Free and Bound Phenolics of Buckwheat Varieties: HPLC Characterization, Antioxidant Activity, and Inhibitory Potency towards α-Glucosidase with Molecular Docking Analysis

Free and bound phenolic fractions from six buckwheat varieties were investigated for their compositions, antioxidant activities, and inhibitory effects on α-glucosidase. The results showed that different buckwheat varieties have significant differences in phenolic/flavonoid contents, and these contents were found in higher quantities in free form than in bound form. HPLC results revealed that rutin, quercetin, and kaempferol-3-O-rutinoside were the most abundant components in free and bound forms, whereas dihydromyricetin was found only in the bound form. Free phenolics showed higher antioxidant activities of DPPH, ABTS+, OH•, and FRAP than those of bound phenolics. Strong inhibitory effects against α-glucosidase by the free/bound phenolic fractions were found in all buckwheat varieties, and free phenolics showed stronger α-glucosidase inhibition than that of the corresponding bound phenolics. More importantly, the main phenolic compounds observed in the buckwheat varieties were subjected to molecular docking analysis to provide insight into their interactions with α-glucosidase. The contributions by individual phenolics to the observed variation was analysed by Pearson correlation coefficient analysis and principal component analysis. The present study provides a comprehensive comparison for the phenolic fractions of buckwheat varieties and identify the main contributors to antioxidant and α-glucosidase inhibitory activity.


Introduction
Nowadays, the incidence of conditions related to metabolic syndrome is rapidly rising worldwide, including oxidative damage, high blood sugar, hypertension, hyperlipidaemic diseases, and so on [1,2].Nutritionists have confirmed that oxidative stress and high-calorie diets were closely related to the occurrence of chronic metabolic syndrome [3].Chemical drugs are somewhat effective in relieving these chronic diseases, but they can also cause drug dependence and serious side effects [4].Phytochemicals, including phenolics/flavonoids, which are found in cereal foods or natural products, have been demonstrated to have important physiological functions, such as weight loss and antioxidant, anti-diabetic, anti-inflammatory, anti-glycosylation, and anti-proliferative effects [5][6][7][8].
Buckwheat (Fagopyrum esculentum Moench), as an important functional cereal food of the Polygonum family, is widely distributed in Asia, Europe, Africa, North America, and Oceania [9].Generally, buckwheat includes two species: common buckwheat (Fagopyrum esculentum Moench) and tartary buckwheat (Fagopyrum tataricum Gaertn) [10].Buckwheat has received much attention not only because of its delicious flavor and nutritional quality in terms of macro-nutrients, but also as a cereal raw material rich in flavonoid compounds, which may reduce chronic conditions including oxidative damage, diabetes, and hypertensive diseases [11][12][13].Researchers have reported that flavonoid contents in buckwheat were 23-45 and 25-50 times greater than those in wheat and corn, respectively [14,15].Moreover, the phytochemical composition of cereal crops mainly depends quantitatively and qualitatively on their genotypes and environmental factors that affect growth [16,17].Although many studies have measured the total phenolic contents and antioxidant capacity in some buckwheat varieties [12,18], information remains limited regarding the characterization and contents of free phenolic (FP) and bound phenolic (BP) fractions of different buckwheat varieties and their corresponding in vitro biological activities (especially anti-diabetic effects).Furthermore, the contributions of the total phenolic contents (TPC), total flavonoid contents (TFC), and the content of individual phenolic on their bio-activities has not been clearly investigated.
The aim of the present work was to systematically investigate the HPLC characterizations, in vitro antioxidant activities, and inhibitory effects against α-glucosidase of FP and BP fractions from six buckwheat varieties.More importantly, the potential inhibitory mechanism against α-glucosidase by the main phenolic compounds in six buckwheat samples was clarified by molecular docking analysis.In addition, the contributions of the individual phenolics to the observed variation were analyzed by Pearson correlation coefficient analysis and principal component analysis.This work may provide a comprehensive comparison for the phenolic fractions of buckwheat varieties and identify the main contributors to antioxidant and α-glucosidase inhibitory activity.

Extraction of FP and BP Fractions
Different buckwheat samples were first freeze-dried with vacuum freeze dryer FD-2B-80 (Shanghai Gipp Electronic Technology Co., Ltd., Shanghai, China).Then, the dried samples were ground to a fine powder by a micronizer and sifted through a 20-mesh sieve.FP and BP fractions were extracted following the reported method [19].In brief, 2 g of the above powder samples were blended with 8 mL of 80% ethanol in a 15 mL tube.The mixture was kept in a water bath at 50 °C for 30 min and then centrifuged at 4000× g for 10 min at 4 °C.The procedure was repeated twice, and then the filtrate was combined.After FP extraction, the residues were used to extract BP.One gram of the above dried residues was hydrolyzed by adding 40 mL of 2 M NaOH at 30 °C for 4 h under a nitrogen atmosphere.Then, the resultant hydrolysate was acidified to pH 2 with 6 M hydrochloric acid.The mixture was first degreased three times with 100 mL hexane.The supernatants were combined, extracted three times with the solvents (diethyl ether:ethyl acetate = 1:1, v/v), and then evaporated under reduced pressure at 30 °C.After removing the diethyl ether and ethyl acetate, the samples were dissolved in 5 mL of 50% ethanol (v/v) to obtain BP fractions, which were stored at −20 °C before analysis.

Determination of Phenolic and Flavonoid Contents
The Folin-Ciocalteau method was applied to determine phenolic content of different fractions [20].Gallic acid was employed as standard.The average value of triplicate data was expressed as mg of gallic acid equivalents per gram sample in dry weight (mg GAE/g DW).
Flavonoid content in both free and bound phenolic fractions were quantified according to the aluminum chloride colorimetric method [21].Rutin was employed as the standard.The average value of triplicate data was expressed as mg rutin equivalents per gram sample in dry weight (mg RE/g DW).

Quantitative Analysis by HPLC
A Hitachi 1200 HPLC system (SHIMADZU, Kyoto, Japan) was used to separate and quantify the phenolic compounds in free and bound phenolic fractions from BW samples.The HPLC system was equipped with a diode array detector (SPD-10A, SHIMADZU).Chromatographic separation was achieved in a Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm, Agilent, Santa Clara, CA, USA).The mobile phase used was 0.1% formic acid-H2O (phase A) and 0.1% formic acid-acetonitrile (phase B) with the gradient program as follows: 15% B at 0-5 min, 15-20% B at 5-10 min, 20-50% B at 10-40 min, 50-80% B at 40-55 min, and 15% B at 55-60 min.The flow-rate was kept at 0.8 mL/min at all times.The other chromatographic conditions used were as follows: the column was operated at 30 °C, the scanning detection wavelength ranged from 200 to 600 nm, and the injection volume was 10.0 μL.All samples were analyzed in triplicate, and the amount of phenolic compound was expressed as micrograms per gram sample in dried weight (μg/g DW).

DPPH Radical Scavenging Activity Assay
The scavenging activity of DPPH free radicals was performed according to the reported method [22].Briefly, 50 μL of the diluted sample extracts was mixed with 400 μL of 100 μM DPPH-methanol solution for 30 min at 25 °C.The absorbance was measured at 517 nm.Trolox solution (0-40 μg/mL) was used as the positive control.The results were expressed as μmol Trolox equivalents (TE)/g sample in DW (μmol TE/g DW).

ABTS •+ Radical Scavenging Activity Assay
The scavenging activity of ABTS •+ radical was measured according to the previously described method [23].Trolox solution (0-40 μg/mL) was used as the positive control.The ABTS •+ value was expressed as μmol Trolox equivalents (TE)/g sample in DW (μmol TE/g DW).

Hydroxyl (OH • ) Radical Scavenging Activity Assay
The scavenging activity of OH • radical was measured using the reported method [24].Briefly, 100 μL of the extract dilutions was mixed with 100 μL of 6 mM FeSO4 solution and 100 μL of 2.4 mM H2O2 solution.After incubating for 10 min at 25 °C, 100 μL of 6 mM salicylic acid was added to the reaction solution.The mixture was further incubated for 30 min at 25 °C; then, the absorbance was read at 510 nm.Trolox solution (0-40 μg/mL) was used as the positive control.The OH • value was expressed as μmol TE/g DW.

Ferric Reducing Antioxidant Power (FRAP) Assay
FRAP assay was performed on the basis of the described method [25].Ferrous sulfate solution (0, 50, 200, 400, 600, 800, and 1000 M) was used to establish the standard curve.The FRAP values were expressed in μM ferrous sulfate equivalents per gram sample in dried weight (μM Fe(Ⅱ)SE/g DW).

Determination of α-Glucosidase Inhibitory Activity
The α-glucosidase inhibitory activity of FP and BP fractions in different samples was measured according to the previously described method [26].Firstly, 100 μL of 1 U/mL α-glucosidase in phosphate buffer solution (PBS, 0.1 M, pH 6.8) was mixed with 50 μL of the test extracts dilutions in a 2 mL tube.After incubation at 37 °C for 10 min, the reaction was begun by adding 100 μL of 5 mM p-NPG solution, and incubated for 20 min at 37 °C.The absorbance of the reaction solution was read at 405 nm in 15 min.The inhibiting activity of α-glucosidase (%) was calculated on the basic of the following equation: where ΔAcont = Abuffer+enzyme−Abuffer, and ΔAs = Aextract+enzyme−Aextract.

Molecular Docking Analysis
The ChemBio3D Ultra (CambridgeSoft Corporation, Massachusetts, United States) was used to draw the 2D structures of the main phenolic compounds in BW samples.It is worth noting that little information was available regarding the structure of α-glucosidase from Saccharomyces cerevisiae.Hence, its homologous structure (isomaltase, PDB ID: 3A4A) obtained from RCSB PDB was usually applied to conduct the docking test [27,28].The Surflex-Dock Geom (SFXC) mode was used to perform docking analysis using SYBYL-X 2.0 software (Tripos, Inc., St. Louis, MO, USA).A docking score file was generated, and a C-score ≥ 4 was considered as a credible result.Several parameters including four score functions (T-score, PMF-score, D-score, and CHEM-score), hydrogen bond distances, and amino acid binding sites were used to explain the active inhibitory mechanisms of the main phenolics against α-glucosidase [29].

Statistical Analysis
All assays were conducted in triplicate.All values were expressed as the average value ± standard deviation (SD).SPSS Statistics version 17.0 (IBM SPSS, Chicago, IL, USA) was used to perform the statistical analyses.IC50 value was measured by Probit analysis on SPSS Statistics version 17.0.Significant differences (p < 0.05) were considered statistically significant.Correlation analysis between the analytes and the investigated bio-activities were evaluated using Pearson correlation.

Total Phenolic Contents (TPC) and Total Flavonoid Contents (TFC)
As shown in Table 1, significant differences were observed with respect to TPC and TFC in different buckwheat samples.The contents of free phenolic (FP) and free flavonoid (FF) in six buckwheat samples ranged between 5.18-13.74mg GAE/g DW and 7.37-26.60mg RE/g DW, respectively, while bound phenolic (BP) and bound flavonoid (BF) contents ranged between 0.63 and 0.96 mg GAE/g DW and 0.72 and 1.38 mg RE/g DW, respectively.It was found that FP and FF were the main contributors to TPC/TFC, accounting for over 90% of contents.Moreover, the FP/FF contents and TPC/TFC of the buckwheat sample from Shanxi were significantly higher (p < 0.05) than those of the samples from other genotypes and regions in China (Table 1).Qin et al. (2010) reported that the FP contents (25.3 mg GAE/g DW) were higher than the BP contents (1.8 mg GAE/g DW) in tartary buckwheat bran [15].Liu et al. (2019) confirmed that the highest phenolic content of 15 buckwheat varieties from China was only 7.32 mg GAE/g DW, which was lower than that of samples from Shanxi (13.74 mg GAE/g DW) [30].In this work, we found that the average TPC and TFC of tartary buckwheat samples (TPC: 9.97 mg GAE/g DW; TFC: 19.26 mg RE/g DW) were significantly higher than those of the common buckwheat samples (TPC: 6.47 mg GAE/g DW; TFC: 10.87 mg RE/g DW) (p < 0.001).Owing to the genotypes and growth-influencing environmental factors of buckwheat varieties, significant differences were seen in TPC/TFC.Many studies have confirmed that phytochemical compositions of cereal crops mainly depend qualitatively and quantitatively on its genotypes and environmental factors that influence growth [30,31].Different lowercase letters (a-e) mean statistically significant differences following different samples at the same fractions (p < 0.05).FP, free phenolic; BP, bound phenolic; TP, total phenolic; FF, free flavonoid; BF, bound flavonoid; TF, total flavonoid.

Quantitative HPLC Analysis of Phenolic Compositions
Phenolic compositions were preliminary identified by comparing retention time (RT), UV spectra, and the MS spectral data of standards (Table S1).The phenolic compositions of FP and BP fractions from six buckwheat samples were quantified by HPLC (Figure 1).As shown in Figure 1 and Table 2, it can be seen that phenolic compounds in buckwheat samples were divided into two categories: flavonoids and phenolic acid groups.The six flavonoid compounds included rutin, dihydromyricetin, kaempferol-3-O-rutinoside, quercetin, apigenin, and kaempferol.Five of these compounds existed in free and bound forms, except dihydromyricetin, which was only detected in BP fractions.Five phenolic acids including gallic acid, 4-hydroxybenzoic acid, 5-caffeoylquinic acid, syringic acid, and ferulic acid were detected in the free and bound fractions.Among them, rutin, kaempferol-3-O-rutinoside, and quercetin were the most predominant compounds that existed in both forms.
Significantly higher quantities of rutin, kaempferol-3-O-rutinoside, ferulic acid, quercetin, apigenin, and kaempferol were found in free form than those in bound form, which was consistent with the report described by Li et al. (2016) [10].However, some phenolic acid compounds including gallic acid, 4-hydroxybenzoic acid, 5-caffeoylquinic acid, and syringic acid were significantly more common in bound form.Samples from Shangdong and Heilongjiang contained high bound gallic acid and syringic acid.Many studies have confirmed that rutin, quercetin, and isoquercitin were the most predominant compounds in buckwheat [32].In the present work, in addition to quercetin and rutin, high kaempferol-3-O-rutinoside contents were also found in all buckwheat samples, which may be due to differences in genotypes and growth-influencing environmental factors [31].Different lowercase letters (a-e) mean statistically significant differences following different samples at the same status (p < 0.05).FP, free phenolic; BP, bound phenolic; TP, total phenolic.N.D. not detected.

Antioxidant Activities
Albishi et al. ( 2013) suggested that at least two test methods should be used to evaluate the in vitro antioxidant activity of samples, owing to different mechanisms involved in determining antioxidant capacity [33].In this study, four independent methods including FRAP and the radical scavenging activities of DPPH, ABTS •+ , and OH • were used to comprehensively evaluate the antioxidant capacity of the FP and BP fractions from different buckwheat varieties.
The results showed that the antioxidant capacities of FP fractions of six buckwheat varieties varied significantly (p < 0.05).Meanwhile, DPPH levels of FP and BP fractions in different buckwheat samples ranged between 17.55-114.02μmol TE/g DW and 4.30-7.68μmol TE/g DW, respectively.For ABTS •+ assays, FP and BP fractions yielded ABTS •+ values of 69.19-175.66μmol TE/g DW and 7.12-10.62μmol TE/g DW, respectively.However, OH • levels of FP fractions in different buckwheat samples ranged between 32.92-82.64μmol TE/g DW.The OH • values of BP fractions showed no significant differences (p > 0.05) among the different buckwheat samples.FRAP values of FP fractions in different buckwheat samples ranged between 29.58 and 84.72 mM FeS(Ⅱ)E/g DW (Table 3).The highest antioxidant capacity of FP fractions was detected in the buckwheat sample from Shanxi (DPPH: 114.02 μmol TE/g DW; ABTS •+ : 175.66 μmol TE/g DW; OH • : 82.64 μmol TE/g DW; FRAP: 84.72 mM FeS(Ⅱ)E/g DW), which was due to its high TPC/TFC and individual phenolic contents.Moreover, the antioxidant activities of FP fractions from different buckwheat samples were significantly higher than those of the BP fractions.More importantly, it was found that the average antioxidant activities (DPPH: 71.54 μmol TE/g DW; ABTS •+ : 138.48 μmol TE/g DW; OH • : 73.39 μmol TE/g DW; FRAP: 58.41 mM FeS(Ⅱ)E/g DW) in tartary buckwheat samples were higher than those in common buckwheat samples (DPPH: 33.79 μmol TE/g DW; ABTS •+ : 89.05 μmol TE/g DW; OH • : 55.65 μmol TE/g DW; FRAP: 37.03 mM FeS(Ⅱ)E/g DW).The results showed that FP contributes to the main antioxidant activities in buckwheat samples.Li et al. (2016) also confirmed that the FP fractions of buckwheat bran samples contributed to the main antioxidant activities [10].In addition, higher phenolic contents resulted in stronger antioxidant activities.Xiang et al. (2019) reported that the phenolic contents of finger millets have a strong positive correlation with the oxygen radical absorbance capacity and ABTS •+ radical scavenging activities (r = 0.948, r = 0.836, respectively; p < 0.01) [34].Different lowercase letters (a-e) mean statistically significant differences following different samples (p < 0.05) at the same status.FP, free phenolic; BP, bound phenolic; TP, total phenolic.N.D. not detected.

Inhibitory Activity against α-Glucosidase
Some chemical drugs are widely applied to manage type II diabetes, but some side effects have been reported in their application [35].Consequently, there is an urgent need to identify natural alternative products without side effects to manage type II diabetes.Many studies have confirmed that α-glucosidase inhibitors from cereal products, which had fewer side effects, played important roles in regulating blood glucose levels [36,37].

Molecular Docking Analysis
In the present work, the inhibitory mechanisms of the main four phenolic constituents in BW samples including quercetin, rutin, kaempferol-3-O-rutinoside, and dihydromyricetin against αglucosidase were further illuminated by molecular docking analysis.Figure 3 and Table 4 show the docking results regarding interactions between the main phenolic molecules and α-glucosidase binding.As shown in  4).
The results clearly showed that the phenolic compound structures influenced the inhibitory effects on α-glucosidase.Among them, the numbers of H-bonds and active sites residues played important roles in exerting the catalytic functions of the complex of the α-glucosidase receptor and ligands.When the main four phenolics were docked with α-glucosidase, the numbers of formed active site residues were ordered as follows: quercetin (7) = rutin (7) > dihydromyricetin (5) = kaempferol-3-O-rutinoside (5); those of the formed H-bonds were as follows: quercetin (10) = rutin (10) > dihydromyricetin (8) > kaempferol-3-O-rutinoside (6).As a result, quercetin showed the strongest α-glucosidase inhibition (Figure 2B).Although quercetin and rutin docked with αglucosidase exhibited equal numbers of H-bonds and active site residues, there were significant differences in the capacity for α-glucosidase inhibition.This may be because these different molecules had different residue interaction sites with α-glucosidase.Both quercetin and rutin interacted with the amino acid residues Glu 411 and Gln 279, indicating that these two residues may be the important catalytic sites of α-glucosidase.However, kaempferol-3-O-rutinoside formed an H-bond with Asp 415, indicating that it may bind to the active site of α-glucosidase to inhibit its catalytic activity.Consequently, kaempferol-3-O-rutinoside exhibited the weakest inhibitory effect against αglucosidase.It was found that Glu 411 bound with each of the four phenolics, implying that it may exert important functions in the catalytic reaction of α-glucosidase.Many studies also verified that Asp 215 and Glu 411 were the important active sites involved in this catalytic reaction [28,34].In addition, the formation of hydrogen bonds between the hydroxyl group at C-3 or C-4′ of the molecules (i.e., quercetin and rutin) and the active site residues may produce a higher inhibitory ability towards α-glucosidase compared to kaempferol-3-O-rutinoside, which was consistent with the results reported by Zeng et al. (2016) [41].Rasouli et al. (2017) also reported that the hydrogen bonds and active site residues formed by the ligand molecules and receptor enzymes exerted important effects on α-glucosidase inhibitory activities [42].

Correlations between the Investigated Bio-Activities and Phenolic Compositions
To explore the effect of the phenolic compounds on the investigated bio-activities in different buckwheat varieties, correlations among the examined variables were elucidated by Pearson correlation coefficient analysis [43].

Principal Component Analysis (PCA)
PCA is widely used to reduce the dimensionality and increase the interpretability of large datasets.To systematically and fully investigate the contributions of the individual phenolics to the variables investigated, PCA was carried out using FP, BP, FF, BF, the individual phenolic contents, antioxidant activities (DPPH, ABTS •+ , OH • , and FRAP values), and α-glucosidase inhibitory activity (IC50) for different buckwheat samples.
PCA yielded two principal components (with an eigenvalue higher than 1) explaining 98.98% of the total variances in the data to simplify the analysis of the results.The loading plot illustrates the relationship between the investigated variables (Figure 4).The two principal components PC1 and PC2 accounted for 83.17% and 15.81% of the total variation, respectively.Among them, PC1 separated the samples based on FP, BP, FF, DPPH, ABTS •+ , OH • , FRAP values, rutin, kaempferol-3-O-rutinoside, dihydromyricetin, and quercetin, which are present in the upper right square.The variables were separated along PC2 by differences observed in 4-hydroxybenzoic acid, ferulic acid, and 5caffeoylquinic acid, which are present in the upper left square.The results demonstrated that FP, BP, FF, rutin, kaempferol-3-O-rutinoside, dihydromyricetin, and quercetin were closely correlated with DPPH, ABTS •+ , OH • , and FRAP values, which was consistent with the results of Pearson correlation coefficient analysis.Therefore, the scatter plot produced by PCA may be used to reduce the dimensionality and interpret the differences among the variables in large datasets.

Table 1 .
Specific information, free and bound phenolic/flavonoid contents of the six buckwheat samples from China

Table 2 .
Individual phenolic compounds contents in free and bound fractions of six buckwheat samples from China.

Table 3 .
The antioxidant activities of free and bound phenolic fractions of different buckwheat samples.

Table 4 .
The analysis results of the main phenolic analytes' ligands docking into α-glucosidase.

Table 5 .
Correlation matrix between the major phenolic compounds and the investigated bioactivities.Correlation was significant at the 0.05 level (two-tailed).** Correlation was significant at the 0.01 level (two-tailed).*** Correlation was significant at the 0.001 level (two-tailed). *