Reversed Phase HPTLC-DPPH Free Radical Assay as a Screening Method for Antioxidant Activity in Marine Crude Extracts

Abstract

EditorialThe large number of marine organisms present in the ocean, together with the diverse marine environments to which they have adapted, provides an enormous and mostly unexploited source of structurally novel and biologically active secondary metabolites [1]. Some of these organisms have provided compounds that have served as important leads in the discovery of new drugs [2], as probes in molecular pharmacology [3], and use as pest control agents [4]. In particular, they are a rich source of antioxidants but there has been limited work in screening organisms containing this rich source of structurally unique natural products for antioxidant activity. For instance, there are a large number of active components in macroalgae with antioxidant properties, including pigments such as polyphloroglucinols and fucoxanthin [5,6], bioactive carbohydrates such as fucoidan and laminarin, minerals, and UV absorbing mycosporine like amino acids [7]. Glutathione, an important antioxidant in plants, animals, fungi and some bacteria, is found in all macroalgae with some species containing as much as 3082 mg/100 g [8].One way to screen crude marine extracts for antioxidant activity or radical-scavenging effects is to use either: (i) a solution based or (ii) a high performance thin layer chromatography (HPTLC) based 2,2diphenyl-1-picrylhydrazyl (DPPH) free radical bioassay [9][10][11][12]. This method is based on the use of stable DPPH• free radical species which when added to a sample, react and neutralize antioxidants present in the sample [13,14]. DPPH• is a deep purple colored free radical that turns into pale yellow when reduced by an antioxidant. If a known amount of DPPH• is added to a sample, the decrease in absorbance at 517 nm due to the disappearance of DPPH• free radical is directly proportional to the amount of antioxidants present. The DPPH• free radical is stable in ethanol solution at room temperature as the odd electron on the nitrogen is able to delocalize through the entire molecule (Figure 1), so the molecules do not dimerise like most other free radicals. This delocalization of electrons gives rise to the intense violet color of DPPH solutions. It is the odd electron on the nitrogen atom in DPPH• that is reduced by receiving a hydrogen atom or electron from an antioxidant present to form its corresponding hydrazine (yellow non-radical species) [15]. The concentration of the DPPH at the end of a reaction will depend on the concentration and activity of the free radical scavengers presents [16]. Figure 1: DPPH -free radical and its reduced formIn order to isolate and identify which are the most potent free radical scavengers present in a sample, the DPPH assay can be combined with either high performance liquid chromatography (HPLC) or high performance thin layer chromatography (HPTLC). Unfortunately, the use of the on-line HPLC method has not been successful as slow reaction kinetics results in inaccurate antioxidant capacity measurements [17]. HPTLC combined with DPPH radical detection of antioxidants in s...