Profiling of phenolic compounds by HPLC-ESI-QqQ-MS in sorghum and buckwheat flours at different germination times

Buckwheat (Fagopyrum esculentum) is well recognized as an excellent antioxidant and hypolipidemic nutrient food [1]. The concentration of natural hydrophilic and lipophilic antioxidants may show strong variations depending on several factors including variety, location, and environmental conditions [2]. Buckwheat seeds serve as a rich source of many phenolic compounds such as rutin, quercitrin, catechin, p-hydroxybenzoic, protocatechuic, gallic, p-coumaric, syringic, caffeic, and ferulic acids [3]. Sorghum (Sorghum Möench) has attracted increasing attention not only because it can be grown in diverse and harsh environments including at high temperature, high altitudes and drought conditions [4], but also for its content in health-promoting bioactive phenolic compounds [5]. The phenolic compounds in sorghum are abundant and diverse, including phenolic acids, flavonoids (such as flavonols, flavones, flavanones and 3- deoxyanthocyanidins) and proanthocyanidins (tannins) [6]. The phenolic profile of sorghum is also unique depending on the genotype, environmental and growth conditions. Germination is the process that consists of the external growth of the parts of the grain bud and root until appropriate enzymes are produced in adequate quantities, also to produce malt. The aim of this research was to study the phenolic compounds in buckwheat and sorghum flour and their evolution at three different times of germination (0, 48 and 72 h). 2. Experimental 2.1 Phenolic extraction To determine the free and bound phenolic fraction of flour samples, the method developed by Verardo and collaborators [7] was applied. Briefly, 2 g of bread were extracted twice in an ultrasonic bath with a solution of ethanol/water (4:1 mL/mL). The supernatants were collected, evaporated and reconstituted with 2 mL of methanol/water (1:1 mL/mL). The extracts were stored at -18 °C until use. To obtain the bound phenolic fraction, residues of free phenolic extraction were digested with 200 mL of 2 mol/L NaOH at room temperature for 4 h by shaking under nitrogen gas. The hydrolyzed solution was acified to pH 2-3 by adding 10 mol/L hydrochloric acid in a cooling ice bath and extracted with 500 mL of hexane to remove the lipids. The final solution was extracted five times with 100 mL of 1/1 diethyl ether/ethyl acetate (mL/mL). The organic fractions were pooled and evaporated to dryness. The phenolic compounds were reconstituted in 2 mL of methanol/water (1:1 mL/mL) 2.2 Phenolic determination Determination of free and bound phenolic compounds was carried out by using a liquid chromatography apparatus HP 1290Series (Agilent Technologies, Palo Alto, CA, USA) equipped with a degasser, a binary pump delivery system, an automatic liquid sampler and coupled to a QqQ mass spectrometer detector. Separation of phenolic compounds was carried out by a C-18 column (Poroshell 120, SB-C18, 3.0 × 100 mm, 2.7 μm from Agilent Technologies, Palo Alto, CA, USA). The gradient elution was with as the mobile phase A acidified water (1% acetic acid) and as mobile phase B acetonitrile. MS analysis was carried out using an electrospray ionization (ESI) interface in negative ionization mode at the following conditions: drying gas flow (N2), 9.0 L/min; nebulizer pressure, 50 psi; gas drying temperature, 350 °C; capillary voltage, 4000 V. The fragmentor and m/z range used for HPLC-ESI/MS analyses were 80 V and m/z 50-1000, respectively [7]. For the quantification of 3-deoxyanthocyanidins, the column was kept at 30 °C. The gradient of mobile phase was as follows: initial, 15% B; 4 min, 25% B; 4.1 min, 100% B; 5 min, 100% B; 5.1 min, 15% B; and 6 min, 15% B. The QqQ-MS system was performed in ESI-positive mode with a collision voltage of 35 V, source temperature of 120 °C, and desolvation temperature of 450 °C. Data were processed by the software MassHunter Workstation Qualitative Analysis Version B.07.00 (Agilent Technologies, Santa Clara, CA, USA) [8]. 3. Results In buckwheat flour, flavonoids were the preponderant free phenolic compounds. In particular flavan-3-ols such as catechin, epicatechin and catechin-glucoside increased their concentration after 48 h of germination and then decrease at 72 h; on the contrary, epicatechin-gallate and epicatechin-O-3,4-dimethyl gallate decreased their concentration during germination. Flavonols, such as rutin and quercitrin, and procyanidins like procyanidin A and procyandin B2, significantly increased (p<0.05) their concentration during germination. Three phenolic acids, swertiamacroside, caffeic acid hexose and 2-hydroxy-3-O-β-D-glucopyranosylbenzoic acid, were also identified in free phenolic profile, and their concentration increased from 0 to 72 h of germination. Only swertiamacroside significantly decreased (p<0.05) from 48 to 72 h. As regard bound phenolic compounds, three hydroxycinnamic acids, p-coumaric, syringic and caffeic acids, were determined; among them only the bound caffeic acid decreased from 0 to 48 h of germination and then increase its concentration at 72 h of germination. A lot of free flavonoids determined in sorghum flour increased their concentration from 0 to 48 h of germination and then decreased after 72 h. Taxifoline hexoside (flavonol), eriodictyol (flavanone), dihydrokaempferol (flavanone), luteolin (flavones), and naringenin (flavanone) showed this trend. Benzoic acid, taxifolin and hispidulin significantly increase (p<0.05) along the germination; on the other hand, 3-deoxyanthocyanidins, N1-N4-dicaffeoyl spermidine, 3,4,5-trimethoxy cinnamic acid and ononin decrease their concentration along the germination. Among bound phenolic compounds determined in sorghum flour, p-coumaric acid, ferulic acid, luteolin and apigenin showed a significant (p<0.05) increased at 48 h of germination and then suffered a decrease at 72 h. Only bound benzoic acid and hispidulin registered a significant increase along the germination and, on the contrary, isoferulic acid and apigeninidin decreased their concentrations. 4. Conclusions These results point out how thegermination process can affect phenolic compounds distribution in buckwheat and sorghum flours. In particular, buckwheat registered a significant increment of both free and bound phenolic compounds along the germination; otherwise, sorghum showed an increment from 0 to 48 h but a further decrement at 72 h of germination. References List reference quoted sequentially in the text and marked as [1]. Reference text 10 pts. Journal title in italics. Paper title is not requested. 1. V. Verardo, D. Arráez-Román, A. Segura-Carretero, E. Marconi, A. Fernáandez-Gutiérrez, M.F. Caboni; Journal of Cereal Science, 52 (2010), pp 170-176. 2. J. Kalinová, E. Dadáková; Cereal Research Communications, 34 (2006), pp 1315-1321. 3. Z. Zhang, C. He, R. Zhu, J. Shen, Y. Yu, Q. Peng, J. Gao, Y. Li, M. Wang; Journal of the American Oil Chemists’ Society, 93 (2016), pp. 1127-1136. 4. S. Schittenhelm, S., S. Schroetter; Journal of Agronomy and Crop Science, 200 (2014), pp. 46–53. 5. L. de Morais Cardoso, S.S. Pinheiro, H.S. Martino, H.M. 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