Palmaria Palmata) Are Effective Antioxidants and Inhibitors of Cell Proliferation in Vitro
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Food and Chemical Toxicology 43 (2005) 1073–1081 www.elsevier.com/locate/foodchemtox Extracts from dulse (Palmaria palmata) are effective antioxidants and inhibitors of cell proliferation in vitro Yvonne V. Yuan *, Meshell F. Carrington, Natalie A. Walsh School of Nutrition, Faculty of Community Services, Ryerson University, 350 Victoria Street, Toronto, ON, Canada M5B 2K3 Received 1 October 2004; accepted 27 February 2005 Abstract Previously, we reported that a 1-butanol soluble extract of the edible red alga Palmaria palmata, known as dulse, exhibited hydroxyl and stable free radical scavenging activity as well as inhibition of lipid peroxidation, attributed to the reducing activity and polyphenol content of the dulse extract. In the present study, we evaluated the antioxidant and antiproliferative activities of two grades of dulse harvested from Canadian Maritime locations differing in UV radiation exposure (i.e. west versus east coasts of Grand Manan Island, New Brunswick). The 1-butanol soluble extract from Grade 1 dulse (reduced UV-exposure) exhibited lower reducing activity versus Grade 2 dulse (greater UV exposure) reflecting a lower requirement for endogenous antioxidant protection. Grade 1 and 2 dulse extracts both inhibited (p 6 0.03) AAPH-induced lipid peroxidation, but had no effect on AMVN-induced lipid peroxidation, demonstratingthe aqueous nature of the antioxidants involved. The Grade 1 and 2 dulse extract inhibition ( p < 0.05) of HeLa cell proliferation was dose-dependent over 0.5–5.0 mg/mL and maximal at 48 and 72 h incubation. The antiproliferative effects of the Grade 1 and 2 dulse extracts in the present study likely reflect the bioactivity of the polyphenol content of these extracts. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Palmaria palmata; Dulse; Antioxidant activity; Lipid oxidation; AAPH; AMVN; Linoleic acid emulsion; HeLa cells; Cell proliferation 1. Introduction taxonomies such as the Laminariales, Ulvales and Por- phyridiales, respectively, are common in the diets of Dietary seaweeds, such as algae from brown (Pha- Asian countries, particularly China, Japan, Korea and eophyta), green (Chlorophyta) and red (Rhodophyta) Indonesia (Anggadiredja et al., 1997; Chan et al., 1997; Yan et al., 1998; Nakayama et al., 1999; Nagata et al., 2003; Skibola, 2004); but less so in European diets Abbreviations: AAPH, 2,20-azo-bis(2-amidinopropane) dihydro- (e.g. France, Iceland, Ireland and Norway) (Mabeau chloride; AMVN, 2,20-azobis(2,4-dimethylvaleronitrile); BHA, butyl- and Fleurence, 1993; Le Tutour, 1990; Le Tutour ated hydroxyanisole; CD, conjugated dienes; EGC, epigallocatechin; et al., 1998) and the Canadian Maritimes (Morgan FBS, foetal bovine serum; GSH, glutathione; MAA, mycosporine-like amino acid; MDA, malondialdehyde; MEM, minimum essential et al., 1980; Yuan et al., 2005). These seaweeds, or sea medium; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium vegetables, are often used as wrappers such as the bromide; ÅOH, hydroxyl radical; PBS, phosphate buffered saline; Japanese sushi seaweed ÔNoriÕ or Korean ÔKimÕ derived ROS, reactive oxygen species; TBARS, 2-thiobarbituric acid reactive from the roasted or toasted red alga, Porphyra tenera; substances; TCA, trichloroacetic acid. condiments and seasonings in soups and salads such as * Correspondingauthor. Tel.: +41 6979 5000x6827; fax: +41 6979 5204. the brown algae in Japanese cuisine: ÔHijikiÕ (Hijikia fusi- E-mail address: [email protected] (Y.V. Yuan). formis), ÔWakameÕ (Undaria pinnatifida)orÔMakonbuÕ 0278-6915/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.02.012 1074 Y.V. Yuan et al. / FoodandChemical Toxicology 43 (2005) 1073–1081 (Laminaria japonica); as well as health food snacks such cules such as ascorbate and glutathione (GSH) when as the European/North American ÔDulseÕ derived from fresh (Burritt et al., 2002; Morgan et al., 1980), as well the red alga, Palmaria palmata. as more stable molecules includingcarotenoids ( Yan Estimates of seaweed intake in Japanese populations et al., 1999; Okai et al., 1996; Morgan et al., 1980), range from 4.6 g/day (0.4–29.2 g) from food-frequency mycosporine-like amino acids (MAA, mycosporine– questionnaire data (Nagata et al., 2003) to 10–25% of glycine and –taurine; Dunlap et al., 1997; Nakayama food intake, or approximately 3–13 g/day (Skibola, et al., 1999; Sekikawa et al., 1986), catechins (Yoshie 2004), with the highest intakes attributed to the Japa- et al., 2000), phlorotannins (Jime´nez-Escriget al., nese of Okinawa (Sho, 2001). Conversely, seaweed in- 2001) and the tocopherols (Morgan et al., 1980). In fact, takes are vastly decreased outside of Japan from an extracts from several brown, green and red alga har- estimated 100% consumption of seaweed by Japanese vested in France (Le Tutour et al., 1998; Le Tutour, livingin Japan to approx. 12–20% by migrant Japanese 1990), Spain (Jime´nez-Escriget al., 2001 ), Indonesia livingin Hawaii ( Teas, 1981). Similarly low to zero sea- (Anggadiredja et al., 1997), Korea (Han et al., 1999), weed intakes can be expected in Western, particularly China (Yan et al., 1998) and Japan (Yan et al., 1999; North American, populations. These estimates of sea- Sekikawa et al., 1986) have demonstrated antioxidant weed intakes in different cultures and populations high- activity in vitro. Moreover, algae have been reported light one clear environmental (i.e. dietary) difference to vary in antioxidant composition due to environmen- between populations which are also known to differ in tal factors such as water depth and UV-exposure chronic disease rates; for example, breast cancer with (Burritt et al., 2002; Karsten and Wiencke, 1999). We re- 42.2 and 13.1 one-year prevalence cases per 100,000 in cently reported hydroxyl and stable free radical scaveng- Japan and China (includingKorea, Mongoliaand Hong ingactivities by a P. palmata extract (Yuan et al., 2005). Kong), respectively versus 125.9 cases in North America Interestingly, this alga is known to vary in pigmentation (NA); and prostate cancer with 10.4 and 0.7 one-year (R-phycoerythrin) dependingon its UV-exposure during prevalence cases in Japan and China, respectively versus growth (Galland-Irmouli et al., 2000); however, it is not 117.2 cases in NA using1990 data ( Pisani et al., 2002). known whether this also influences the antioxidant The potential beneficial association between con- capacity of P. palmata. sumption of seaweed and breast cancer can be traced Taken together, these lines of evidence suggest a po- back to the ancient Egyptian ÔEbers PapyrusÕ dating tential for edible seaweeds as sources of dietary antiox- from approx. 1534 B.C., which mentioned that seaweed idants and anticarcinogens. There is a paucity of data was administered as a treatment to breast cancer pa- however, characterizingthe antioxidant and anticarci- tients (Teas, 1981). Indeed, a growing body of research nogenic potential of North American algae such as P. in rodent models has demonstrated the anticarcinogenic palmata (order Palmariales, family Palmariaceae). effects of dietary kelps, such as Laminaria angustata,and Therefore, the objectives of this study were to character- other red and green seaweed species against mammary ize the antioxidant activity of dulse extracts from two (Maruyama et al., 1991; Yamamoto et al., 1987), intes- different locations in the Canadian Maritimes, known tinal (Yamamoto and Maruyama, 1985) and skin carci- to vary in UV-exposure, and to assess the influence of nogenesis (Higashi-Okai et al., 1999). Mammalian cell these extracts on epithelial cancer cell (HeLa cells) pro- culture and cell-free studies have begun to elucidate liferation in vitro. the underlyingmechanisms of the potential protective effects of seaweed constituents against carcinogenesis, includingantimutageniceffects of kelp extracts against 2. Materials and methods breast and colon cancer inducers (Reddy et al., 1985); inhibition of telomerase activity (Eitsuka et al., 2004) 2.1. Materials and anti-inflammatory and anti-proliferative activities by a variety of red algal extracts (Berge´ et al., 2002). Two grades of certified organic (Organic Crop One of the mechanisms thought to contribute to the Improvement Association International, OCIA) dulse inhibitory effect of dietary kelp against chemically-in- (P. palmata) harvested in Spring2001 were obtained duced mammary carcinogenesis was the enhancement from Atlantic Mariculture Ltd. (Grand Manan, New of antioxidant enzyme activity (i.e. glutathione peroxi- Brunswick (NB), Canada). Grade 1 dulse was harvested dase) and reduction in lipid peroxides in the livers of from Dark Harbour on the western side of Grand Man- treated rats (Maruyama et al., 1991). A similar antioxi- an Island, NB and consisted of intact dulse fronds dant effect was observed with a sulfoglycolipid fraction (whole leaf); whereas the Grade 2 dulse was harvested isolated from the red alga, Porphyridum creuntum, which from the ÔPassageÕ area on the eastern side of Grand inhibited the production of superoxide anion radicals by Manan Island and consisted of comminuted dulse cultured peritoneal activated leukocytes (Berge´ et al., flakes. Grade 1 ÔDark HarbourÕ dulse is a dark red- 2002). Edible seaweeds contain labile antioxidant mole- dish-purple, thicker and more flavourful than the Grade Y.V. Yuan et al. / FoodandChemical Toxicology 43 (2005) 1073–1081 1075 2 ÔPassageÕ dulse, which is noted to be a bit saltier tast- 1.25 mL of H2O + 0.25 mL 0.1% FeCl3 Æ 6H2O and left ing, lighter