2.1. Total Phenolics and Flavonoids Content
The phenolic content in the tartary buckwheat was examined first (
Table 1). The free phenolic content (4,820–9,590 μmol of gallic acid eq./100 g DW) was 93–98-fold higher (
P < 0.05) than the respective bound phenolic content (71–394 μmol of gallic acid eq./100 g DW) in the six samples tested, showing that most phenolics in tartary buckwheat were present in the free form. Meanwhile, the free phenolic content of tartary buckwheat was higher than that of corn (23-45-fold), wheat (25-50-fold) [
9], cranberry (2-13-fold) and apple (3-6-fold) [
7]. In our study, the bound phenolic content of Diqing tartary buckwheat grown at Sichuan (394 ± 3 μmol of gallic acid eq./100 g dried grain) was the highest among the six samples and slightly higher than that of rice (346 ±13 μmol of gallic acid eq./100 g DW) [
9], indicating that the bound phenolics in tartarty buckwheat should not be neglected. The total phenolic content of the tested buckwheat flour ranged from 5,150 to 9,660 μmol of gallic acid eq./100 g DW, which suggests that the levels of phenolics in tartary buckwheat are higher than those found in some fruits, vegetables and other cereals when expressed on a per 100 g dry weight basis. For example, the total phenolic content of tartary buckwheat was much higher than that of cranberry, apple [
7], raspberry [
19], honey [
20], corn, wheat, oats and rice [
9], suggesting that tartary buckwheat may serve as an excellent dietary source of phenolics. In this study, tartary buckwheat, Xingku No.2 from Sichuan had the highest phenolic content, followed by Diqing from Gansu. Interestingly, the phenolic content of Qiqing variety from Gansu was almost two times higher than that of the Xingku No.2 variety from Gansu. These results may be due to the interaction among the environmental conditions. Because low temperature may increase production of phenolics by enhancing synthesis of phenylalanine ammonia lyase (PAL) in plants, while high altitude and long sunlight hours with higher UV radiation positively affect the activity of phenolics synthase [
18]. Meanwhile, small amounts of precipitation could enhance the defense system of plant against stress, leading to an increased phenolic content [
21].
The free phenolic content of each tested sample was significantly higher than bound phenolics (
P < 0.05).
Table 1 clearly showed that most tartary buckwheat phenolics were in the free fraction, which was consistent with the results reported by Hung and Morita [
22], who found that common buckwheat phenolics existed predominantly in the free form. By contrast, the phenolics in wheat, rye, corn, oat and rice exist primarily in the bound form [
9,
23]. Research suggests that free phenolics may be digested in the upper gastrointestinal tract, while bound phenolics may reach the colon and exert their health benefits [
9]. Therefore, phenolics in tartary buckwheat may be more readily available in the upper gastrointestinal tract compared to wheat, corn, rice and oat. Therefore, the unique phenolics in tartary buckwheat complement those in wheat, corn, rice and oat when consumed together. Moreover, bound phenolic compounds of tartary buckwheat tested cannot be ignored as their content was much higher than that of rice that contained more bound phenolics than free phenolics.
In the tartary buckwheat samples tested, the free flavonoid contents were higher than the bound flavonoid contents (
Table 1). This was different from the flavonoid distribution in common buckwheat [
22], wheat, rice, corn and oat [
9], whose bound flavonoid contents was higher than the free flavonoids. The free flavonoids ranged from 76% in Diqing from Sichuan and Ningxia to 95% in Xingku No.2 from Ningxia (
Table 1). The free flavonoid content of Xingku No.2 from Ningxia was significantly higher than those of the other tested samples (
P < 0.05) (
Table 1); therefore, it may play more preventive and protective role in upper gastrointestinal tract compared to other samples. The bound flavonoid contents of Diqing from Ningxia and Sichuan were similar and both were significantly higher than that of other samples (
P < 0.05) (
Table 1), and they may be more beneficial in delivering bound flavonoids to the colon compared to other samples. Total flavonoid contents ranged from 2,077–3,149 μmol of rutin eq./100 g DW, which were much higher than that of common buckwheat [
15] and tartary buckwheat in western Himalaya [
18]. Total flavonoid contents followed a similar pattern as free flavonoid contents in all tested samples, because of the large contribution from free flavonoids. Flavonoids are important phytochemical components of tartary buckwheat and they have potent antioxidant and anticancer activity [
9].
Table 1.
Phenolic and flavonoid content of tartary buckwheat.
Table 1.
Phenolic and flavonoid content of tartary buckwheat.
Variety | Location | Phenolic content | Flavonoid content |
---|
(μmol of gallic acid eq./100 g DW) | (μmol of rutin eq./100 g DW) |
---|
Free | Bound | Total | Free | Bound | Total |
---|
Xingku No.2 | Sichuan | 9590 ± 428 a | 71 ± 10 d | 9660 ± 433 a | 1980 ± 210 bc | 97 ± 12 d | 2077 ± 198 c |
Ningxia | 8410 ± 621 b | 353 ± 16 ab | 8760 ± 614 abc | 3014 ± 188 a | 135 ± 23 d | 3149 ± 187 a |
Gansu | 4820 ± 260 d | 333 ± 26 b | 5150 ± 283 d | 2161 ± 170 b | 318 ± 14 c | 2479 ± 157 bc |
Diqing | Sichuan | 7310 ± 412 c | 394 ± 3 a | 7700 ± 414 c | 1719 ± 77 c | 541 ± 5 a | 2260 ± 81 bc |
Ningxia | 8150 ± 337 bc | 253 ± 13 c | 8400 ± 342 bc | 1871 ± 124 bc | 593 ± 85 a | 2464 ± 151 bc |
Gansu | 8950 ± 138 ab | 310 ± 20 b | 9260 ± 118 ab | 2109 ± 84 bc | 425 ± 26 b | 2534 ± 102 b |
2.2. Phenolic Compound Profiles
Both phenolic acids and flavonoids, which are phenolics, are natural products commonly found in many cereal grains. Ferulic, vanillic and syringic acids were found as the major phenolic acids in wheat [
24] and flavonoids were also found in wheat, rice, corn and oat [
9]. Common buckwheat was found to contain rutin, phenolic acids and tocopherols [
15,
25]. In this study, the phenolic acids such as
p-hydroxybenzoic, ferulic, protocatechuic,
p-coumaric, gallic, vanillic, caffeic and syringic acids and the flavonoids such as rutin, quercetin and catechin were detected in the free and bound phenolic extracts of tartary buckwheat using a high-performance liquid chromatography (HPLC) system (
Table 2). As shown in
Table 2,
p-hydroxybenzoic, ferulic, protocatechuic acid were the prominent phenolic acids in tartary buckwheat, which accounted for 83–88% of total phenolic acid, and the concentration of rutin was the highest among the three flavonoid compounds, followed by quercetin, and catechin was the lowest. The total concentrations of phenolic acids and flavonoids in the free phenolic extracts were significantly higher than that in the bound phenolic extracts in each tartary buckwheat sample (
P < 0.05). These results were consistent with the results of total phenolic content determined by the Folin-Ciocalteu method. Among the eight phenolic acids,
p-hydroxybenzoic, ferulic, protocatechuic,
p-coumaric, gallic and vanillic acids were present in all six tartary buckwheat samples, whereas caffeic and syringic acids were detected in individual samples (
Table 2), which differs from the phenolic acid composition of common buckwheat because protocatechuic acid was not detected in the latter [
5,
22,
26]. Variety and growing location influenced the phenolic acid and flavonoid concentration in this study. The total phenolic acid and flavonoid content of Xingku No.2 were higher than those of Diqing (
Table 2). The total phenolic acid content of Xingku No.2 and Diqing were 10.66–19.91 mg/100g DW and 8.91–14.99 mg/100g DW, respectively, and the flavonoid content of Xingku No.2 and Diqing were 1,653–1,991 mg/100g DW and 1,385–1,897 mg/100g DW, respectively.
Table 2.
Flavonoid and phenolic acid composition in tartary buckwheat seed.
Table 2.
Flavonoid and phenolic acid composition in tartary buckwheat seed.
Variety | Location | Free (mg/100 g DW) | Bound (mg/100 g DW) | Total (mg/100 g DW) |
---|
(A) Rutin composition |
Xingku No.2 | Sichuan | 1444.59 ± 1.75 a | 3.28 ± 0.06 d | 1447.87 ± 1.69 a |
Ningxia | 1213.98 ± 9.05 e | 2.94 ± 0.04 e | 1216.92 ± 9.09 e |
Gansu | 1344.47 ± 5.86 b | 3.83 ± 0.04 b | 1348.30 ± 5.90 b |
Diqing | Sichuan | 1322.00 ± 10.59 c | 3.59 ± 0.06 c | 1325.59 ± 10.65 c |
Ningxia | 517.45 ± 4.34 f | 1.09 ± 0.03 f | 518.54 ± 4.32 f |
Gansu | 1247.01 ± 6.74 d | 11.49 ± 0.04 a | 1258.50 ± 6.77 d |
(B) Quercetin composition |
Xingku No.2 | Sichuan | 478.76 ± 2.39 d | 0.61 ± 0.01 c | 479.37 ± 2.40 d |
Ningxia | 425.09 ± 4.03 e | 0.56 ± 0.01 d | 425.65 ± 4.03 e |
Gansu | 621.82 ± 2.28 b | 0.72 ± 0.02 b | 622.54 ± 2.29 b |
Diqing | Sichuan | 538.42 ± 2.60 c | 0.61 ± 0.02 c | 539.03 ± 2.61 c |
Ningxia | 857.23 ± 3.66 a | 0.39 ± 0.01 e | 857.62 ± 3.66 a |
Gansu | 626.59 ± 3.14 b | 0.86 ± 0.03 a | 627.46 ± 3.12 b |
(C) Catechin composition |
Xingku No.2 | Sichuan | 4.40 ± 0.03 a | 7.61 ± 0.03 d | 12.01 ± 0.05 b |
Ningxia | 3.74 ± 0.03 b | 6.34 ± 0.05 e | 10.08 ± 0.05 d |
Gansu | 3.13 ± 0.04 c | 16.84 ± 0.04 a | 19.96 ± 0.01 a |
Diqing | Sichuan | 0.95 ± 0.02 f | 8.83 ± 0.04 c | 9.78 ± 0.06 e |
Ningxia | 2.95 ± 0.03 d | 5.94 ± 0.04 f | 8.89 ± 0.06 f |
Gansu | 2.31 ± 0.04 e | 9.02 ± 0.07 b | 11.34 ± 0.06 c |
(D)
p-Hydroxybenzoic acid composition |
Xingku No.2 | Sichuan | 5.39 ± 0.15 b | 0.11 ± 0.01 d | 5.51 ± 0.14 b |
| Ningxia | 2.95 ± 0.03 c | 0.14 ± 0.01 c | 3.10 ± 0.03 c |
| Gansu | 8.56 ± 0.32 a | 0.21 ± 0.01 a | 8.78 ± 0.31 a |
Diqing | Sichuan | 5.64 ± 0.07 b | 0.10 ± 0.01 e | 5.74 ± 0.07 b |
Ningxia | 2.22 ± 0.00 c | nd | 2.22 ± 0.00 c |
Gansu | 5.00 ± 0.03 b | 0.19 ± 0.01 b | 5.18 ± 0.04 b |
(E) Ferulic acid composition |
Xingku No.2 | Sichuan | 6.4 ± 0.42 a | 0.89 ± 0.01 b | 7.29 ± 0.39 a |
Ningxia | 2.07 ± 0.01 e | 0.78 ± 0.01 d | 2.85 ± 0.01 e |
Gansu | 1.00 ± 0.01 f | 0.86 ± 0.00 c | 1.86 ± 0.01 f |
Diqing | Sichuan | 4.31 ± 0.09 b | 0.61 ± 0.01 e | 4.92 ± 0.07 b |
Ningxia | 2.73 ± 0.21 d | 0.48 ± 0.02 f | 3.21 ± 0.20 d |
Gansu | 3.73 ± 0.12 c | 0.98 ± 0.04 a | 4.71 ± 0.11 c |
(F) Protocatechuic acid composition |
Xingku No.2 | Sichuan | 3.16 ± 0.11 a | 1.47 ± 0.01 b | 4.63 ± 0.13 a |
Ningxia | 1.81 ± 0.02 b | 1.41 ± 0.02 c | 3.21 ± 0.02 c |
Gansu | 1.58 ± 0.03 c | 2.15 ± 0.02 a | 3.73 ± 0.04 b |
Diqing | Sichuan | 1.32 ± 0.05 d | 0.4 ± 0.00 e | 1.73 ± 0.06 f |
Ningxia | 1.6 ± 0.01 c | 0.5 ± 0.03 d | 2.1 ± 0.04 e |
Gansu | 1.14 ± 0.01 e | 1.49 ± 0.04 b | 2.64 ± 0.05 d |
(G)
p-Coumaric acid composition |
Xingku No.2 | Sichuan | 0.72 ± 0.03 a | 0.26 ± 0.01 a | 0.98 ± 0.04 a |
Ningxia | 0.23 ± 0.00 d | nd | 0.23 ± 0.00 e |
Gansu | 0.5 ± 0.02 b | 0.18 ± 0.02 b | 0.68 ± 0.03 |
Diqing | Sichuan | 0.38 ± 0.01 c | 0.11 ± 0.00 c | 0.49 ± 0.01 c |
Ningxia | 0.18 ± 0.02 e | 0.11 ± 0.01 c | 0.29 ± 0.02 d |
Gansu | 0.51 ± 0.01 b | nd | 0.51 ± 0.01 c |
(H) Gallic acid composition |
Xingku No.2 | Sichuan | 0.62 ± 0.01 a | nd | 0.62 ± 0.01 a |
Ningxia | 0.48 ± 0.01 c | nd | 0.48 ± 0.01 c |
Gansu | 0.48 ± 0.00 c | nd | 0.48 ± 0.00 c |
Diqing | Sichuan | 0.48 ± 0.00 c | nd | 0.48 ± 0.00 c |
Ningxia | 0.49 ± 0.02 c | nd | 0.49 ± 0.02 c |
Gansu | 0.55 ± 0.05 b | nd | 0.55 ± 0.05 b |
(I) Caffeic acid composition |
Xingku No.2 | Sichuan | 0.49 ± 0.00 a | nd | 0.49 ± 0.00 a |
Ningxia | nd | 0.12 ± 0.00 b | 0.12 ± 0.00 b |
Gansu | 0.23 ± 0.01 c | 0.12 ± 0.00 b | 0.35 ± 0.00 c |
Diqing | Sichuan | nd | nd | nd |
Ningxia | 0.32 ± 0.02 b | nd | 0.32 ± 0.02 d |
Gansu | 0.19 ± 0.01 d | 0.17 ± 0.02 a | 0.36 ± 0.03 b |
(J) Vanillic acid composition |
Xingku No.2 | Sichuan | nd | 0.21 ± 0.01 d | 0.21 ± 0.01 f |
Ningxia | 0.53 ± 0.01c | 0.14 ± 0.01 e | 0.67 ± 0.02 c |
Gansu | nd | 0.52 ± 0.00 a | 0.52 ± 0.00 d |
Sichuan | 1.17 ± 0.01a | 0.43 ± 0.01 b | 1.6 ± 0.01 a |
| Ningxia | nd | 0.28 ± 0.02 c | 0.28 ± 0.02 c |
| Gansu | 0.6 ± 0.00b | 0.43 ± 0.01 b | 1.04 ± 0.01 b |
(K) Syringic acid composition |
Xingku No.2 | Sichuan | 0.18 ± 0.01 a | nd | 0.18 ± 0.01 a |
Ningxia | nd | nd | nd |
Gansu | 0.12 ± 0.00 b | nd | 0.12 ± 0.00 b |
Diqing | Sichuan | nd | nd | nd |
Ningxia | nd | nd | nd |
Gansu | nd | nd | nd |
Comparing the three growing locations, the total phenolic acid content of both Xingku No.2 and Diqing from Sichuan were significantly higher than those from Gansu and Ningxia, while the flavonoid content of Xingku No.2 and Diqing from Gansu were significantly higher than those from Sichuan and Ningxia (P < 0.05).
2.3. Antioxidant Properties
Tartary buckwheat showed significant DPPH
● and ABTS
●+ scavenging activities and effectiveness in preventing the bleaching of β-carotene in a β-carotene-linoleate model system (
Figure 1,
Figure 2,
Figure 3). As can be seen, the radical scavenging capacity of free phenolic compounds was greater than that of the bound phenolic compounds. On average, free phenolics contributed more than 99% of the total radical scavenging capacity. Therefore, the free phenolic extracts were considered to be the major contributors of the total radical scavenging capacity. In this study, DPPH
● and ABTS
●+ scavenging activity was in the range from 2.3 × 10
4 to 3.3 × 10
4 μmol Trolox eq./100g DW (
Figure 1) and 1.2 × 10
5 to 1.4 × 10
5 μmol Trolox eq./100 g DW (
Figure 2), respectively. The free radical scavenging capacity of tartary buckwheat was greater than that of wheat [
12] and bio-fortified carrots [
27], suggesting that tartary buckwheat may serve as excellent dietary source of free radical scavengers. In terms of effectiveness at preventing the bleaching of β-carotene, the antioxidant activity coefficient (AAC) of the free phenolics was higher than that of the bound phenolics in the tartary buckwheat, and the AAC of free and bound phenolics ranged from 518 to 701 (
Figure 3A) and from 178 to 501 (
Figure 3B), respectively. In a previous report, the AACs of common buckwheat seed, wheat germ, sunflower seed and blueberry were 125, 236, 298 and 796, respectively [
28]. These data indicated that the AAC of free phenolics of tartary buckwheat was comparable to, or even higher than certain fruits and grains, although the concentration of free phenolics in this study was lower than that of fruits and grains. The results also suggest that tartary buckwheat has a higher comparative effectiveness at preventing the bleaching of β-carotene in the β-carotene-linoleate model system.
Figure 1.
DPPH radical scavenging activity of two tartary buckwheat varieties grown at 3 locations (μmol Trolox eq /100 g DW).
Figure 1.
DPPH radical scavenging activity of two tartary buckwheat varieties grown at 3 locations (μmol Trolox eq /100 g DW).
Figure 2.
ABTS●+ scavenging activity of two tartary buckwheat varieties grown at 3 locations (μmol Trolox eq /100g DW).
Figure 2.
ABTS●+ scavenging activity of two tartary buckwheat varieties grown at 3 locations (μmol Trolox eq /100g DW).
Figure 3.
Antioxidant activity coefficient (AAC) of free (A) and bound (B) phenolics of two tartary buckwheat varieties grown at three locations.
Figure 3.
Antioxidant activity coefficient (AAC) of free (A) and bound (B) phenolics of two tartary buckwheat varieties grown at three locations.
2.4. Effects of V and E on Tartary Buckwheat Phenolics Content and Antioxidant Properties
The above results support the assumption that variety and environment may have remarkable effects on the phenolics and antioxidant properties of tartary buckwheat. To separate and quantify the contribution of V, E and V × E interactions on tartary buckwheat antioxidant property and phenolics content variance, a 2 × 3 factorial designed ANOVA was conducted on the data from the two tartary buckwheat varieties grown in three locations. The magnitude of variance proportion (percent total mean squares) attributed to V, E and V × E indicates their relative significance in determining each antioxidant property. Results showed that V, E and V×E significantly influenced antioxidant properties of tartary buckwheat except the ABTS
●+ scavenging activity, AAC and TFC (
P < 0.05) (
Table 3). As for AAC, E contributed the highest proportion (77%) of total variance for free phenolics and V × E contributed the highest proportion (77%) of total variance for bound phenolics. In terms of TFC, E and V × E contributed 52% and 27% of total variance, respectively. For the antioxidant activity, E contributed the highest proportion of total variance, ranging from 40 to 77%, whereas V contributed 31–33% and V × sE contributed 20–77%. For the phenolics data, E contributed the highest proportion of total variance, ranging from 6 to 79%, V and V × E ranged from 3.5 to 75% and 5.8 to 71%, respectively. Although revealing significant information for determining the separate effects of V and E on antioxidant properties, the scope of these results was limited by the small number of samples involved. The contributions of V, E and V × E on antioxidant properties of several more tartary buckwheat varieties should be investigated to increase the scope of our results in further study.
Table 3.
Proportions of variance attributed to variety (V), environment (E) and V × E interaction for 2 tartary buckwheat varieties grown in 3 locations a.
Table 3.
Proportions of variance attributed to variety (V), environment (E) and V × E interaction for 2 tartary buckwheat varieties grown in 3 locations a.
Antioxidant property | Variance component |
---|
V | E | V × E |
---|
DPPH● scavenging activity (μmol Trolox eq./100 g DW) | 33.25 ** | 40.23 ** | 20.37 ** |
ABTS●+ scavenging activity (μmol Trolox eq./100 g DW) | 31.47 * | 7.42 | n |
AAC (free phenolics) | 2.45 | 77.36 ** | 6.33 * |
AAC (bound phenolics) | 0.41 | 16.23 ** | 77.11 ** |
Total Phenolic Content (μmol of gallic acid eq./100 g DW) | 3.48 * | 18.89 ** | 71.12 ** |
Total Flavonoid Content (μmol of rutin eq./100 g DW) | 3.40 | 52.01 ** | 27.14 ** |
Total phenolic acid (mg/100g DW) | 18.86 ** | 66.45 ** | 14.68 ** |
Rutin (mg/100 g DW) | 6.96 ** | 46.86 ** | 46.13 ** |
Quercetin (mg/100 g DW) | 19.40 ** | 72.87 ** | 7.69 ** |
Catechin (mg/100 g DW) | 29.47 ** | 50.79 ** | 19.72 ** |
p-Hydroxybenzoic acid (mg/100 g DW) | 5.24 ** | 79.19 ** | 5.75 * |
Ferulic acid (mg/100 g DW) | 4.08 ** | 55.83 ** | 40.08 ** |
Protocatechuic acid (mg/100 g DW) | 75.67 ** | 5.97 ** | 18.28 ** |
Growing environment (E) may be a significant factor affecting some antioxidant properties for tartary buckwheat flour. The effects of environmental parameters including mean temperature, amount of precipitation, sunlight hours and altitude on the antioxidant properties of tartary buckwheat were investigated. Correlation analysis found significant positive correlations between altitude and rutin or total phenolic acid content for both Xingku No.2 and Diqing (
P < 0.05). No significant correlation among the other three environmental parameters and antioxidant property was detected for the two varieties tested (
P < 0.05) (
Table 4). The effects of altitude on polyphenol and antioxidant property of tartary buckwheat have been reported in a recent study [
18]. Our results support the notion that higher altitude, which is often linked to higher UV radiation, causes an increase in rutin content.
Table 4.
Correlation analysis of growing conditions and antioxidant properties and phenolics for two tartary buckwheat varieties grown in 3 locations.
Table 4.
Correlation analysis of growing conditions and antioxidant properties and phenolics for two tartary buckwheat varieties grown in 3 locations.
Environment | Variety | Antioxidant property and phenolic content |
---|
parameter | DPPH | AACF | AACB | TPC | TFC | TPA | R | Q | HA | FA | PA |
---|
MT (°C) | XK | −0.72 | −0.22 | −0.26 | 0.80 | 0.56 | −0.56 | −0.50 | −0.98 * | −0.99 * | 0.25 | −0.29 |
DQ | 0.13 | 0.46 | 0.91 | −0.61 | −0.32 | −0.83 | −0.78 | 0.64 | −0.73 | −0.76 | −0.65 |
AOP (mm) | XK | 0.85 | 0.98 * | 0.99 * | 0.38 | −0.94 | 0.94 | 0.96 * | 0.06 | 0.23 | 0.88 | 0.99 * |
DQ | 0.93 | 0.75 | −0.63 | −0.62 | −0.84 | 0.74 | 0.79 | −0.90 | 0.84 | 0.82 | −0.58 |
SH (h) | XK | −0.98 * | −0.57 | −0.75 | 0.34 | 0.92 | −0.93 | −0.90 | −0.72 | −0.83 | −0.33 | −0.77 |
DQ | −0.44 | −0.11 | 0.99 * | −0.07 | 0.25 | −0.99 * | −0.99 * | 0.96 * | −0.99 * | −0.99 * | −0.12 |
A (m) | XK | 0.99 * | 0.77 | 0.9 | −0.07 | −0.99 * | 0.99 * | 0.98 * | 0.51 | 0.64 | 0.57 | 0.91 |
DQ | 0.67 | 0.37 | −0.91 | −0.2 | −0.51 | 0.97 * | 0.98 * | −0.99 * | 0.99 * | 0.99 * | −0.15 |