3.3. Quantification of Free and Bound Phenolic Compounds in Buckwheat Fractions
Free phenolic compounds were quantified through of calibration curves of standards. A total of 25 free phenolic compounds were quantified in buckwheat meals/flours: de-hulled grain meal, bran meal, middling flour, and light flour (Table 4
The most concentrated free phenolic compound in all buckwheat flours was epiafzelchin–epicatechin-O-dimethylgallate, whose content was 13.11 mg/kg d.w. in light flour, 93.83 mg/kg d.w. in de-hulled grain meal, 176.67 mg/kg d.w. in middling flour, and 216.94 mg/kg d.w. in bran meal. The second most concentrated in buckwheat flours was rutin, whose content was from 7.03 mg/kg d.w in light flour, 87.33 mg/kg d.w. in de-hulled grain meal, 148.63 mg/kg d.w. in middling flour, to 214.99 mg/kg d.w in bran meal. Thus, the most abundant free flavonoids are present in buckwheat bran meal, followed by middling flour, de-hulled buckwheat meal, and light flour. Besides, 2-hydroxy-3-O-β-d-glucopyranosylbenzoic and protocatechuic-4-O-glucoside acid appear in buckwheat fractions in significant quantities, whose values were 2.67–2.93 mg/kg d.w. in light flour, 32.71–65.56 mg/kg in de-hulled grain meal, 42.17–79.69 mg/kg d.w. in bran meal, and 78.22–120.56 mg/kg d.w. in middling flour. Therefore, the highest content of phenolic acids appears in middling flour, followed by bran meal, de-hulled grain meal, and light flour. The third most abundant phenolic compound in middling and de-hulled grain meal was protocatechuic-4-O-glucoside acid (120.59 and 65.56 mg/kg d.w.), whereas in light flour was swertiamacroside (4.23 mg/kg d.w.), and in bran meal was epicatechin-O-3,4-dimethylgallate (98.07 mg/kg d.w.).
The total free phenolic content in buckwheat flours was decreasing in the following order: bran meal > middling flour > de-hulled buckwheat meal > light flour (1242.49, 901.10, 520.74, and 46.36 mg/kg d.w.). These results are due to the most abundant free phenolic compounds being flavonoids, which corresponded to 66–79% of total free phenolic compounds, and these are found in higher concentration in outer layers than in inner layers of buckwheat grain [2
]. For that reason, bran meal contains the highest content of free phenolic compounds, followed by middling flour, as it contains seed coat.
The concentration of free phenolic compounds obtained in buckwheat was compared with that obtained previously in other works. Verardo et al. (2011) [5
] quantified the individual free phenolic compounds in de-hulled buckwheat grain, where rutin was the most concentrated, whose value was 35.12% higher than that obtained in the present work and total content of free phenolic compounds was 48.39% higher than in the present work. Nevertheless, the most concentrated free phenolic compound in our work was epiafzelchin–epicatechin-O
-dimethylgallate, whose value was 50% higher than that obtained by Verardo et al. (2011) [5
]. These differences of concentration could be due to the different buckwheat cultivar. Besides, Inglett et al. (2011) [18
] quantified the free flavonoid content in different buckwheat flours (fancy, farinetta, supreme, and whole), fancy corresponded with light flour, supreme flour is similar to bran meal, farinetta consists of a fine granulated mixture of aleurone layer of hulled achene and achene embryo, a composition similar to middling flour [19
]. The value of free flavonoids obtained in our study in light flour, de-hulled grain meal, bran meal, and middling flour (34.47 mg/kg d.w., 371.25 mg/kg d.w., 982.23 mg/kg d.w., and 598.83 mg/kg d.w) were in the same order of magnitude than that obtained in fancy (71.40 mg/kg d.w.), whole buckwheat flour (417.03 mg/kg d.w.), supreme (525.27 mg/kg d.w.), and farinetta (671.50 mg/kg d.w.) by Inglett et al. (2011) [18
Hung et al. (2008) [2
] reported the content of rutin in the free form obtained in different buckwheat flour fractions, and its concentration was 2.5–3 mg/kg d.w. in the innermost layers, whereas in the outer layers, it was 274-337.8 mg/kg. These results were similar to those obtained in the present work in the light flour (7.03 mg/kg dw.) and bran meal (214.99 mg/kg d.w.). Kalinová et al. (2019) [21
] reported the free phenolic compounds in the seed coat (553.18 mg/kg d.w.), in the endosperm (2.59 mg/kg d.w.), and in the groat (139.66 mg/kg d.w.). These values were lower than those obtained in bran meal, light flour, and de-hulled grain meal, and also, the content of rutin in seed coat (54.23 mg/kg d.w.) represents a quart of the phenolic bran meal (214.99 mg/kg d.w.) obtained in our study. This could be due to the different cultivar and/or the different methodology of determination of phenolic compounds (by MS detection a higher number of compounds are determined).In addition, Liu et al. (2019) [22
] reported the concentration of rutin in common buckwheat (62.19 mg/kg d.w.) that was in the same order as that obtained in de-hulled grain meal (87.33 mg/kg d.w.). According to the results obtained in these previous works, it has shown that rutin in the free form is concentrated in the outer layers, which is in concordance with our results.
The Table 5
reports the content of bound phenolic compounds in buckwheat flours. Bound phenolic compounds composition in buckwheat flours was similar than that obtained in free phenolic fraction; nevertheless, flavonoids such as isorientin, epiafzelchin–epiafzelchin–epicatechin, Procyanidin B2-dimethylgallate, hyperin, and (epi)afzelchin-(epi)catechin were not detected in bound fraction, whereas some phenolic acids such as syringic and p-coumaric acid, procyanidin A, and myricetin were determined only in bound fraction.
Catechin was the most concentrated bound phenolic compound in all buckwheat flours, representing 25–30% of total bound phenolic compounds, and its concentration was 54.67 mg/kg d.w. in light flour, 95.45 mg/kg d.w. in de-hulled grain meal, 200.17 mg/kg d.w. in middling flour, and 207.74 mg/kg d.w. in bran meal, respectively. The second component most abundant was epicatechin, whose content was 34.67 mg/kg d.w. in light flour, 41.55 mg/kg d.w. in de-hulled grain meal, 59.08 mg/kg d.w. in bran flour, and 97.50 mg/kg d.w. in middling flour. The third most abundant phenolic compound in de-hulled grain meal and bran meal was syringic acid (35.62 mg/kg d.w. and 85.86 mg/kg d.w.), whereas in middling flour it was caffeic acid hexose (56.73 mg/kg d.w.), and in light flour it was swertiamacroside.
The total bound phenolic content in buckwheat flours was increasing in the following order: light flour < de-hulled grain meal < bran meal < middling flour (207.74, 389.51, 689.81, and 704.47 mg/kg d.w.). Therefore, the highest concentration of bound phenolic compounds is in middling and bran meal due to these compounds being linked to the cell wall of buckwheat layers. Flavonoids represented 59–65% of the bound phenolic fraction. Whereas, phenolic acids represented 35–41% of bound phenolic fraction.
Concentrations of catechin, epicatechin, syringic, and total bound phenolic compounds in de-hulled whole buckwheat flour obtained by Verardo et al. (2011) [5
] were 23.88%, 48.54%, and 53.18% higher than those obtained in the present work. Inglett et al. (2011) [18
] reported the content of total bound flavonoid in buckwheat flour fractions obtained was 59.25 mg/kg d.w. in fancy, 389.68 mg/kg in farinetta, 530.21 mg/kg in supreme, and 613.77 mg/kg d.w. in whole flour, which are in the same order of magnitude as that obtained in our work. Nevertheless, in this study, the highest bound phenolic content was obtained in whole buckwheat flour, whereas in our work, the maximum value of phenolic content corresponded with the middling flour. This could be due to the different cultivar or because Inglett et al. (2011) [18
] could include the hull in the buckwheat grain flour.
The total content of flavonoids was from 157.84 mg/kg d.w. in light flour to 1422.52 mg/kg d.w. in bran meal, whereas the content of phenolic acids was from 96.261 mg/kg d.w. in light flour to 548.63 mg/kg d.w. in middling flour. Total phenolic content was from 254.10 mg/kg d.w. in light flour to 1932.30 mg/kg d.w. in bran meal (Table 6
). According to the results, the total phenolic content was increasing in the following order: light flour < de-hulled grain meal < middling flour < bran meal Therefore, middling flour and bran meal possess the highest phenolic content due to bran and the aleurone layer being richer in many phenolic compounds than the others buckwheat flours [23
]. Total flavonoid obtained in de-hulled grain meal, bran meal, and middling flour was 49.22%, 71.21%, and 27.83% higher than that obtained in whole grain meal, supreme, and farinetta by liquid chromatography-electrospray ionization- mass spectrometry (LC–ESI-MS) [18
]. According to Guo and co-workers, free rutin was determined in a range of 51–81% [24