Basella alba L. (Malabar Spinach) as an Abundant Source of Betacyanins: Identification, Stability, and Bioactivity Studies on Natural and Processed Fruit Pigments.

The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.

Identification and reactivity of pigments in prominent vegetable leaves of Basella alba L. var. 'Rubra' (Malabar spinach).

The unique profiles of betacyanins as well as their stability and antioxidant activity in purple leaf extracts of the fast-growing, soft-stemmed vine Basella alba L. var. 'Rubra', known as Malabar spinach, are partly characterized for the first time. The distribution of gomphrenin and its acylated derivatives in the leaves is completely different from the profiles of the pigments in the fruits. The most abundant acylated pigment in leaves (24%) turned out 6'-O-E-sinapoyl-gomphrenin (gandolin), however, the most significant difference in the pigment profiles is a presence of two novel pigments tentatively identified as highly abundant 6'-O-(3,4-dimethoxy-E-cinnamoyl)-gomphrenin and 6'-O-(3,4,5-trimethoxy-E-cinnamoyl)-gomphrenin as well as their isoforms. Significant degradation of the pigments in the fruit extracts under the impact of selected metal cations and UV-Vis irradiation as well as high protective activity of the leaf extract matrix were observed. Partial chromatographic purification of the leaf extract resulted in an increase of the pigment concentration which was correlated positively with the increased antioxidant activity of obtained fractions.

Influence of Citrates and EDTA on Oxidation and Decarboxylation of Betacyanins in Red Beet (Beta vulgaris L.) Betalain-Rich Extract.

The influence of stabilizing activity of citric buffers on betacyanins, as well as their thermal dehydrogenation and decarboxylation in a beetroot betalain-rich extract (BRE), was studied at pH 3-8 and temperature 30, 50 and 85 °C with an additional effect of EDTA. In acetate/phosphate buffers, the highest stability is observed at pH 5 and it decreases toward pH 3 as well as pH 8, which is more remarkable at 85 °C. For the citrates, a contradictory effect was observed. Citric buffers tend to stabilize the substrate pigments and their intermediary products in acidic solutions, although increase their reactivity at pH 6-8. The highest impact of EDTA addition on pigment retention in acetate buffers is observed at 85 °C and pH 3-5 as well as 8, reflecting the preserving activity of EDTA at the most unfavorable conditions. At lower temperatures, pigment stability in more acidic conditions is still at higher levels even without addition of citrates or EDTA. The most striking effect on generation of betanin derivatives during heating is 2-decarboxylation which preferentially proceeds in the most acidic environment and this generation rate at 85 °C is much higher in the citrate buffers compared to acetates.

Structural Studies on Diverse Betacyanin Classes in Matured Pigment-Rich Fruits of Basella alba L. and Basella alba L. var. 'Rubra' (Malabar Spinach).

Identification of betacyanins in Basella alba L. and Basella alba L. var. 'Rubra' fruits was performed by low- and high-resolution mass spectrometry (LRMS and HRMS) as well as 1H, 13C and two-dimensional NMR which revealed hitherto completely not known betacyanin classes in the plant kingdom. Especially, the presence of unique nitrogenous acyl moieties in the structures of the pigments was ascertained by the HRMS Orbitrap detection. Except for detected polar betacyanin glycosylated derivatives, presence of a series of previously not reported pigments such as malonylated betanidin 6-O-ß-glusosides with their acyl migration isomers along with the evidence of the 3''-hydroxy-butyrylated betacyanins is reported. The first complete NMR data were obtained for novel and principal acylated gomphrenins with hydroxycinnamic acids: 6'-O-E-caffeoyl-gomphrenin (malabarin), 6'-O-E-sinapoyl-gomphrenin (gandolin), 6'-O-E-4-coumaroyl-gomphrenin (globosin) and 6'-O-E-feruloyl-gomphrenin (basellin).

Dehydrogenation of Betacyanins in Heated Betalain-Rich Extracts of Red Beet (Beta vulgaris L.).

Betacyanins are a group of water-soluble red-violet compounds containing nitrogen in their structure. These are biosynthesized in red beetroot (Beta vulgaris L.), a widely consumed vegetable that contains significant amounts of nutritious and bioactive compounds which are also found in dietary supplements. This contribution presents results of betacyanin thermal oxidation (resulting in dehydrogenation) interrelated with decarboxylation in selected acetate/phosphate buffers at pH 3-8 and at 85 °C, which may be of particular significance for formulation and performance of foods. Most of the reaction products were detected at the highest concentrations in the acidic solutions (pH 3-4). The main dehydrogenation reaction pathways were monitored by LC-DAD-MS/MS and were associated with decarboxylation of the principal extract pigments, betanin/isobetanin and neobetanin, at carbon positions C-2 and C-17. Additional reactions are accompanied by the 2,15-decarboxylation processes at different dehydrogenation levels with 15-decarboxy-betanin and 2,15-bidecarboxy-betanin, structurally elucidated by NMR analysis, as the distinct indicators of this route type. For other novel pigments detected, 2,15-bidecarboxy-xanbetanin, 2,15-bidecarboxy-xanneobetanin and 2,15,17-tridecarboxy-neobetanin, additional high resolution mass spectrometric analyses were performed and confirmed their molecular formulas.