Highland Barley and Its By-Products Enriched with Phenolic Compounds for Inhibition of Pyrraline Formation by Scavenging α-Dicarbonyl Compounds

Pyrraline, a typical kind of advanced glycation end product, has been found to contribute to the development of pathologies associated with ageing and diabetes mellitus. In the study, phenolic compounds extracted from highland barley whole grain (HBWG) and vinasse (HBVN) were used to inhibit pyrraline formation in a simulated food. The optimal extraction condition for HBWG and HBVN was using 8 mL of 50% acetone solution at 50 °C for 60 min. The extraction and identification of phenolic compounds from HBWG and HBVN were performed by UPLC–PAD–MS/MS. The inhibitory effects of pyrraline in the simulated food were 52.03% and 49.22% by HBVN and HBWG, respectively. The diphenyl picrylhydrazyl radical- and ferric-reducing ability of plasma assays was used to evaluate the antioxidant activity of the extracts. The main inhibition pathways and molecular mechanism of phenolic compounds on pyrraline regulation were explored by scavenging α-dicarbonyl compounds. The study demonstrated that highland barley and its by-products can potentially be used as a functional food to regulate pyrraline formation during food processing.


Introduction
The Maillard reaction, also known as nonenzymatic browning or protein glycation, was originally discovered by Louis Maillard [1]. A series of chemical reactions occur between sugars and proteins, resulting in a yellowish-brown color change during food heating, processing, and storage [2]. The Maillard reaction leads to a lot of positively food-related colors, flavors, and aromas, which also occur in vivo. It is seen in conjunction with accelerated aging and diabetic complications [3,4]. However, controlling the negative effects of the Maillard reaction on food quality and safety is an urgent problem. Because of the potential health risks associated with advanced glycation end products (AGEs), people are increasingly concerned about the level of foodborne AGEs.
Many studies have shown that the Maillard reaction is initiated with the attachment of the aldehyde function of acyclic glucose to a protein amino group using nucleophilic addition to form an aldimine of a Schiff base [5]. The stable Amadori product is formed by Schiff base rearrangement [6], which could undergo multiple chemical rearrangements to form high reactivity of carbonyl compounds. These compounds react again with free amino groups of proteins, resulting in the production of advanced glycation end products (AGEs) [7]. Several AGEs have been proposed, such as N ε -(carboxymethyl)-L-lysine [8], N ε -carboxymethyl arginine [9], perlysines [10], crosslines [11], imidazolone [7], and vesperlysines [12]. Pyrraline, ε-2-(formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine, is one of the major compounds of AGEs formed in the final stage of the Maillard reaction [8]. It is generally accepted that the main compounds of AGEs are pathophysiologically derived from the ingestion of dietary N ε -carboxymethyllysine, but pyrraline studies are lacking.
α-Dicarbonyl compounds are the typical degradation products of reducing sugars and Amadori products for AGE formation during food processing, such as 3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal (MGO) [13]. These compounds induce inter-and intra-crosslinks in proteins, leading to their structural and functional changes. Pyrraline may be formed from α-dicarbonyl compounds and the ε-amino group of amino acid or proteins. Portero-Otin et al. [14] reported higher levels of urinary pyrraline in patients with diabetes. We quantified pyrraline in milk and milk powder using core-shell nanoparticles as absorbents coupled with high-performance liquid chromatography, which was constructed of metal-organic frameworks as core and molecularly imprinted polymers as shell [15]. Convenient and fast optical-based sensing technology was used for the sensitive detection of pyrraline in our lab [16]. However, there was a lack of research on the inhibition of pyrraline during food processing. Therefore, we focused on the inhibition of foodborne pyrraline by α-dicarbonyl compounds.
Highland barley, the largest coarse cereal in northwest China, has attracted much attention for its unique nutritional value as a bioactive phytochemical [17]. Recently, whole grain intake has been found to reduce the risk of chronic diseases possibly due to its rich bioactive phytochemicals. Many studies have shown that bioactive phytochemicals could combat oxidative stress in the body by maintaining a balance between oxidants and antioxidants [18,19]. Emerging academic research has confirmed that highland barley whole grain (HBWG) contains an abundance of native phytochemicals. In particular, an average phenolic content of 333.9−460.8 mg is the most abundant in HBWG [20]. The addition and synergism of these phytochemicals endow HBWG with potential functions, such as antihyperglycemic, antihyperlipidemic, and anticancer activities [21,22]. Highland barley vinasse (HBVN) is the residue of highland barley grains after fermentation and wine making, which is usually discarded as waste or used to produce animal feed. The potential applications of HBWG and HBVN in the inhibition or degradation of pyrraline-induced chronic disease have rarely been studied.
In the study, phenolic compounds extracted from HBWG and HBVN were used to inhibit foodborne pyrraline associated with the Maillard reaction in food processing. The extraction and identification of phenolic compounds from HBWG and HBVN were studied by an ultraperformance liquid chromatography-photodiode array detector coupled with mass spectrometry/mass spectrometry (UPLC-PAD-MS/MS). The inhibition effect of HBWG and HBVN on pyrraline was performed by a simulation system based on glucose and L-lysine. The diphenyl picrylhydrazyl radical-and ferric-reducing ability of plasma assays was used to evaluate the antioxidant activity of HBWG and HBVN. The main pathway and the molecular mechanisms of phenolic compounds on pyrraline inhibition were explored. Several studies have shown that α-dicarbonyl compounds are major intermediates in the formation of AGEs by protein glycation [23,24], leading to associated α-oxoaldehyde-derived AGE formation. Therefore, the pathway of α-dicarbonyl compounds is an important topic of discussion in the study.

Samples
The samples of HBWG and HBVN were from the local factory. The extraction of the whole grain of highland barley was based on the methods described by Meneses et al. [25] with some modifications. HBWG and HBVN weighed with 1 g samples each were extracted with 8 mL of 50% acetone solution at 50 • C for 60 min. The extract was centrifuged at 5000 rpm for 10 min, the supernatant was collected, and the insoluble component was extracted twice using the same method. The supernatant was merged and dried by a rotary evaporator. After nitrogen blowing, it was dissolved in 1 mL of ethanol and filtered through a 0.2 µm microfiltration membrane for UPLC-PAD-MS/MS analysis.

Inhibition of Pyrraline Formation by Phenolic Compounds from HBWG and HBVN
An inhibition study was performed by adding HBWG and HBVN (1 mL) to the simulation system, respectively. We used the typical simulation system of L-lysine and glucose, and the preparation process for the model system was as follows [13]: 0.1 mol/L Llysine and glucose were mixed with 0.1 mol/L phosphate-buffered solution (PBS, pH 7.4). The mixtures were incubated at 80 • C for 40 min and cooled immediately for further analysis. To evaluate the inhibition effect, the simulation systems were compared with a control experiment without the addition of HBWG.

Antioxidant Activities of HBWG and HBVN
The radical-scavenging activity was studied using the DPPH and FRAP methods. In the dark, 150 µL of HBWG or HBVN was mixed with DPPH (2.85 mL, 6.6 × 10 −5 M) for 2 h, and then the absorbance was measured with UV detection at 515 nm. The inhibition effect of the DPPH method was calculated using Equation (1) [26]: where As and Ac are the absorbance values of the sample and blank, respectively. In the FRAP method, we used S0116 test kits for analysis of radical-scavenging activity. In the acidic conditions, ferric tripyridyl triazine TPTZ-Fe (III) was reduced to ferrous tripyridyl triazine TPTZ-Fe (II) by antioxidant activity. The concentration of TPTZ-Fe (II) was determined with UV detection at 593 nm.

Statistical Analysis
The results are expressed as the means ± standard deviation from three independent determinations. Analysis of variance was used to establish any significant differences (p < 0.05) between the applied treatments using the SPSS software package (version 17.0, SPSS Inc., Chicago, IL, USA).

Optimization of Different Extraction Conditions of Phenolic Compounds from HBWG and HBVN
The total content of phenolic compounds from HBWG and HBVN was evaluated by gallic acid with different extraction solvents. Preliminarily, our group studied that acetone was a good solvent for the extraction of phenolic compounds [27]. Therefore, we chose 40%, 50%, 60%, 70%, 80%, and 90% acetone for the extraction solvents and compared them with water ( Figure 1A). When using 50% acetone as the extraction solvent, the extracted amount of phenolic compounds from HBWG was 725.97 mg/100 g, which was higher than that of the other solvents. When the acetone concentration was 50-70%, the extraction efficiency of phenolic compounds from different sources was relatively high, especially in the substrate containing protein, because acetone aqueous solution could effectively degrade the complex of polyphenol and protein. However, when the amount of acetone increased, other substances in HBWG might have been extracted, which hindered the extraction of polyphenols. When the acetone content was 50%, the extraction content of phenolic compounds in HBVN was 146.3 mg/100 g ( Figure 1A). However, it had a negative effect at acetone concentrations greater than 50%. In the protein-based matrix, 50-95% acetone aqueous solution had relatively good extraction efficiency for different sources of phenolic compounds. Because acetone aqueous solution can effectively degrade the polyphenol-protein complex, when the amount of acetone increased, other impurities were extracted from the samples to inhibit the extraction of phenolic compounds. Therefore, we chose 50% acetone for the extraction solvent. creased, other substances in HBWG might have been extracted, which hindered the extraction of polyphenols. When the acetone content was 50%, the extraction content of phenolic compounds in HBVN was 146.3 mg/100 g ( Figure 1A). However, it had a negative effect at acetone concentrations greater than 50%. In the protein-based matrix, 50-95% acetone aqueous solution had relatively good extraction efficiency for different sources of phenolic compounds. Because acetone aqueous solution can effectively degrade the polyphenol-protein complex, when the amount of acetone increased, other impurities were extracted from the samples to inhibit the extraction of phenolic compounds. Therefore, we chose 50% acetone for the extraction solvent. The extraction times were optimized by 1, 2, 3, 4, 5, and 6 ( Figure 1B). When the extraction time was 4, the extraction amount of phenolic compounds in HBWG reached a maximum value of 544.33 mg/100 g. With increasing extraction times, the content of phenolic compounds from HBWG increased, but there was no significant difference with an extraction time of 4. It can be seen from Figure 1B that the extraction amount of phenolic compounds did not increase significantly with the continuous increase in extraction time. When the extraction times were 4, the extraction amount of phenolic compounds in HBVN reached a maximum value. When the extraction times increased, the extraction amount of phenolic compounds did not increase significantly. Therefore, we chose the optimal extraction time of 4. Figure 1C shows that the content of phenolic compounds increased with increasing extraction temperature. When the extraction temperature was 50 °C, the extraction amount of phenolic compounds in HBWG reached a maximum value of 538.11 mg/100 g. As the temperature continued to rise, the extraction amount of phenolic compounds in HBWG decreased, which may be due to the degradation of phenolic substances caused by the temperature increase. Therefore, 50 °C was selected as the optimal extraction temperature of phenolic compounds in HBWG. When the extraction temperature was 50 °C, the extraction content of phenolic compounds in HBVN reached a maximum of 124.00 mg/100 g, and the content of phenolic compounds decreased with increasing temperature The extraction times were optimized by 1, 2, 3, 4, 5, and 6 ( Figure 1B). When the extraction time was 4, the extraction amount of phenolic compounds in HBWG reached a maximum value of 544.33 mg/100 g. With increasing extraction times, the content of phenolic compounds from HBWG increased, but there was no significant difference with an extraction time of 4. It can be seen from Figure 1B that the extraction amount of phenolic compounds did not increase significantly with the continuous increase in extraction time. When the extraction times were 4, the extraction amount of phenolic compounds in HBVN reached a maximum value. When the extraction times increased, the extraction amount of phenolic compounds did not increase significantly. Therefore, we chose the optimal extraction time of 4. Figure 1C shows that the content of phenolic compounds increased with increasing extraction temperature. When the extraction temperature was 50 • C, the extraction amount of phenolic compounds in HBWG reached a maximum value of 538.11 mg/100 g. As the temperature continued to rise, the extraction amount of phenolic compounds in HBWG decreased, which may be due to the degradation of phenolic substances caused by the temperature increase. Therefore, 50 • C was selected as the optimal extraction temperature of phenolic compounds in HBWG. When the extraction temperature was 50 • C, the extraction content of phenolic compounds in HBVN reached a maximum of 124.00 mg/100 g, and the content of phenolic compounds decreased with increasing temperature ( Figure 1C). Therefore, 50 • C was selected as the optimal extraction temperature for HBVN extraction.
When the extraction solvent volume was 8 mL, the extraction content of phenolic compounds in HBWG and HBVN reached a maximum of 536.53 mg/100 g and 132.21 mg/100 g, respectively ( Figure 1D). The extraction volume of phenolic compounds did not increase significantly with increasing solvent volume. Too much extraction solvent would lead to waste of raw materials and environmental pollution. Therefore, the optimal extraction volume was 8 mL.
As shown in Figure 1E, when the extraction time of HBWG and HBVN was 60 min, the extraction amounts of phenolic compounds reached a maximum value of 514.60 mg/100 g and 131.31 mg/100 g, respectively. The content of phenolic compounds in HBWG decreased after continuous heating, which may be due to the degradation of phenolic compounds caused by the long heating time.

Antioxidant Activity of HBWG and HBVN Extracts
Many studies have reported that AGE formation is viewed as a major contribution to oxidative damage in some tissues in chronic diseases, such as the pathogenesis of diabetic complications of eye lenses and nerves [28]. Therefore, oxidative stress is thought to be closely related to AGE formation, and antioxidant compounds are considered potential inhibitors of AGE formation [29]. The radical-scavenging activity uses fresh and dry HBVN to compare the antioxidant capacity. Fresh HBVN is wet and not conducive to preservation. After spray drying, HBVN is beneficial for long-term storage and utilization. Therefore, we used 0.05 g/mL of fresh and dry HBVN and 0.05 mg/mL of HBWG in the study. Figure 2 shows the scavenging activity of DPPH by using HBWG and fresh and dry HBVN extracts, at 83%, 75%, and 44%, respectively, which is higher than that using DDW at 27%. The antioxidant capacities of HBWG and fresh and dry HBVN extracts were 10.11, 7.94, and 3.57 mg FE(II)/g EXTRACTS for the FRAP method, respectively.
( Figure 1C). Therefore, 50 °C was selected as the optimal extraction temperature for HBVN extraction.
When the extraction solvent volume was 8 mL, the extraction content of phenolic compounds in HBWG and HBVN reached a maximum of 536.53 mg/100 g and 132.21 mg/100 g, respectively ( Figure 1D). The extraction volume of phenolic compounds did not increase significantly with increasing solvent volume. Too much extraction solvent would lead to waste of raw materials and environmental pollution. Therefore, the optimal extraction volume was 8 mL.
As shown in Figure 1E, when the extraction time of HBWG and HBVN was 60 min, the extraction amounts of phenolic compounds reached a maximum value of 514.60 mg/100 g and 131.31 mg/100 g, respectively. The content of phenolic compounds in HBWG decreased after continuous heating, which may be due to the degradation of phenolic compounds caused by the long heating time.

Antioxidant Activity of HBWG and HBVN Extracts
Many studies have reported that AGE formation is viewed as a major contribution to oxidative damage in some tissues in chronic diseases, such as the pathogenesis of diabetic complications of eye lenses and nerves [28]. Therefore, oxidative stress is thought to be closely related to AGE formation, and antioxidant compounds are considered potential inhibitors of AGE formation [29]. The radical-scavenging activity uses fresh and dry HBVN to compare the antioxidant capacity. Fresh HBVN is wet and not conducive to preservation. After spray drying, HBVN is beneficial for long-term storage and utilization. Therefore, we used 0.05 g/mL of fresh and dry HBVN and 0.05 mg/mL of HBWG in the study. Figure 2 shows the scavenging activity of DPPH by using HBWG and fresh and dry HBVN extracts, at 83%, 75%, and 44%, respectively, which is higher than that using DDW at 27%. The antioxidant capacities of HBWG and fresh and dry HBVN extracts were 10.11, 7.94, and 3.57 mg FE(II)/g EXTRACTS for the FRAP method, respectively.   Figure 3 shows that the inhibitory effect of the HBWG extract was compared with that of aminoguanidine on pyrraline in the simulated food system. The inhibitory effects were studied at extract concentrations of 0.02, 0.04, and 0.08 mg/mL. When the HBWG addition was 0.04 mg/mL, pyrraline in the simulation system was reduced significantly with the best inhibition effect. The inhibition rate of pyrraline was 52.03% by HBWG extracts in the simulated system, which was not significantly different from that in the positive control aminoguanidine group. This result indicates that there are more kinds of phenolic compounds and that there is a higher content of phenolic compounds in HBWG. that of aminoguanidine on pyrraline in the simulated food system. The inhibitory effects were studied at extract concentrations of 0.02, 0.04, and 0.08 mg/mL. When the HBWG addition was 0.04 mg/mL, pyrraline in the simulation system was reduced significantly with the best inhibition effect. The inhibition rate of pyrraline was 52.03% by HBWG extracts in the simulated system, which was not significantly different from that in the positive control aminoguanidine group. This result indicates that there are more kinds of phenolic compounds and that there is a higher content of phenolic compounds in HBWG. The fresh and dry HBVN extracts were used to inhibit pyrraline formation in the model system. When the content of fresh HBVN was 0.04 g/mL, pyrraline in the model system was reduced significantly. The inhibition effect of fresh HBVN on pyrraline in the model system reached 49.22%, which was coincident with the positive control group of aminoguanidine. However, pyrraline inhibition was 11.77% by using dry HBVN with a concentration of 0.04 g/mL. This result indicates that there was a higher content of phenolic compounds in fresh HBVN than dry HBVN. However, phenolic compounds may be degraded by using high-temperature drying technology. This conclusion is consistent with the antioxidant activity of fresh and dry HBVN. The fresh and dry HBVN extracts were used to inhibit pyrraline formation in the model system. When the content of fresh HBVN was 0.04 g/mL, pyrraline in the model system was reduced significantly. The inhibition effect of fresh HBVN on pyrraline in the model system reached 49.22%, which was coincident with the positive control group of aminoguanidine. However, pyrraline inhibition was 11.77% by using dry HBVN with a concentration of 0.04 g/mL. This result indicates that there was a higher content of phenolic compounds in fresh HBVN than dry HBVN. However, phenolic compounds may be degraded by using high-temperature drying technology. This conclusion is consistent with the antioxidant activity of fresh and dry HBVN.

Scavenging Experiment on α-Dicarbonyl Compounds by Phenolic Compounds from HBVN Extracts
During the nonenzymatic reaction system, the carbonyl group of the reducing sugar interacted with the ammonia group of the protein to form a Schiff base, which produced α-dicarbonyl compounds, such as GO, MGO, and 3-DG by Amadori rearrangement. These highly reactive α-dicarbonyl compounds reacted with ammonia groups of proteins to form AGEs. Therefore, we focused on phenolic compounds to scavenge and inhibit α-dicarbonyl compounds in the model system. Table 3 shows the main phenolic compounds that scavenge and inhibit α-dicarbonyl compounds at a concentration of 5 mM with 0.1, 0.2, and 0.5 mL. The resulting compound revealed a good inhibitory effect on pyrraline formation induced by α-dicarbonyl compounds. When using 0.5 mL of phenolic compounds, the scavenging effects on GO, MGO, and 3-DG were 43-54%, 42-57%, and 37-51%, respectively, indicating a good scavenging capacity of phenolic compounds for α-dicarbonyl compounds. When using a low concentration, the scavenging capacity was lower than when using a concentration of 0.5 mL (5 mM). The increase in concentration of phenolic compounds increased the scavenging capacity for α-dicarbonyl compounds. The major phenolic compounds had a good inhibitory effect of 40-55% on pyrraline formation with a concentration of 0.5 mL (5 mM). The inhibitory effects of phenolic compounds at three concentrations on food model systems in Table 3 show that the increase in concentration of phenolic compounds increased the inhibitory ability for pyrraline, which is consistent with the scavenging experiment.

Conclusions
In this study, the main phenolic compounds were extracted from HBWG and HBVN to assess the inhibitory effect on pyrraline formation. The optimal extraction condition for HBWG and HBVN was using 8 mL of 50% acetone solution at 50 • C for 60 min. The inhibitory effects of 52.03% and 49.22% exhibited good inhibitory effects by HBVN and HBWG. Separation and identification of the main phenolic compounds from HBWG and HBVN were performed by UPLC-PAD-MS/MS. Furthermore, the mechanism underlying the inhibition of HBVN extraction on the formation of pyrraline was discussed in detail. During simulated food processing, HBWG and HBVN extracts might inhibit the formation of foodborne pyrraline by two pathways: (1) scavenging of intermediate products of pyrraline formation, such as α-dicarbonyl compounds, and (2) inhibition of oxidative stress. This study not only was conducive to the recycling of resources but also provided a promising strategy for inhibiting the foodborne pyrraline formed in food processing with the addition of proper concentrations of HBWG and HBVN. Highland barley and its by-products enriched with phenolic compounds could potentially regulate pyrraline formation and show promise for future applications in the inhibition of other foodborne AGEs to effectively reduce glycation in foods and benefit those with chronic diseases.