The polyphenol content of the extract was quantified by Folin–Ciocalteu reagent (FCR) and such an assay was performed for the entire BR bulb. The results were evaluated according to the calibration series prepared, through application of the gallic acid standard. The various sections of the BR constituted significant sources of polyphenol. Indeed, the entirety of the BRE contained 93 mg (expressed as mg of gallic acid equivalent per 100 g of BRE). The content of polyphenolic substances in the BRE, as prepared in the ethanol solution (70%), reached similar values to those in the literature. This BRE was seen to remain stable under conditions of storage at low temperature (4–8 °C) in darkness for at least one month, with no significant change in polyphenol content
[20][21].
The antioxidant activity of the BRE was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), a scavenging method widely deployed for evaluating antioxidant activity for relatively short durations, compared to other methods. The main attribute of AO is the neutralization of free radicals by donating an electron or hydrogen atom. In particular, polyphenols more often act as direct radical scavengers of the lipid peroxidation chain reaction (chain-breakers). The cessation of the reaction chain and the formation of a stable radical occurs upon the supply of an electron from the chain-breaker to the free radical, which it neutralizes
[22][23]. The reducing ability of the DPPH radicals was determined by diminishing its absorbance at 517 nm, as induced by the AOs. The subsequent measurements revealed that the antioxidant activity of the BRE corresponded to 114 mg per the equivalent amount of 100 g of ascorbic acid. The value for antioxidant activity, herein converted to the amount of ascorbic acid, is influenced by various aspects such as the type of BR storage, conditions, the environment of its cultivation, and the technique of extract preparation. This explains the wide variance reported in other publications, i.e., from 20 to 170 mg (expressed as mg of ascorbic acid equivalent per 100 g of BRE)
[24][25][26].
Figure 1 shows a representative total ion chromatogram (TIC) for the BRE from mass spectrometry in positive ion mode. On integration, the TIC exhibited several peaks evaluated as ms/ms fragmentation obtained arising through collision-induced dissociation. The peak at 2.98 min, a [M + H]
+ molecular peak of
m/
z 549.1336, presented a fragment of
m/
z 387.0809 and was consistent with previous findings on the fragmentation of neobetanin (
Figure 2A) published in references
[27][28]. The molecular ion at 2.49 min had a value for
m/
z of 551.1509, which, on dissociation, yielded fragments of
m/
z 389.0976 (
Figure 2B) typical for betanin or isobetanin (2.61 min)
[27][28][29]. In coelution with betanin and isobetanin, the following were identified: 2′-
O-glucosyl-betanin (t
R = 2.52 min; [M + H]
+ = 713.2034) and 2′-
O-glucosyl-isobetanin (t
R = 2.63 min; [M + H]
+ = 340.1132)
[28]. Tentative identification was also performed on feruloylglucose at 3.55 min ([M + H]
+ = 357.1175), 5,5′,6,6′-tetrahydroxy-3,3′-biindolyl at 3.74 min ([M + H]
+ = 297.0874), betavulgarin at 5.35 min ([M + H]
+ = 313.0704), and cochliophilin A at 6.23 min ([M + H]
+ = 283.0604)
[27][28].
Figure 2. Positive electrospray tandem mass spectra for (A) neobetanin, and (B) betanin or isobetanin. The daughter ion of m/z 387.0809 was obtained by fragmentation of the parent ion of m/z 549.1228 of neobetanin. The daughter ion of m/z 389.0976 corresponds to protonated aglycone, and was obtained by fragmentation of the parent ion of m/z 551.1516 of betanin or isobetanin. The collision energy was 10 eV and 30 eV for (A) and (B), respectively.