Chemical assays can be used to estimate the antioxidant potential of seaweeds however a greater understanding of the possible antioxidant effects in vivo can be determined using cells in culture or animal cell models. Tables 1 and 3 detail the overall antioxidant effects of seaweed extracts in cells in culture and animal models, respectively and tables 2 and 4 detail studies which have investigated the ability of seaweed extracts to protect against oxidant-induced DNA damage in cells in culture and in animal models, respectively.
6.1 Seaweeds and seaweed extracts investigated in cell and animal model systems Japan is the world leader in the field of seaweed research therefore the majority of seaweeds that have been added to cells or administered to animals for an investigation of their antioxidant effects are those which are endemic to the coasts of Japan and neighbouring countries such as China and Korea (Tables 1-4). The majority of seaweeds investigated were brown or red species as brown species are known to contain the highly antioxidant compounds, phlorotannins and red seaweeds contain high concentrations of sulphated-polysaccharides.
In general, an extract of the seaweed was prepared prior to its addition to cells or use in animal feeding trials. A simple solvent extract was investigated in a number of studies. The solvents used in the preparation of antioxidant extracts from seaweeds should ideally be food grade when they are to be used in animal feeding trials. Extracts prepared using water have been added to cells in culture (Fallarero et al., 2003, Yang et al., 2012) and have also been fed to rats (Sathivel et al., 2008).
Seaweed extracts, prepared using ethanol, have also been fed to rats (Raghavendran et al., 2005; Raghavendran et al., 2007). Methanol, which is toxic and not food grade, was used to prepare extracts from seaweeds which were subsequently fed to fish (Nagarani et al., 2012) and added to human keratinocytes, HaCaT cells (Ko et al., 2011). Prior to their use in a cell model or animal system, solvent-derived extracts are generally dried to a powder which removes all but trace amounts of the solvent. These extracts are then generally added into the animal feed directly, however; a study by Schultz Moreira et al. (2011) incorporated the seaweed Himanthalia elongata into a reconstituted pork product for a rat feeding trial (Table 3).
Enzymes have also been used to extract antioxidants from seaweed for an investigation of the seaweeds cellular antioxidant effects. The two main classes of enzymes which have been used are proteases and carbohydrases. The advantages of preparing an enzymatic extract are an increased extraction yield. A range of carbohydrases (AMG, Celluclast, Termamyl, Ultraflo, Viscozyme and Maltogenase) and proteases (Protamex, Kojizyme, Neutrase, Flavourzyme and Alcalase) have been used in the preparation of extracts which were subsequently added to cells in culture (Table 2). The use of enzymatic antioxidant extracts in animal trials has not been reported.
Several purified compounds including phlorotannins, sulphated polysaccharides, carotenoids, mycosporine-like amino acids (MAA), monoterpene lactone (loliolide) and phycobiliprotein have been isolated from seaweed and added to cells in culture (Table 1 and 2). Ecklonia cava is a particularly rich source of phlorotannins and contains a diverse range of these compounds including Triphlorethol-A, 1,3,5-trihydroxybenzene, pyrogallol-phloroglucinol-6,6'-bieckol, phloroglucinol, eckol and dieckol. Diphlorethohydroxycarmalol from Ishige okamurae is another phlorotannin that has been tested in cells in culture (Tables 1 fucoidan has been extracted from seaweed (Chotigeat et al., 2004) in comparison to
6.2 Antioxidant effects of seaweed extracts
The antioxidant effects of seaweed and seaweed extracts have been investigated in a broad range of cell types including human skin, fibroblast, lung and lymphocytes;
rodent brain, T-cells, lung, lymphocytes and macrophages and monkey kidney cells (Table 1 and 2). Their antioxidant activities were also assessed using animal models which mostly involved the use of rat or mouse feeding trials, however in a limited number of studies the seaweed extracts were administered to the animals intravenously (Tables 3 and 4). The antioxidant effects of the seaweed extracts were then determined in various cells harvested from the animals following the desired feeding period. As is the case in many pharmaceutical and toxicological studies, the majority of studies selected hepatocytes as the main cell type for investigation which is due to the presence of major drug and xenobiotic-metabolizing enzymes such as the cytochrome P450 enzyme superfamily (Guo et al., 2011). Additional target organs investigated included brain, kidney and plasma cells.
Seaweed and seaweed extracts were found to ameliorate the increase in reactive oxygen species (ROS) generation induced by oxidants H2O2, UV-B and methyl mercury in human and animal cell models. Seaweed, delivered orally, intravenously or topically also increased the activity of the antioxidant enzymes including CAT, SOD, GPx in the cells of animal models and prevented the decrease in antioxidant enzyme activity induced by xenobiotics such as ethanol, tetrachloride, paracetamol, γ-radiation and cyclosporine A (Table 3). Seaweed protected against the DNA damage induced by H2O2, UV-B and γ-radiation in various cell types in culture (Table 2) and against the DNA damage induced by mercury, H2O2, cyclophosphamide and paracetamol in the cells of fish, mice and rats (Table 4).
H2O2 is the most commonly employed oxidant in studies investigating DNA damage because it is a physiological oxidant, naturally produced in oxidative metabolism, and its damaging mechanism, especially to DNA, is well established (Barbouti et al., 2001). Similarly the damaging effects of UV radiation which is caused either by direct DNA damage through interaction with DNA bases or indirect damage through the formation of ROS such as singlet oxygen and H2O2 has been established (Abdel-Malek et al., 2009). The main mechanism in the cellular protective ability of antioxidants is through the chelation of transition metals such as iron and copper. Seaweeds contain a number of metal chelating components such as
polysaccharides, proteins (Murphy et al., 2007) and polyphenols (Perron &
Brumaghim, 2009).
6.3 Human studies
A limited number of studies have investigated the antioxidant effects of seaweed and seaweed extracts in human trials (Table 3). The daily consumption of 48 g of a seaweed supplement containing equal parts sea tangle (Laminaria japonica) and sea mustard (Undaria pinnatifida) resulted in enhanced cellular antioxidant enzyme activity and reduced lipid oxidation in red blood cells isolated from diabetic patients (Kim et al., 2008). Similarly, consumption of 1.5 g of fermented Laminaria japonica for each day over a five week period resulted in an increased antioxidant enzyme activity in the hepatocytes of patients suffering from high levels of γ-glutamyltransferse (γ-GT), a condition that is associated with a number of diseases (Kang et al., 2012). It has also been found that the topical application of MAA extracted from Porphyra umbilicalis reduced UV-A induced lipid oxidation in human skin cells (Schmid et al., 2006).