Antiproliferative and antioxidant properties of an enzymatic hydrolysate from brown alga, Ecklonia cava

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Abstract

The potential antiproliferative and antiradical activities of an enzymatic extract of Ecklonia cava together with its crude polysaccharide (CpoF) and crude polyphenolic fractions (CphF) were evaluated in vitro. Tested extracts showed strong selective cell proliferation inhibition on all cancer cell lines tested, especially CphF extract, containing high polyphenol amount, showed 5.1 μg/ml of IC50 value on murine colon cancer (CT-26) cell line. According to the nuclear staining experiment, antiproliferative effect of CphF was associated with apoptotic cell demise in CT-26. In addition, The CphF at 5 μg/ml scavenged 70% of DPPH radical, which is much higher than those of BHA and BHT at same concentration. Further more CphF exhibited interesting antiradical properties, expressed by its capacity to scavenge superoxide anion (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OHradical dot). In reducing power assay, CphF extract at 5 μg/ml was found to be as high as that of BHT at same concentration. Also, in total antioxidant assay the effect of CphF at 50 μg/ml was equivalent or slightly higher than those of commercial counterparts at 5 μg/ml concentration. Taken together, the CphF may be a promising alternative to synthetic substances as natural compound with high antiproliferative and antiradical activity.

Introduction

Seaweeds as well-balanced, harmless, natural sources with a high degree of bioavailability of trace elements are strongly advised for fast grown children and pregnant women (Booth, 1964). In contrast to their use as a source of food, marine algae are widely used in the life science as the source of compounds with diverse structural forms and biological activities. Over the years marine algal species offer the biological diversity for sampling in discovery-phase of new drug development (Munro et al., 1987, Munro et al., 1999). Therefore, it is clearly documented that, pre-clinical pharmacological research with new marine compounds continued to be extremely active in resent history (Mayer and Gustafson, 2003).

The formation of cancer cell in human body can be directly induced by free radicals. Further more, ionizing radiation, which causes free radicals, is well documented as a carcinogen. Therefore, radical scavenging compounds such as vegetables and fruits can indirectly reduce cancer formation in human body. It has been reported that, increased consumption of fruits and vegetables, which are rich with free radical scavenging activity, leads up to a doubling of protection against many common types of cancer formations (Nandita and Rajini, 2004, Chu et al., 2002, Cooke et al., 2002).

Recent years, several algal species also have been reported to prevent oxidative damage by scavenging free radicals and active oxygen and hence able to prevent the occurrence of cancer cell formation. Therefore, algal species as alternative materials to extract natural antioxidative compounds have attracted much attention of biomedical scientists. There are some evidences that seaweeds contain compounds with a relatively high antioxidant and antiproliferative activity. Therefore, extraction of bioactive natural compounds from seaweeds is desired, but little has happened in this area to systematically study their potentiality. Polyphenols in marine brown algae are called phlorotannins and known to act as potential antioxidants. Phlorotannins are formed by the polymerization of phloroglucinol (1,3,5-trihydroxybenzene) monomer units and synthesized in the acetate–malonate pathway in marine alga (Ragan and Glombitza, 1986). Furthermore, sulfated polysaccharides isolated from marine alga also have been shown to exert radical scavenging activities in vitro and in vivo.

However, biochemical scientists have several techniques to extract bio-active compounds from algal biomass. As one of the techniques, enzymatic hydrolysis of algal biomass gains more advantages over other conventional techniques. Enzymes can convert water-insoluble materials into water soluble materials, also this method do not adapt any toxic chemicals. Interestingly, this technique gains high bioactive compound yield and shows enhanced biological activity in comparison with water and organic extract counterparts (Heo et al., 2005).

In this study, Ecklonia cava, which was collected along Jeju Island coast of Korea, was enzymatically hydrolyzed using AMG 300L and after ethanol precipitation (Matsubara et al., 2000, Kuda et al., 2002) the crude polysaccharide (CpoF) and crude polyphenolic fractions (CphF) were evaluated for their suppressive effect on tumor cell growth, antioxidant and radical scavenging activities.

Section snippets

Reagents

1,1-Diphenyl-2-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), thiobarbituric acid (TBA), trichloroacetic acid (TCA), naphthylethylene diamine dihydrochloride, xanthine, xanthine oxidase from butter milk, nitro blue tetrazolium salt (NBT), butylated hydroxytoluene (BHT), butylated hydroxyanisol (BHA), disodium salt (ferrozine), ferric chloride (FeCl3 · 6H2O), potassium ferricyanide [K3Fe(CN)6], ferrous chloride (FeCl2 · 4H2O), FeSO4 · 7H2O, Folin–Ciocalteu reagent, linoleic acid and

Inhibitory effect of E. cava hydrolysate on the growth of tumor cells

In this study, four tumorigenic cell lines including, murine colon cancer cell line (CT-26), human leukemia cell line (THP-1), mouse melanoma cell line (B-16) and human leukemia cell line (U-937) were chosen to determine the antiproliferative activity of E. cava extracts. Also, under same experimental conditions the extracts were evaluated on chinese hamster lung fibroblast cell line (V-79-4) in order to examine its cytotoxicity effect on normal cells.

Cultures of CT-26 cells were treated with

Discussion

Jeju Island is located in the southwest sea of the Korean peninsula and is highlighted for its uniqueness. Especially in the coastal area of this island the seawater level fluctuate rapidly. Therefore, the algal species present along the shoes of Jeju Island may require high endogenous antioxidant protection as an adaptative response to this especial environment. Recently it has been reported several biologically important seaweed species from Jeju Island (Athukorala et al., 2003, Athukorala et

Acknowledgements

This work was supported by the a grant of Regional Industry Development Research Program funded by Ministry of Commerce, Industry and Taerim Trade Co., Ltd. Also, this work was supported by the New Frontier Education Center for Eco-Friendly Marine Industry in 2005.

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