Identification and antioxidant activity of several pigments from the residual green tea (Camellia sinensis) after hot water extraction.

Antioxidant activity of green tea extract or tea-derived polyphenols has been extensively studied. However, antioxidant activity in the non-polyphenolic fraction of green tea has been poorly analyzed. In the present study, we analyzed the antioxidant activity of the non-polyphenolic fraction of the residual green tea (Camellia sinensis) after hot water extraction using the aluminum chloride method. The non-polyphenolic fraction of residual green tea caused a significant suppression against hydroperoxide generation from oxidized linoleic acid in a dose-dependent manner. When the concentrate of the non-polyphenolic fraction was applied to a silica gel TLC plate and developed, six color spots were observed, which were considered to be chlorophylls a and b, pheophytins a and b, carotenoids, such as beta-carotene and lutein according to their specific colors, Rf values of silica gel TLC and spectrophotometric properties. Among these pigments, pheophytins a and b showed relatively abundant amounts, and the second major group of the pigment was chlorophylls a and b, and carotenoids such as beta-carotene and lutein indicated lower concentrations. Although all these pigments exhibited significant antioxidant activities, the ranks of suppressive activity against hydroperoxide generation were chlorophyll a > lutein > pheophytin a > chlorophyll b > beta-carotene > pheophytin b. These results suggest that the non-polyphenolic fraction of residual green tea has a potent suppressive activity against hydroperoxide generation from oxidized linoleic acid, which is derived from the antioxidant activities of chlorophylls a and b, pheophytins a and b, beta-carotene and lutein. This finding also implies that the combined intake of polyphenols in water-soluble fraction and antioxidative pigments in the non-polyphenolic fraction of green tea will be more efficient to prevent life style-related chronic diseases.

Protective effects of alpha-tocopherol and beta-carotene on para-nonylphenol-induced inhibition of cell growth, cellular respiration and glucose-induced proton extrusion of bacteria.

para-Nonylphenol (NP) showed a dose-dependent inhibition against the cell growth of Bacillus subtilis, Micrococcus luteus, Pseudomonas aeruginosa and Staphylococcus aureus at 5-100 microM. However, other typical plastic-derived endocrine disruptors such as bisphenol A and di-2-ethylhexyl phthalate (DEHP) did not significantly affect the cell growth of these bacteria at 5-100 microM. The NP-induced cell growth inhibition was restored when concomitantly supplemented with lipophilic antioxidants such as alpha-tocopherol and beta-carotene, but not with hydrophilic antioxidants, ascorbic acid and (-)-epigallocatechin gallate (EGCG). NP also suppressed in a dose-dependent manner cellular oxygen consumption and glucose-induced proton extrusion of these bacteria at 10-100 microM. Both effects were prevented when added with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG. The significance of these results is discussed from the viewpoint of environmental microbiology and a possible biochemical mechanism of the inhibitory effect of NP is suggested.

Protective effect of antioxidants against para-nonylphenol-induced inhibition of cell growth in Saccharomyces cerevisiae.

The cell growth-modulating activity of an endocrine disruptor, p-nonylphenol (NP), was estimated using the yeast Saccharomyces cerevisiae as a simple model of eukaryotic cells. NP caused a dose-dependent suppressive effect on cell growth of S. cerevisiae at 10, 25 and 50 microM. The NP-induced cell growth inhibition was restored when concomitantly lipophilic antioxidants such as alpha-tocopherol and beta-carotene were supplied, but not the hydrophilic antioxidants ascorbic acid or (-)epigallocatechin gallate (EGCG). The cellular oxygen consumption of S. cerevisiae was also inhibited in a dose-dependent fashion by the extracellular addition of NP, and pretreatment with alpha-tocopherol and beta-carotene suppressed NP-induced inhibition of cellular oxygen consumption, but ascorbic acid and EGCG were not effective. Furthermore, NP caused a marked generation of radical oxygen species (ROS) in S. cerevisiae, which was suppressed by treatment with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG. However, NP did not show a significant inhibitory effect on cell growth and survival of mitochondria-deficient petite mutant cells and they showed a relatively weak ROS-generating activity compared with parent yeast cells. These results suggest that NP-induced inhibition of cell growth and oxygen consumption in S. cerevisiae might be possibly associated with ROS generation in yeast mitochondria. The significance of this finding is discussed from the viewpoint of NP-induced oxidative stress against eukaryotic cells.

Enhancing effect of a plastic plasticizer, di-2-ethylhexyl phthalate on umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002).

Di-2-ethylhexyl phthalate (DEHP) is the most extensively used phthalate ester as a plasticizer for plastic products made of polyvinyl chloride (PVC), and previous mutagenic and genotoxic studies have shown positive and negative results of DEHP-induced genotoxicity. To elucidate this discrepancy, we reestimated the genotoxicity of DEHP in more detail using the umu C gene expression system in Salmonella typhimurium (TA 1535/pSK 1002) which reflects SOS response against genotoxin-induced DNA damage. Although DEHP itself did not have a significant effect on umu C gene expression in tester bacteria at 0.5 to 4 mM, higher concentrations of DEHP (2 and 4 mM) caused a weak induction of umu C gene expression in the presence of commercially available S-9 mixture. Rat liver S-9 fraction alone also showed a similar weak inducing activity in the absence of substrates for drug-metabolizing enzymes. When DEHP was preincubated with S-9 fraction of various rat organs and applied to the umu C gene expression assay, S-9 fraction of rat pancreas had the strongest inducing activity, and S-9 fractions of liver and intestine homogenates showed weak but significant activities. However, S-9 fractions of lung and kidney homogenates did not exhibit any significant activities. These S-9 fractions have proportional lipase activities comparable with umu C gene expression activities. Furthermore, when DEHP was treated with highly purified lipase from porcine pancreas, a significant umu C gene expression was observed and this expression was enhanced in the presence of 1 or 5 mM bile acids such as choric acid and deoxychoric acid. These results suggest that DEHP itself has no or very low genotoxicity, but enzymatic and non-enzymatic factors in specific tissues induce DEHP-dependent genotoxic activity.

Potent suppressive effect of a Japanese edible seaweed, Enteromorpha prolifera (Sujiao-nori) on initiation and promotion phases of chemically induced mouse skin tumorigenesis.

Potent antigenotoxic and anti-tumor promoting activities of a Japanese edible seaweed, Enteromorpha prolifera (Sujiao-nori in Japanese) were previously identified using an in vitro cell culture experiment (Y. Okai, K. Higashi-Okai, S. Nakamura, Y. Yano, S. Otani, Cancer Lett. 87 (1994) 25-32). However, in vivo anti-carcinogenic activity of this seaweed has not been elucidated until now. In the present study, the anticarcinogenic activity of E. prolifera was analyzed using an initiation and promotion model experiment of mouse skin tumorigenesis caused by 7,12-dimethylbenz[a]anthracene (initiator) and 12-O-tetradecanoylphorbol-13-acetate (promoter). (1) Application of the extract of E. prolifera prior to the treatment with a tumor initiator or promoter caused a significant suppression against skin tumorigenesis, and the combined application of the extract prior to both treatments with initiator and promoter exhibited much stronger suppression against the same skin tumorigenesis. (2) As a possible active principle for the anticarcinogenic activity of the extract, we propose a chlorophyll-related compound, pheophytin-a, which has been recently identified in the extract of this alga as an antigenotoxic substance (Y. Okai, K. Higashi-Okai, J. Sci. Food Agric. 74 (1997) 531-535), and showed significant suppressive effects in the same tumorigenesis experiment. These results suggest that E. prolifera has a potent suppressive activity against chemically induced mouse skin tumorigenesis through the suppression at the initiation and promotion phases, and that pheophytin-a might be partially associated with the in vivo anticarcinogenic activity.

Potent suppressive activity of chlorophyll a and b from green tea (Camellia sinensis) against tumor promotion in mouse skin.

Potent antigenotoxic and anti-tumor promoting activities of chlorophyll a from green tea (camellia sinensis) have been shown using in vitro cell culture experiments (Okai Y. et al. (1996) Mutation Res., 370, 11-17). In the present study, the authors analyzed in vivo effects of chlorophyll a and b from green tea on tumor promotion in mouse skin in the following ways. 1. When chlorophyll a and b from green tea were applied before each treatment by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on BALB/c mouse skin initiated by 7, 12-dimethylbenz [a] an-thracene (DMBA), they caused significant suppression in a dose-dependent manner against BALB/c mouse skin tumorigenesis. 2. Chlorophyll a and b showed significant suppressive effects against TPA-induced inflammatory reaction such as edema formation in BALB/c mouse ear skin in a dose-dependent fashion. These results suggest that chlorophyll a and b possess potent suppressive activities against tumor promotion in mouse skin.

Potent suppressive activity of pheophytin a and b from the non-polyphenolic fraction of green tea (Camellia sinensis) against tumor promotion in mouse skin.

Chlorophyll-related compounds pheophytin a and b have been recently identified as antigenotoxic substances in the non-polyphenolic fraction of green tea (Camellia sinensis), which suppressed umu C gene expression in tester bacteria induced by various genotoxins (Okai and Higashi-Okai, Cancer Lett. 118 (1997) 117-123). In the present study, the authors analyzed in vivo and in vitro effects of pheophytin a and b from the non-polyphenolic fraction of green tea on tumor promotion in mouse skin as follows. (1) When pheophytin a and b from green tea were topically applied prior to each treatment with a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on BALB/c mouse skin initiated by 7,12 dimethylbenz[a]anthracene (DMBA), they caused suppression in a dose-dependent fashion against skin tumorigenesis. (2) Pheophytin a and b exhibited significant suppressions against TPA-induced inflammatory reaction, such as edema formation, in BALB/c mouse ear skin in a dose-dependent manner. (3) Pheophytin a and b from green tea showed inhibitory effects against early induction of ornithine decarboxylase (ODC) in BALB/c mouse skin fibroblasts caused by TPA. These results suggest that pheophytin a and b from the non-polyphenolic fraction have potent suppressive activities against tumor promotion in mouse skin.

Potent anti-inflammatory activity of pheophytin a derived from edible green alga, Enteromorpha prolifera (Sujiao-nori).

Recently, a chlorophyll-related compound, pheophytin a, has been identified from an edible green alga, Enteromorpha prolifera (Sujiao-nori in Japanese) as a potent suppressive substance against genotoxin-induced umu C gene expression in a tester bacteria (Okai and Higashi-Okai, 1997, J. Sci. Food Agricul. 71, 531-535). In the present study, anti-inflammatory effects of pheophytin a from Enteromorpha prolifera have been analyzed using in vitro and in vivo experiments. 1. Pheophytin a suppressed the production of superoxide anion (O2-) in mouse macrophages induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) using the cytochrome C reduction method. 2. Pheophytin a caused a suppressive effect against formyl-Met-Leu-Phe, (FMLP)-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) in Boyden's chamber experiment. 3. Pheophytin a exhibited a significant suppression against TPA-induced inflammatory reaction such as edema formation in BALB/c mouse ear. These results suggest that pheophytin a from Enteromorpha prolifera has a potent anti-inflammatory activity.

Enhancing effect of polysaccharides from an edible brown alga, Hijikia fusiforme (Hijiki), on release of tumor necrosis factor-alpha from macrophages of endotoxin-nonresponder C3H/HeJ mice.

An enhancing activity for the release of tumor necrosis factor-alpha from macrophages of C3H/HeJ mice was detected in the hot water-soluble extract of an edible brown alga, Hijikia fusiforme (Hijiki in Japanese). This activity was divided into the polysaccharide and nonpolysaccharide fractions, with the former showing much higher activity than the latter. The active components in the polysaccharide fraction were further purified by ion-exchange column chromatography and gel permeation system of high-performance liquid chromatography; they were identified as polysaccharides with apparent molecular mass of about 2,000 and 70 kDa and were designated Hijiki-derived polysaccharides I and II (HPS-I and HPS-II), respectively. They also enhanced macrophage-dependent suppression against the growth of EL-4 tumor cells in an in vitro culture experiment, with HPS-I exhibiting much higher immunologic activity than HPS-II. Furthermore, other comparative experiments confirmed that the immunoenhancing activities of polysaccharides from H. fusiforme are associated with the functions of polysaccharides themselves, but not with the artificial activity induced by contaminated endotoxins. Some biochemical properties of immunoenhancing polysaccharides were partially characterized, and the significance of this finding is discussed from the viewpoint of the protective role of edible seaweeds against carcinogenesis.

Potent suppressive activity of nonpolyphenolic fraction of green tea (Camellia sinensis) against genotoxin-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002), tumor promotor-dependent ornithine decarboxylase induction of BALB/c 3T3 fibroblast cells, and chemically induced mouse skin tumorigenesis.

Many experimental studies for anticarcinogenic activity of green tea (Camellia sinensis) and tea-derived polyphenols have been carried out. However, the anticarcinogenic activity of the nonpolyphenolic fraction of green tea has been poorly elucidated. To study this problem, the effect of the nonpolyphenolic fraction of green tea leaves was analyzed using in vitro and in vivo experiments associated with tumor initiation and promotion as follows: 1) The nonpolyphenolic fraction caused a strong suppressive effect on umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by genotoxic substances such as 2-amino-6-methyldipirido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-aminoanthracene (2-AA) in the presence of a hepatic metabolizing enzyme mixture. 2) The same fraction showed a dose-dependent inhibition of ornithine decarboxylase (ODC) in BALB/c 3T3 fibroblasts induced by a tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA). 3) The same fraction also exhibited a significant suppression against mouse skin tumorigenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) (initiator) and TPA (promotor) through inhibition at both stages of tumor initiation and promotion. These results suggest that the nonpolyphenolic fraction of green tea has a potent suppressing activity against carcinogenesis associated with tumor initiation and promotion.

Potent suppressing activity of the non-polyphenolic fraction of green tea (Camellia sinensis) against genotoxin-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002)--association with pheophytins a and b.

Antigenotoxic and antimutagenic activities of green tea extract and tea-derived polyphenols have been studied using in vitro and in vivo experiments. However, antigenotoxic substances in the non-polyphenolic fraction of green tea have been poorly elucidated. In the present study, the effect of the non-polyphenolic fraction of green tea on genotoxin-induced umu C gene expression was analyzed using a tester bacteria, and potent antigenotoxic substances in the non-polyphenolic fraction were identified. The non-polyphenolic fraction of green tea showed strong suppressive activities against umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1) or mitomycin C (MMC) in the presence or absence of S9 metabolizing enzyme mixture. The non-polyphenolic fraction of green tea exhibited major two-color bands in a silica gel TLC and they were identified as chlorophyll-related compounds, pheophytins a and b, judged by their specific colors, Rf values in silica gel TLC and absorption spectra. These pigments showed significant suppressive activities against umu C gene expression in tester bacteria induced by Trp-P- and MMC in a dose-dependent manner. These results suggest that the non-polyphenolic fraction of green tea contains pheophytins a and b as potent antigenotoxic substances.

Possible immunomodulating activities of carotenoids in in vitro cell culture experiments.

Immunomodulating activities of beta-carotene and carotene-associated carotenoids such as canthaxanthin (beta, beta-carotene-4,4 dione) and astaxanthin (3,3'-dihydroxyl beta, beta-carotene 4,4-dione) were analyzed by in vitro cell culture experiments. (i) beta-Carotene, canthaxanthin and astaxanthin caused significant stimulatory effects on the cell proliferative response of spleen cells and thymocytes from BALB/c mice at the concentrations of 2 x 10(-8) to 10(-7) M, although they showed the activities different from each other. (ii) Astaxanthin exhibited the highest activity on the polyclonal antibody (immunoglobulin M and G) production of murine spleen cells at the concentrations of 2 x 10(-8) to 10(-7) M but beta-carotene did not cause a significant effect at a low concentration (2 x 10(-8) M) although stimulated at a high concentration (2 x 10(-7) M). Canthaxanthin expressed moderate activities at the same concentrations. (iii) All tested carotenoids significantly enhanced the release of interleukin-1 alpha and tumor necrosis factor-alpha from murine peritoneal adherent cells at the concentrations of 2 x 10(-8) to 10(-7) M and the ranks of cytokine-inducing activities were astaxanthin > canthaxanthin > beta-carotene. These results indicate that carotenoids such as beta-carotene, canthaxanthin and astaxanthin have possible immunomodulating activities to enhance the proliferation and functions of murine immunocompetent cells.

Suppressive effects of chlorophyllin on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promoter-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells.

The potentially protective role of chlorophyllin, the sodium and copper salt of chlorophyll a against the initiation and promotion stages in carcinogenesis was studied by in vitro short-term assays. Chlorophyllin showed a dose-dependent suppressive effect on 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1)-induced umu C gene expression of Salmonella typhimurium (TA 1535/pSK 1002) in the presence of metabolizing enzyme mixture. The similar inhibitory effect of chlorophyllin was detected in mitomycin C (MMC)-dependent umu C gene expression in the absence of metabolizing enzyme mixture. Furthermore chlorophyllin also exhibited a dose-dependent inhibition on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity of 3T3 fibroblast cells at the same concentrations. However, when chlorophyll a isolated from Japanese tea leaves was applied on the same assay systems as a comparative experiment, chlorophyll a showed much weaker activity compared with that of chlorophyllin. The significance of this finding is discussed from the viewpoint of the protective role of chlorophyllin against carcinogenesis.

Suppressive effects of retinoids, carotenoids and antioxidant vitamins on heterocyclic amine-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002).

Effects of retinoids, carotenoids and antioxidant vitamins were studied by mutagen-induced umu C gene expression system in Salmonella typhimurium (TA 1535/pSK 1002). Retinol (vitamin A), retinol acetate and retinoic acid showed remarkable inhibitory activities, whereas retinol palmitate exhibited significant but weak activity for umu C gene expression in tester bacteria induced by 3-amino-3,4-dimethyl-5H-pyrido[4.3-b]indol (Trp-P-1) in the presence of hepatic metabolizing enzymes (S9 mixture). Carotenoids having provitamin A activity (beta-carotene and canthaxanthin) exhibited moderate suppressive effects on the same experimental system. The ranks of suppressive activities were retinol > retinol acetate > retinoic acid > canthaxanthin > beta-carotene > retinol palmitate and their doses for inhibition by 50% (ID50) were estimated to be 1.2 x 10(-7), 3.0 x 10(-7), 5.4 x 10(-7), 1.5 x 10(-6), 4.0 x 10(-5) and 6.0 x 10(-5) M, respectively. However, they did not cause significant inhibition on umu C gene expression induced by direct-acting mutagen (adriamycin or mitomycin C) in the absence of S9 mixture. Inhibition of umu gene expression appears to be due to inhibition of P450-mediated metabolic activation of the heterocyclic amine Trp-P-1. Ascorbic acid (vitamin C) showed weak but significant suppressive activity at high-dose concentrations (3 x 10(-6) - 10(-4)M). However, alpha-tocopherol did not exhibit significant suppression at all dose concentrations. The significance of the experimental results is discussed from the viewpoint of the chemoprevention against genotoxicity associated with carcinogenesis.

Identification of antimutagenic substances in an extract of edible red alga, Porphyra tenera (Asakusa-nori)

Recently, a relatively strong antimutagenic activity has been detected in the extract of Porphyra tenera (Asakusa-nori in Japanese) which showed a suppressive effect on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002 (Okai et al. (1994) Cancer Lett., 87, 25-32). In the present paper, we analyzed the active principles for the antimutagenic activity in an extract of Porphyra tenera and detected three color spots on a silica gel TLC plate which indicated very similar Rf values and absorbance spectra of standard pigments such as beta-carotene, chlorophyll a and lutein. The seaweed pigments recovered from preparative silica gel TLC corresponding to beta-carotene, chlorophyll a and lutein exhibited significant suppressive activities against mutagen-induced umu C gene expression and combined treatment with these pigments showed an additive effect compared with single treatment with each pigment. Furthermore, the standard pigments prepared from other biological sources also exhibited similar anti-mutagenic activities. The significance of this finding is discussed from the protective role of seaweed pigments against mutagenesis probably associated with carcinogenesis.

All-trans beta-carotene enhances mitogenic responses and ornithine decarboxylase activity of BALB/c 3T3 fibroblast cells induced by tumor promoter and fetal bovine serum but suppresses mutagen-dependent umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002).

Although previous epidemiological studies have indicated that beta-carotene is an important agent for the chemical prevention against carcinogenesis, a recent prospective study has strikingly suggested that supplementation with beta-carotene significantly increased the incidence of some types of cancer (The alpha-Tocopherol and beta-Carotene Cancer Prevention Study Group, New Engl. J. Med., 330 (1994) 1031-1035). To analyze the discrepancy of this problem, the authors analyze the effects of beta-carotene on biochemical and biological events associated with carcinogenesis by in vitro experiments. (1) All-trans beta-carotene enhanced the proliferation and DNA synthesis of BALB/c 3T3 cells induced by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and fetal bovine serum, although beta-carotene itself did not show mitogenic activity. (2) All-trans beta-carotene caused a remarkable stimulation for the early induction of ornithine decarboxylase (ODC) activity after the stimulation of TPA and fetal bovine serum. (3) All-trans beta-carotene exhibited significant antimutagenic activity which suppresses umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by a typical mutagen, 2-aminoanthracene (2-AA). These experimental results suggest that all-trans beta-carotene might cause beneficial and harmful effects on different phases of carcinogenesis.

Suppressive effects of the extracts of Japanese edible seaweeds on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promotor-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells.

Some of epidemiological data indicated that ubiquitous consumption of seaweeds in Japan may be a possible protective factor against some types of tumor. To analyse this problem, the authors studied the antimutagenic and antitumor promotion activities in methanol-soluble extracts of typical edible seaweeds which showed suppressive effects on 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1)-induced umu C gene expression in SOS response of Salmonella typhimurium (TA 1535/pSK 1002) and 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells. Although eight varieties of edible seaweeds including chlorophyta, Phaenophyta and Rhodophyta showed significant antimutagenic and antipromotion activities, they expressed the activities different from each other. Among these seaweeds, Enteromorpha prolifera ('Sujiaonori' in Japanese) and Porphyra tenera ('Asakusanori') showed relatively strong suppressive activities in both antimutagenic and antipromotion assays compared with other seaweeds. These seaweeds contained considerable amounts of beta-carotene as a possible active principle with anticarcinogenic activity. This compound was partially associated with the antimutagenic activity in the seaweed extract, but did not contribute to the antipromotion activity of seaweed extract under our experimental conditions. These results strongly suggest that Japanese edible seaweeds have possible antimutagenic and antipromotion activities probably associated with antitumor activity.

Identification of heterogenous antimutagenic activities in the extract of edible brown seaweeds, Laminaria japonica (Makonbu) and Undaria pinnatifida (Wakame) by the umu gene expression system in Salmonella typhimurium (TA1535/pSK1002).

A significant antimutagenic activity was found in the hot water-soluble extract from a common edible brown alga, Laminaria japonica (Makonbu in Japanese) which showed suppressive effects on umu gene expression of the SOS response against DNA damage in Salmonella typhimurium (TA1535/pSK1002). The extract showed a drastic antimutagenic activity against 2-acetylaminofluorene (2-AAF)- or 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1)-induced mutagenesis which requires liver-metabolizing enzymes, whereas the same extract exhibited weak but significant inhibitory effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)- or furylfuramide (AF-2)-induced mutagenesis in the absence of liver-metabolizing enzymes. Among these antimutagenic activities, the minor activity was found in the polysaccharide fraction of the extract which showed roughly equal antimutagenic activities against all the mutagens tested. The major activity was detected in the nonpolysaccharide fraction which exhibited a relatively strong antimutagenic activity against 2-AAF- or Trp-P-1-induced mutagenesis but a weak activity against MNNG- or AF-2-induced mutagenesis. The nonpolysaccharide fraction was further separated into high- or low-molecular-weight fractions and the latter fraction showed a much stronger activity than the former fraction. In addition, similar antimutagenic activities were detected in polysaccharide and nonpolysaccharide fractions from the extract of the other edible brown alga, Undaria pinnatifida (Wakame in Japanese). These experimental results indicate that the hot water-soluble extract of Laminaria japonica or Undaria pinnatifida contains heterogenous antimutagenic activities against typical genotoxic substances. The significance of this finding is discussed from the viewpoint of the protection against genotoxic substances by traditional edible seaweeds in Japan.