Betalain pigments: isolation, characterization and in vitro antioxidant and anti-inflammatory properties

Betalains are water-soluble natural pigments which can be classified as red-violet colour betacyanins and yellow-orange colour betaxanthins. Apart from their tinctorial applications, betalains have recently drawn attention due to their biological activities such as antioxidant, anti-inflammatory, and antimicrobial properties which have been associated with health benefits and functional applications. However, the antioxidant and anti-inflammatory properties of individual betalains have not been fully elucidated with one of the reasons being the lack of purified compounds. Therefore, the aim of this study was to optimize betalain extraction while introducing a novel purification method of betalains as well as conducting a comparative assessment of the antioxidant and anti-inflammatory activities of individual betalains. Low ethanol-water concentrations were found to be most preferable solvent mixtures to extract and support stability of betalains over the 4 weeks of studied period. A flash chromatography-based separation, which is a convenient and fast method to isolate unstable materials, was exploited for the first time to purify betalains from beetroot and prickly pear. Proposed method was successful to obtain high yields of individual betalains betanin, neobetanin, vulgaxanthin I and indicaxanthin in high purity, which were used for biochemical characterization and cellular assays. As the results demonstrate, all purified betalains exhibited antioxidant and free radical scavenging activities, with betanin showing much stronger capacity compared to the other betalains. In addition, all betalains suppressed LPS-induced macrophage inflammatory response determined via expression of interleukin-6 (IL-6), IL-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2) while showing moderate cell-protective properties via induction of nuclear factor erythroid 2-related factor 2 (Nrf2) targets. When compared, betacyanins displayed better antioxidant and anti-inflammatory properties than betaxanthins. In summary, this research demonstrated that betalains are potent anti-inflammatory and antioxidant compounds, emphasizing their potential for applications beyond colouring purposes. Further, based on the multiple regression analysis, betalains could be assumed as the main contributors to antioxidant activity of betalain extracts while different betalains contribute differently towards the antioxidant and anti-inflammatory activities which can be related to structural differences.