Background
Refrigerated, ready-to-eat products, especially dairy foods, have become increasingly popular in recent years because of their convenience. Many pathogenic organisms spoil such foods, reducing their shelf life and often leading to food poisoning. It has been estimated that as many as 30% people in industrialized countries suffer from a food poisoning every year [
1,
2]. In addition to microbial contamination, all packed and refrigerated food also undergoes gradual changes during storage, due to auto oxidation which releases reactive oxygen species (ROS) including free radicals like superoxide anion (O
2
•-) and hydroxyl radicals (OH
•) and non-free radical species like singlet oxygen (
1O
2) and hydrogen peroxide (H
2O
2) [
3,
4] into the food. These ROS induce peroxidation of lipids (polyunsaturated fatty acids) generating secondary oxidants like heptanol and hexanal [
5], which contributes to oxidative rancidity, deteriorating the flavor of the food [
6]. These not only cause a loss in food quality but are also believed to be associated with carcinogenesis, mutagenesis, arthritis, diabetes, inflammation, cancer and genotoxicity [
7‐
9]. To overcome these problems a wide range of synthetic antimicrobial agents (sodium benzoate, calcium benzoate, sorbate) and synthetic antioxidants (butylhydroquinone, propyl gallate, butylated hydroxy toluene (BHT), butylated hydroxy anisole (BHA) [
10], have been used as food preservatives. However, these preservatives can cause liver damage and are suspected to be mutagenic and neurotoxic. Hence, most consumers prefer additive-free foods [
11,
12] or a safer approach like the utilization of more effective antioxidants and antimicrobials of natural origin [
8,
13]. Recently, various phytochemicals like polyphenols, which are widely distributed in plants, have been reported to act as free radical scavengers and antimicrobial agents [
14,
15]. Marine plants, like seaweeds, also contain high amounts of polyphenols. For example, high concentrations of polyphenols such as catechin, epicatechin, epigalloctechin gallate and gallic acid are reported in the seaweed
Halimada (Chlorophyceae) [
16]. Since many types of seaweed have still to be investigated, we were prompted to take up this study. The Gulf of Mannar is a Marine Biosphere Reserve situated along the east coast of India and Sri Lanka, an area of about 10,500 sq. km which has a luxuriant growth of about 680 species of seaweed belonging to the Rhodophyta, Pheaophyta and Chlorophyta, in both the inter-tidal and deep water regions. Seaweed constitutes a commercially important marine renewable resource.
Sargassum, Padina, Dictyota and
Gracilaria sps. Are used by common people as fertilizers, food additives and animal feed [
17]. The sulphated polysaccharides of
Sargassum act as a potent free radical scavenger and anticancer agent [
18,
19].
Gelidella and
Gracilaria sps are widely used for the production of agar and for the treatment of gastrointestinal disorders [
20]. The methanolic extract of brown seaweeds such as
Ecklonia cava [
21] and
Hizikia fusiformis [
22] exhibit potent antioxidant activity. Although seaweeds possess wide application in food and in the pharmaceutical industry, the antioxidant and antimicrobial activity of many types of seaweed in the South Indian coastal area are still unexplored. The main objective of the present study is to evaluate the antioxidant and antimicrobial activities of seaweeds obtained from the Thondi, South Coastal Area of Tamil Nadu, India.
Discussion
Free radicals are highly reactive molecules with an unpaired electron and are produced by radiation or as by-products of metabolic processes. They initiate chain reactions which lead to disintegration of cell membranes and cell compounds, including lipids, proteins and nucleic acids. Besides damage to living cells, free radicals are the major cause of food deterioration through lipid oxidation, which ultimately affects the organoleptic properties and edibility of foods. Hence, intervention of an antioxidant may have a therapeutic effect and also maintain the freshness of food products. Antioxidant compounds scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus reduce the level of oxidative stress and slow/prevent the development of complications associated with oxidative stress-related diseases [
35] Many synthetic antioxidants have shown toxic and mutagenic effects, which have shifted attention towards naturally occurring antioxidants. A great number of naturally occurring substances like seaweeds have been recognized to have antioxidant abilities [
36].
1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a stable nitrogen centered free radical which can be effectively scavenged by antioxidants [
37]. Hence it has been widely used for rapid evaluation of the antioxidant activity of plant and microbial extracts relative to other methods [
38]. DPPH is also considered as a good kinetic model for peroxyl radicals [
39]. The ability of seaweeds to scavenge DPPH radicals was determined by the decrease in its absorbance at 517 nm. The present investigation has shown that the extracts of all the seaweeds exhibited DPPH scavenging activity, the most effective being
G.acerosa which exhibited significantly higher DPPH scavenging activity (72.5% inhibition) followed by
Haligra species (55% inhibition) when compared with the highest concentration of the standard BHT (72% inhibition) (Fig
1). The result is indicative of the hydrogen donating ability of
G. acerosa, since the effects of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen donating ability [
40]
In addition, the ability to scavenge the DPPH radicals is related to the inhibition of lipid peroxidation [
41]. Hence the ability of the seaweeds to prevent lipid peroxidation was assessed in RBC by inducing lipid peroxidation with H
2O
2. During lipid peroxidation, low molecular weight end products, probably malonaldehyde, are formed by oxidation of polyunsaturated fatty acids and react with two molecules of thiobarbituric acid to give a pinkish red chromagen. As shown in Fig
2, at a concentration of 100 μg/ml,
G. acerosa inhibited lipid peroxidation to 65.6% similar to the reference compound BHT which showed 64.9% inhibition.
OH
• has a short half-life and is the most reactive and damaging ROS. It causes oxidative damage to DNA, lipids and proteins [
42]. The extract was examined for its ability to scavenge OH
• radicals generated by the Fenton reaction. When the seaweed extract and standard BHT (100 μg/ml) were added to the reaction mixture they removed hydroxyl radicals and prevented the degradation of 2-deoxyribose-2-ribose.
G. acerosa exhibited the greatest scavenging effect of OH
• among the seaweeds but less than the standard BHT. OH
• is known to be capable of abstracting hydrogen atoms from membranes and they bring about peroxidic reactions of lipids [
43]. It is thus anticipated that
G. acerosa would show antioxidant effects against lipid peroxidation on biomembranes and would scavenge OH
• radicals at the stages of initiation and termination.
NO radicals play an important role in inducing inflammatory response and their toxicity multiplies only when they react with O
2
•- radicals to form peroxynitrite, which damages biomolecules like proteins, lipids and nucleic acids [
44]. Nitric oxide is generated when sodium nitroprusside reacts with oxygen to form nitrite. Seaweeds inhibit nitrite formation by competing with oxygen to react with nitric oxide directly. The methanolic extract of
G.acerosa and Haligra sp at 100 μg/ml exhibited 39.8% and 33.3% inhibition which was comparable to the standard BHT, which exhibited 39.6% inhibition at 150 μg/ml. The present results suggest that
G.acerosa and Haligra sp might be potent and novel therapeutic agents for scavenging of NO and the regulation of pathological conditions caused by excessive generation of NO and its oxidation product, peroxynitrite
Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. H
2O
2can cross cell membranes rapidly and once inside the cell it can probably react with Fe
2+ and possibly Cu
2+ to form hydroxyl radicals and this may be the origin of many of its toxic effects [
45]. It is therefore advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. Figure
5 illustrates that the strongest anti- H
2O
2 activity was observed for
G.acerosa at 100 μg/ml when compared with the standard BHT while
T conoides appeared to be a very weak scavenger of hydrogen peroxide. The result suggest that
G acerosa can be a better antioxidant for removing H
2O
2 and thus protecting food systems
The reducing ability of a compound greatly depends on the presence of reductones, which have exhibit antioxidative potential by breaking the free radical chain by donating a hydrogen atom [
46]. For the measurement of reductive ability, we investigated the Fe
3+ to Fe
2+ transformation in the presence of an alcoholic extract using the method of Oyaizu et al [
29]. Figure
6 illustrates that
G. acerosa (100 μg/ml) showed higher absorbance when compared with the control and the same dosage as the standard BHT. The reducing capacity of
G. acerosa is a significant indicator of its potential antioxidant activity. The results of the antioxidant assays indicate that
G. acerosa is the best source of antioxidant compounds among the seaweeds investigated.
Apart from damage caused by free radicals, another common problem during food preservation is contamination by microbes. It not only affects the quality of food, but also causes serious health problems to those who consume the contaminated food. Of the ten seaweeds screened
Haligra sps (50 mg/ml) showed antibacterial activity against
S. aureus (MTCC 96) which was significantly (P < 0.05) higher than the standard antimicrobial agent sodium benzoate (200 mg/ml). The inhibition of growth of
S.aureus by
Haligra sp reveals that it can be used as an antibacterial agent against
S.aureus which causes vomiting, diarrhoea, abdominal cramps and prostration and which also spoils raw meats, poultry, dairy products, salads, shrimp and ham [
47].
The total phenolic content expressed as gallic acid equivalents was higher for the
G. acerosa (0.616 ± 0.0063 g/g) and
Haligra sps (0.440 ± 0.0043 g/g) extracts than for the other seaweeds. Plant phenolics in general are effective free radical scavengers and antioxidants. Phenolic compounds are commonly found in the edible brown, green and red seaweeds in which the antioxidative property has been correlated to their phenolic content [
48‐
50]. Some authors claim that there is no correlation between the total phenolic content and the radical scavenging capacity [
51], so it was very important to examine the correlation between the total phenolic contents and total antioxidant capacity of the studied seaweeds. The results of the present study reveal that there is a strong correlation between antioxidant activity and phenolic content. It is believed that the antioxidant properties of phenolics are a result of their ability to act as reducing agents, hydrogen donors, and free radical quenchers and phenolics can also act as metal chelators which prevent the catalytic function of metal in the process of initiating radicals [
35]. It is possible that the antioxidant and antimicrobial activity of both the seaweed extract (
G. acerosa and Haligra) can be the result of their high concentration of phenolic compounds.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KPD and SKP conceived of the study, and participated in its design and coordination. PK carried out the assays. NS participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.