraction (see Fig 1) were added to the assay system in di†ering amounts. The value shown represent the mean ^ SD of triplicate assays. TABLE 5 E†ects of DEAE-fractionated polysaccharides from H fusiforme on the release of interleukin-1a from mouse macrophagesa T est sample Control (PBS) ] Polysaccharide 10 lg ml~1 25 lg ml~1 100 lg ml~1 E coli lipopolysaccharide (50 lg ml~1) IL 1a concentration (pg ml~1) 7 ^ 0É3 75 ^ 14 191 ^ 28 364 ^ 40 12 ^ 1 a The active fractions (nos 13È18) of DE 52 column chromatography puriÐed polysaccharide fraction were added to the culture system of mouse macrophages in di†ering amounts. The values are expressed as the mean ^ SD of triplicate assays. Immunodulating activities of edible brown alga The e†ect of the polysaccharide on the release of TNF-a was also examined. As indicated in Table 6, the polysaccharide-treated cells released much more TNF-a than the control. These results indicate that polysaccharide from H fusiforme has a possible enhancing activity for the release of cytokines such as IL-1a and TNF-a from mouse macrophages. DISCUSSION The results of this study indicate that the hot-watersoluble extract of an edible brown alga, H fusiforme, contains immunomodulating polysaccharides which induced proliferative responses of spleen cells from C3H/HeJ mice. This activity was associated with the response of the B cell population, but not with that of the T cell population. Enhancing e†ects were also observed on the polyclonal antibody production of B lymphocytes. Furthermore, macrophages were stimulated to release typical cytokines such as IL-1a and TNF-a. The immunomodulating activities by the polysaccharide fraction are associated with the polysaccharides themselves, but not with contaminating endotoxins for the following reasons. First of all, to exclude the artiÐcial activity by contaminating endotoxins from the true immunological activity, the present study was carried out in an endotoxin-nonresponder strain of mice. As shown in Table 2, the polysaccharide fraction from H fusiforme caused a marked proliferative response of spleen cells, but a similar concentration of E coli lipopolysaccharide exhibited a very weak response. Furthermore, the experimental system for the antibody production was designed to remove the endotoxin-induced antibody production according to a previous report by Ishizaka (1983) which showed that the spleen cells of endotoxin-nonresponder mice are completely nonresponsive to endotoxins under high cell density conditions. In the present study, the poly- 61 saccharide fraction from H fusiforme stimulated polyclonal antibody production of spleen cells, but a similar concentration of lipopolysaccharide did not cause signiÐcant antibody production (Table 4). In addition, the polysaccharide fraction exhibited a stimulatory e†ect on the release of cytokines from macrophages, but a relatively high concentration of lipopolysaccharide did not cause signiÐcant stimulation (Tables 5 and 6). To conÐrm that the immunomodulating activities were associated with the polysaccharides themselves, the polysaccharide fraction was treated with sodium periodate which destroys the structure of polysaccharides. This treatment caused a drastic decrease in stimulatory e†ects such as proliferative response of spleen cells and the release of cytokines from macrophages (data not shown). To analyse the possibility of the association of the proteins with the immunomodulating activities, the protein content of the polysaccharide fraction was measured. However, the protein content was too low to detect its amount which indicated that the protein content was less than 0É02% in total by mass. Furthermore, treatment with proteases such as trypsin, chymotrypsin and papain did not a†ect the immunomodulating activities of the polysaccharide fraction from H fusiforme (data not shown). These results suggest that the immunomodulating activities in the polysaccharide fraction from H fusiforme are associated with the polysaccharides themselves, but not with the contaminating endotoxins or proteins. Previously, several polysaccharide or polysaccharide-rich fractions from extracts of brown algae with antitumor activity have been reported. To compare these polysaccharides with polysaccharides in the present paper, we preliminarily characterised the physicochemical properties of the polysaccharides from H fusiforme. Fujihara et al (1984) reported the sodium alginate from Sargassum fulvellum and it contained as a major component, uronic acid (about 85% of total mass) which is composed of mannuronic acid and gluronic acid. However, the TABLE 6 E†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) ] Polysaccharide 10 lg ml~1 25 lg ml~1 100 lg ml~1 E coli lipopolysaccharide (50 lg ml~1) 9^1 38 ^ 5 105 ^ 18 231 ^ 12 14 ^ 2 a The active fractions (nos 13È18) of DE 52 column chromatography puriÐed polysaccharide fraction were added to the phagocytic cell culture. The values are expressed as the mean ^ SD of triplicate assays. Y Okai et al 62 content of uronic acid in the polysaccharides of H fusiforme was much lower than that of the previous report (less than 30% of total mass) and a signiÐcant amount of mannuronic acid and gluronic acid could not be detected. The same study group has shown that sulphinated galactofucans from Sargassam kjellmanianum contain a considerable amount of neutral sugars composed of the major sugar monomer, galactose and fucose (20È39% and 48È77% in total neutral sugars, respectively) (Nagumo et al 1988). The polysaccharides from H fusiforme are composed of the major neutral sugars, mannose and galactose. Recently Yoshizawa et al (1993) reported macrophage-stimulating activities of porphyran-rich fractions from a red alga, Porphyra yezoensis which include dominantly a basic disaccharide repeating unit of 3-O-(3,6-anhydro-L-galactopyranosyl)b-D-galactopranose. Although the detailed physicochemical analysis of the polysaccharides from H fusiforme has not been carried out to date, the preliminary analysis suggests that the polysaccharides from H fusiforme might have di†erent structures compared with algal polysaccharides reported previously. The results seem to o†er a possible explanation for the anti-tumour e†ects by edible seaweeds, but immunomodulating activity by seawood may be insufficient to explain the anti-tumour e†ect completely. Other activities derived from seaweed besides immunomodulating activity may be associated with anti-tumour e†ects. Recently, signiÐcant antigenotoxic activities were detected in the hot-water-soluble extracts from edible brown algae which were divided into the polysaccharide and nonpolysaccharide fractions (Okai et al 1993 ; Okai and Higashi-Okai 1994). The authors also found that methanol-soluble extract of edible seaweeds caused suppressive e†ects on tumour promotor-induced biochemical activation in in vitro experiments (Okai et al 1994). Although it cannot be determined at this stage which activity plays a dominant role in protection against carcinogenesis, the additive or synergistic e†ects of these activities in seaweed extracts seem to contribute to protection. 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