Antioxidant and antiproliferative activities of extracts from a variety of edible seaweeds

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Abstract

Dietary Laminaria and Porphyra sp. have been reported to reduce the risk of intestinal or mammary cancer in animal studies. Algal anticarcinogenicity may involve effects on cell proliferation and antioxidant activity. Thus, in the present study, we evaluated the effect of red alga, dulse (Palmaria palmata) and three kelp (Laminaria setchellii, Macrocystis integrifolia, Nereocystis leutkeana) extracts on human cervical adenocarcinoma cell line (HeLa cells) proliferation using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The 1-butanol soluble fractions from the methanol extracts of these algae were also evaluated for reducing activity and total polyphenol content. After 72 h incubation, HeLa cell proliferation was inhibited (p < 0.05) between 0% and 78% by P. palmata; 0% and 55% by L. setchellii and 0% and 69% by M. integrifolia and N. leutkeana at 0.5–5 mg/mL algal extract. Algal extract reducing activities were as follows: P. palmata > M. integrifolia > L. setchellii > N. leutkeana; and total polyphenol contents were: P. palmata > M. integrifolia = N. leutkeana > L. setchellii. The antiproliferative efficacy of these algal extracts were positively correlated with the total polyphenol contents (p < 0.05), suggesting a causal link related to extract content of kelp phlorotannins and dulse polyphenols including mycosporine-like amino acids and phenolic acids.

Introduction

Edible seaweeds, including algae from the Protista orders: Laminariales (brown), Chlorophyta (green) and Rhodophyta (red) have a long history of use in the diets of Pacific and Asian cultures compared to those of Europe, and the Canadian and US Maritimes. These seaweeds include red algae in Japanese and Korean cuisine [‘Nori’ or ‘Kim’, ‘Laver’ (Porphyra tenera)] or European/North American diets [‘Dulse’ (Palmaria palmata)]; brown kelps in Japanese cuisine [‘Hijiki’ (Hijikia fusiformis), ‘Wakame’ (Sea Mustard, Undaria pinnatifida), ‘Makonbu’ (Sea Tangle, Laminaria japonica)] or Chinese cuisine [‘Hai dai’ (Laminaria sp.)]; green algae in Hawaiian cuisine [‘Limu palahalaha’ (Ulva sp.)]. Within the traditional Japanese diet, seaweeds are commonly used as sushi wrappings, seasonings, condiments and vegetables and can thus constitute between 10% and 25% of food intake by most Japanese (Skibola, 2004, Teas, 1981). On the other hand, Japanese migrants in Hawaii and populations of most Western (i.e. North American and European) cultures have very low to zero seaweed intakes (Teas, 1981). When considered in combination with international diet-related chronic disease incidences, a significant environmental (i.e. dietary) difference between populations varying in seaweed consumption is revealed. For example, breast cancer rates are 42.2 and 13.1 one yr prevalence cases/100,000 in Japan and China vs 125.9 and 106.2 cases in North America (NA) and Europe; and prostate cancer rates are 10.4 and 0.7 one yr prevalence cases in Japan and China vs 117.2 and 53.1 cases in NA and Europe (Pisani et al., 2002).

The epidemiological data are supported by rodent model studies demonstrating protective effects of dietary kelps and other red and green algae against mammary (Funahashi et al., 2001, Yamamoto et al., 1987, Teas et al., 1984), intestinal (Lee and Sung, 2003, Yamamoto and Maruyama, 1985) and skin carcinogenesis (Yamamoto et al., 1986, Higashi-Okai et al., 1999). Moreover, cell culture and cell-free studies have begun to elucidate the mechanisms underlying the potential anticarcinogenic effects of seaweed constituents, such as the antimutagenicity of kelp and red algal extracts against breast and colon cancer inducers (Reddy et al., 1984) as well as aflatoxin B1 and N-methyl-N′-nitro-N-nitroguanidine (Cho et al., 1997); inhibition of hyaluronidase activity (Shibata et al., 2002) and anti-inflammatory and -proliferative activities by a variety of red algal (Bergé et al., 2002) and kelp extracts (Ellouali et al., 1993). An algal antioxidant-mediated mechanism was hypothesized as a contributing factor in the inhibition of mammary carcinogenesis by dietary kelp in the presence of enhanced antioxidant enzyme activity and reduced lipid peroxides in livers of treated rats (Maruyama et al., 1991). Moreover, antioxidant and/or antimutagenic effects of dietary seaweeds have been observed in rodent model studies of colon and skin carcinogenesis, wherein treated animals exhibited suppression of tumor initiation (Lee and Sung, 2003, Higashi-Okai et al., 1999). Indeed, seaweeds are noted to contain not only labile antioxidants (i.e. ascorbate, glutathione) when fresh (Morgan et al., 1980, Indergaard and Minsaas, 1991, Kakinuma et al., 2001, Burritt et al., 2002), but also, more stable molecules such as carotenoids (Morgan et al., 1980, Okai et al., 1996, Yan et al., 1999), mycosporine-like amino acids (Nakayama et al., 1999) and a variety of polyphenols (e.g. catechins, phlorotannins; Nakamura et al., 1996, Yoshie et al., 2000).

We recently reported that dulse extracts were not only effective hydroxyl and stable free radical scavengers, but also inhibitors of lipid oxidation and cell proliferation in vitro (Yuan et al., 2005a, Yuan et al., 2005b). The antioxidant activity of P. palmata extracts may be associated with the presence of a unique class of secondary metabolites, the mycosporine-like amino acids (MAAs) which function as UV-absorbing sunscreen molecules in the Rhodophyta (Yuan et al., 2005b, Karsten and Wiencke, 1999). The MAAs, such as mycosporine-glycine, palythine and palythene, consist of glycine residue and hydroxylated sidechain functions on an aromatic methyl ester core. Algal tissue levels of these metabolites have been reported to have a positive correlation with UV exposure during growth (Karsten and Wiencke, 1999). It is noteworthy that UV sunscreen protection is not unique to the Rhodophyta however, as Phaeophyceae, such as the Laminariales, are known to synthesize UV-inducible polyphenols, i.e. phlorotannins, which absorb in the UVB range (280–320 nm; Swanson and Druehl, 2002). Moreover, kelp tissue levels of phlorotannins have been reported to increase during short-term exposure of Macrocystis integrifolia to UVA (320–400 nm) and UVB irradiation (Swanson and Druehl, 2002). While several workers have reported antioxidant activity of phlorotannin-containing extracts from a variety of kelps from China and Spain (Jiménez-Escrig et al., 2001, Yan et al., 1996), it is not known whether kelp extracts also influence the proliferation of cancer cell lines.

The evidence outlined above suggests a protective role for edible seaweeds against oxidative stress and cell proliferation. However, there is little data elucidating the antioxidant and anticarcinogenic activities of NA algae such as the red alga, P. palmata (order Palmariales, family Palmariaceae) and the brown kelps, Laminaria setchellii (Laminariales, Laminariaceae), M. integrifolia and Nereocystis leutkeana (Laminariales, Lessionaceae). These edible seaweed varieties are harvested and packaged for retail sale from the East (Grand Manan Island, New Brunswick; P. palmata) and West Coasts of Canada (Vancouver Island, British Columbia; all three kelps). Therefore, the objectives of the present study were to evaluate the antioxidant potential of extracts from these algae, and to assess the effect of these extracts on epithelial cancer cell (HeLa cells) proliferation in vitro.

Section snippets

Materials

Certified organic (Organic Crop Improvement Association International) dulse (P. palmata) harvested in Spring 2001 was provided by Atlantic Mariculture Ltd. (Dark Harbour, Grand Manan Island, New Brunswick, Canada). Several kelps (L. setchellii, M. integrifolia and N. leutkeana) were purchased from Canadian Kelp Resources (Bamfield, Vancouver Island, BC). HeLa cells were obtained from American Type Culture Collection (ATCC CCL-2, Manassas, VA). Solvents (1-butanol, ethyl acetate, hexane,

Results

Table 1 summarizes the reducing activity and total polyphenol content of the dulse and kelp extracts. The reducing activity (expressed as ascorbic acid equivalents) of the red alga, dulse extract was greater than that of any of the brown kelps (Table 1). Within the kelps, M. integrifolia had the greatest reducing activity, followed by L. setchellii and then, N. leutkeana. The reducing activity of M. integrifolia was 1.50-fold greater than that of N. leutkeana. The total polyphenol content

Discussion

The present study is the first to report on the antiproliferative activities of extracts from the edible kelp species, L. setchellii (Oar weed, Split laminaria), M. integrifolia (Macro or Giant kelp) and N. leutkeana (Bull kelp) using an epithelial adenocarcinoma cell line (HeLa cells) model. We extend these findings by reporting the reducing activity and total polyphenol content of 1-butanol soluble extracts of these brown kelps compared to the red alga P. palmata (dulse). Previous work from

Acknowledgements

This research was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant. The authors thank M. Carrington and M. De Jesus for their technical support.

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