Fagopyrum 19: 89-93 (2002)

Radical-scavenging activity in common buckwheat (Fagopyrum esculentum Moench) harvested in the Kyushu region of Japan
Toshikazu MORISmTA*l,2, Takahiro HARAl, Ikuo SUDAI and Takahisa TETSUKAI
1 National Agricultural Research Center for Kyushu Okinawa Region , National Agricultural Research Organization , Nishigoshi, Kikuchi, Kumamoto 861-1192, Japan 2 Institute ofRadiation Breeding, National Institute ofAgrobiological Science.Ohmiya, Naka, lbaraki 319-2293, Japan

Received April 24, 2002 ; accepted in revised form August 10, 2002

Key words: common buckwheat, Kyushu, polyphenol, radical-scavenging activity, varietal difference

ABSTRACT
Radical-scavenging activity in the flour obtained from 32 cultivars of buckwheat grown and harvested in the Kyushu region of Japan was investigated by a method using 1,I-diphenyl-2-picrylhydrazyl (DPPH). The radical-scavenging activity of DPPH in buckwheat flour extract by an 80% ethanol solution varied in the range of 0.497 to 1.233 umolTrolox equivalent/ml (1 ml equivalent to 100 mg of buckwheat flour), and the varietal difference was statistically significant (P<O.OI). Japanese buckwheat varieties showed radical-scavenging activity in the range of 0.581 to 0.922 umol -Trolox equivalent/ml. Among those tested, 2 varieties originating from Nepal possessed the highest radicalscavenging activity, suggesting that they would be suitable resources for breeding buckwheat with a high radicalscavenging activity. In addition, the radical-scavenging activity of DPPH in all varieties was correlated with total polyphenol content (r=0.975, P<O.Ol). content, but not rutin content. Thus, many studies have reported on varietal and environmental differences in the antioxidative activity in buckwheat; however, little information exists on the antioxidative activity in buckwheat harvested in the Kyushu region of southwestern Japan (Fig. 1). On the other hand, numerous tests have been used to evaluate the antioxidative activity of buckwheat samples. Oomah and Mazza (1996) evaluated the antioxidative activity of buckwheat by using a carotene-bleaching method. Watanabe et al. (1995) used high-performance liquid chromatography (HPLC) of methyllinoleate hydroperoxides. Other methods for determining antioxidative activity include the active oxygen method using an oil system, the ferric thiocyanate and thiobarbituric acid method using a linoleic acid-ethanol system (Suda et al., 1994; Suda et al., 1996), and electron spin resonance spectrometry (Suda et al., 1998). These methods require technical skill, long experimental time, expensive equipment, and a plethora of tools. A spectrophotometric assay using 1,I-diphenyl-2-picrylhydrazyl (DPPH), which forms a stable free radical in an ethanol solution, has recently been used to determine antioxidative activity in various kinds of plant species. Using this method, Przybylski et al. (1998) reported that the DPPH radicalscavenging activity of extracts from buckwheat groats increased when more polor solvents were used for extraction. Suda et al. (1999) measured the DPPH radicalscavenging activity in the ethanol extract from agricul-

INTRODUCTION
Antioxidants in dietary plants have attracted attention because they protect the human body from oxidative damage caused by free radicals. Buckwheat is known to contain various antioxidative compounds, such as vitamins B I, B2 , and E, and phenolic compounds, such as rutin, quercetin, and proanthocyanidines (condensed tannins) (Watanabe et al., 1995; Watanabe et al., 1997; Watanabe, 1998). Furthermore, buckwheat grain has a higher antioxidative activity than other cereal grains (Zielinski and Kozlowska, 2000). There have been some reports concerning the varietal differences in the antioxidative components in buckwheat. Kitabayashi et al. (1995) and Ohsawa and Tsutsumi (1995) have reported varietal differences in the rutin content in buckwheat flour. Varietal and environmental variations in buckwheat flour have also been found in vitamin E (Honda, 1995) and phenolic acid (Oomah et al., 1996). Watanabe et al. (1995) have reported that the antioxidative activity of ethanol extract from buckwheat seeds varies according to the cultivar and is correlated with the polyphenol content. They also showed that antioxidative compounds responsible for a high activity in buckwheat hulls are phenolic compounds that include flavonoids, while those in buckwheat groats are catechins (Watanabe et al., 1997; Watanabe, 1998). Furthermore, Oomah and Mazza (1996) reported a good correlation between antioxidative activity and flavonoid
*Corresponding author

89

90

Morishita el al.

maturity. After the harvest, the seeds were dehulled and then milled with a Brabender test mill (Quadrumat Junior, Brabender, Germany), and the flour obtained was passed through a 60-mesh sieve.

Japan
(J

Kyushu region

Weather survey The temperature and solar radiation were surveyed at the National Agricultural Research Center for Kyushu Okinawa Region during the growing period. Cumulative temperatures were calculated for total daily average temperatures for each variety. Cumulative solar radiations were calculated for total daily solar radiation. Preparation of buckwheat flour extract Buckwheat flour in a screw-capped test tube in the amount of 0.6 g was extracted with 6 ml of 80% ethanol solution warmed to 80aC. After incubation for 30 min, the test tube was cooled and centrifuged at 3,000 rpm (05PR-22, Hitachi Inc., Japan) for 10 min. The supernatant that was obtained was used as buckwheat flour extract. These procedures are shown in Fig. 2. Measurement of DPPH radical-scavenging activity Buckwheat flour extract was diluted to one-fourth concentration with 80% ethanol. Reagent DPPH (Wako Pure Chemical Industries Ltd., Japan) was dissolved in 99.9% ethanol at a final concentration of 0.4 mM (W IV). The reaction mixture consisted of 0.30 ml of 0.4 mM DPPH solution, 0.3 ml of a 200 mM 2-morpholinoethanesulpholic acid (MES) buffer (pH 6.0), 0.30 ml of 20% ethanol, 0.24 ml of 80% ethanol, and 0.06 ml of diluted buckwheat flour extract. The reaction was started by the addition of a diluted extract. After the test tube kept for 20 min, the absorbance of the reaction mixture was measured at 520 nm (Beckman DU 70, Beckman Inc., USA). Another reaction was done simultaneously using 80% ethanol (control) or Trolox instead of buckwheat flour extract. A Trolox" (6hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich Com., USA) solution was prepared by dissolving in 80% ethanol to a final concentration of 0.2 mM. The DPPH radical-scavenging activity was evaluated by the decrease of absorbance at 520 nm and expressed as a Trolox equivalent per ml of sample solution by using a calibration curve of Trolox. These procedures are shown in Fig. 3. Determination of total polyphenol content Total polyphenol content was determined by a modification of a Folin-Ciocalteu method already reported (Furuta et aI., 1998). Namely, 1.0 ml of Folin Ciocalteu solution was mixed with 1.0 ml of sample solution. After 3 min, 1.0 ml of 10% sodium carbonate solution was added and mixed. The mixture was allowed to stand for

Fig. 1. Map of Japan . Kumamoto Prefecture is in black. The National Agricultural Research Center for the Kyushu Okinawa Region is indicated by a white circle.

Buckwheat flour (0.6 g)

1<-

80 % EtOH (6 ml)

Incubate for 30 min under 80C Centrifuged at 3000 rpm for 10 min

I
Precipitate Supernatant

I
Distilled 4 times using 80 % EtOH

I
Assay sample Fig. 2. Procedure for preparing an assay sample from buckwheat flour.

tural products and foods. Oki et al. (2001) reported that the DPPH radical-scavenging activity in fried chips was highly correlated with the superoxide radical-scavenging activities . In this study, 80% ethanol extracts were prepared from groats of various cultivars of buckwheat harvested in the Kyushu region of Japan, and their DPPH radicalscavenging activities were investigated and compared. In addition, the relationship between the radical-scavenging activity and total polyphenol content was examined.

MATERIALS AND METHODS

Plant materials and preparation of buckwheat flours Thirty-two diploid common buckwheat varieties (Fagopyrum esculentum Moench) originating from Japan, Russia, China, Nepal, Canada, and France were used. They were sown at the National Agricultural Research Center for the Kyushu Okinawa Region (Kumamoto, Japan) on August 20, 1998. Each variety had 2 replications in a randomized block design and was harvested at

Radical-scavenging activity in buckwh eat

91

0.4 mM (WN) DPPH solution in EtOH (0.30 ml) 200 mM MES buffer (pH 6.0) (0.30 ml) 20 % EtOH (0.30 ml)

80 % EtOH (0.24 ml)

I
I

<-

Assay sample (0 .06 ml)

(r=0.975, P<O.OI) was seen between radical-scavenging activity and total polyphenol content (Fig. 4). Table 3 shows the results of the analysis of variations, indicating that the differenc e in varieties (genotype) was statistically significant for both DPPH radical-scavenging activity and polyphenol content.

Mixing by vortex

DISCUSSION
The Kyushu region is warmer than other buckwheat cultivating areas in Japan. Autumn varieties grown in this region characteristically have long growing periods and high yields (Morishita and Tetsuka, 2001); they are cultivated from August to the beginning of December and are a major source of the buckwheat supply in Japan. However, there have only been a few reports on the nutrition and physiology of buckwheat cultivated in the Kyushu region, and there has been no research on their antioxidative activity. Buckwheat can be cultivated until the end of November in Kumamoto Prefecture, which is in the center of the Kyushu region (Fig. 1). In previous reports on varietal differences in antioxidative activity of buckwheat flour, Watanabe et a!' (1995) studied 10 cultivars from Japan and Russia and found a varietal difference in antioxidative activity. Oomah and Mazza (1996) reported that variations in antioxidative activity were mainly due to the composite effect of cultivar and environment differences. However, these reports did not evaluate the antioxidative activity of autumn varieties. In the present experiment, using 32 varieties from many regions and different ecotypes, a wide varietal difference in antioxidative activity of buckwheat harvested in the Kyushu region was discovered using a DPPH radical-scavenging assay (Table 2). This range of variation was wider than in previous reports . The Nepal varieties showed a higher DPPH radical-scavenging activity than those of other origins. Kitabayashi et a!. (1995) reported that the varieties from Nepal had a high rutin content. Although the involvement of rutin in the radical-scavenging activity of local varieties grown in the Kyushu region has not been shown in this study, the varieties from Nepal, which contained rutin or unknown substances, were assumed to have materials with a high radical-scavenging activity , making them suitable for buckwheat breeding. A report by Baniya et a!' (1995), which examined the agronomic and morphological traits of the Nepal varieties, showed that they had a wide diversity of buckwheat and were a potential source for improving buckwheat traits . We also confirmed the superior traits of Nepal varieties as a good source for improving buckwheat quality . The present work clearly indicates that the DPPH radical-scavenging activity of buckwheat varieties grown in the Kyushu region is highly correlated with the polyphenol content. These results agree with those of

Still standing for 20 min

I
Measurement at 520 nm Fig. 3. Procedure for measuring DPPHradical-scavenging activity. I hr. The supernatant obtained by centrifugation at 3,000 rpm (05PR-22, Hitachi Inc., Japan) for 10 min was measured at an absorbance of 750 nm (Beckman DU 70, Beckman Inc., USA). Gallic acid was used as the standard.

Analysis of variance The analysis of variance of DPPH radical-scavenging activity and polyphenol content was performed for a twofactor factorial experiment.

RESlTLTS
Table I shows the cumulative temperature and solar radiation during the growing period (from seeding to maturation) for each variety of buckwheat. Their values varied from 1246.1 to 1944.3C and from 764.2 to 1204.2 MJ/m2 , respectively . Table 2 shows the DPPH radical-scavenging activity in the buckwheat varieties evalutaed in this study. The activity varied from 0.497 to 1.233llmol-Trolox equivalent/ml (here, I ml of extract was equivalent to 100 mg of buckwheat flour). Among them, "Phapal", which originated from Nepal, had the highest radical-scavenging activity at 1.233 umol-Trolox equivalent/m!. Another variety from Nepal, "Mitephapal", also showed a high radical-scavenging activity, indicating that varieties from Nepal belong to a group with a high radical-scavenging activity. The second group having a high radical-scavenging activity included varieties originated from Canada and France. "Uchimoko 9", from China, showed the lowest radical-scavenging activity at 0.497 umol-Trolox equivalent/m!. Japanese buckwheat varieties showed radical-scavenging activity in the range of 0.581 to 0.922 umol-Trolox equivalent/m!. Among the Japanese varieties, "Hashigamiwase" had the highest radical-scavenging activity at 0.922 umol-Trolox equivalent/ml, and "Kuzuu zairai" had the lowest activity at 0.581 umol-Trolox equivalent/ m!' In all varieties tested, a significant positive correlation

92

Morishita et al.

Table I. Cumulative temperature and solar radiation during the growing period for each variety of buckwheat.

Table 2. flour.

DPPH radical-scavenging activity in buckwheat

": Mean values of two blocks.

Watanabe et al. (1995) on the buckwheat varieties grown in the Tohoku region in northern Japan. It is interesting that the radical-scavenging activity in buckwheat seeds increases with an increase in the phenolic content. The most likely polyphenol contributors responsible for radical-scavenging activity are rutin, quercetin, and pro-

anthocyanidine . To breed buckwheat varieties with a high radical-scavenging activity, further study will be required to determine which buckwheat varieties in the Kyushu region contain predominant radical-scavengers and to analyze the inheritance and environmental effects that limit antioxidative activity.

Radical-scavenging activity in buckwheat Table 3 . Analysis of variance of DPPH radical-scavenging activity and polyphenol content of buckwheat tlour. Radical-scavenging activity df Total Cultivars Block Error 63 31 1 31 SS 1.3713 1.1719 0 .0223 0.1772 MS 0.0218 0 .0378 0 .0223 0.0057 6 .6'* 3 .9 Polyphenol content

93

SS 69551.4 60280.8 481.3 8789.3

MS 1104.0 1944.5 481.3 283 .5

F
6.9**

1.7

"': Significant at 0.01 level.

1. 4 ,....--

- --

-,

...... . >.... 8
11l
(J)

;>.

R =0.975**
1.2

.... .... u ----l:::

bJl-

o o

English summary) Ohsawa, R. and T. Tsutsumi, 1995 . Intervarietal variations of rutin content in common buckwheat flour (Fagopyrum esculentum Moench.) . Euphytica 86 : 183-189. Oki, T., M. Masuda, S. Furuta, Y. Nishiba and I. Suda, 2001. Radical scavenging of fried chips made from purple-fleshed sweet potato. Nippon Shokuhin Kagaku Kogaku Kaishi 48 : 926-932. (in Japanese with an Engli sh summary)

c:: 11l .... > bJl ... 1.0 c:: ::l (J) 0' > (J)

....
VJ I

11l

11l

0 ..... 0 0.8 ....

><

"1:l ..... 11l 0

.... I

Japan Nepal

.... e

::r:: 0..
0..
Q

::i.
~

0.6
tl

"" Russia v Canada o France tl China

Oomah, B.D., C.G. Campbell and G. Mazza, 1996. Effects of c ultivar and environment on pheno lic acids in buckwheat. Euphytica 90 : 73-76. Oomah, B.D. and C . Mazza, 1996. Flavonoids and antioxidative activi ties in buckwheat. J. Agr. Food Chern . 44 : 1746-1750. Przybylski, R., YC. Lee and N.A.M. Eskin, 1998. Antioxidant and radical-scavenging activities of buckwheat seed components. J. Am . Oil Chern . 75 : 1595-1601. Suda, 1., S. Furuta and Y . Nishiba, 1994. Fluorometric determination of a 1,3-diethyl-2 -thiobarbituric acid-malondialdehyde adduct as an index of lipid of pero xidation in plant materials. Biosci. Biotech. Biochern. 58 : 14-17. Suda, I., S. Furuta and Y. Nishiba, 1996 . A simple and rapid method of evaluate the antioxidative activity and its application to colored vegetables. Kyushu Agric. Res. 58 : 31. (in Japanese)

0.4

150

200

250

300

350

Total po l yp heno l content

(Jlg-GA ecu iva l enr /ml )

Fig. 4 . R e lat io n sh ip between DPPH radical-scavenging activity and polyphenol content in buckwheat flour.

REFERENCES
Baniya, B.K. , D.M.S . Dongol and N.R. Dhungel, 1995. Further characterization and evaluation of Nepalese buckwheat (Fagopyrum spp.) landraces. Current Advances in Buckwheat Research (Proc. 6th Inti . Symp. Buckwheat at Ina): 295- 304 . Furuta, S., I. Suda, Y. Nishiba and O. Yamakawa, 1998. High tert butylperoxy radic al scavenging activities of sweet potato cultivars with purple flesh. Food Sci., Technol. Int. Tokyo, 4: 33-35. Honda, Y., 1995. Var ietal difference of the content of vitamin E homo logues in buckwheat. Current Advances in Buckwheat Research (Proc. 6th IntI. Symp. Buckwheat at Ina) : 777-781. Kitabayashi, H., A. Ujihara, T. Hirose and M. Minami, 1995. Varietal differences and heritabi lity for rutin content in common buckwheat, Fagopyrum esculentum Moench. Breeding Science 45: 75-79. Morishita, T. and T . Tetsuka, 2001. Year -to-year variation and varietal difference of agronomic characters of common buckwheat in the Kyushu area. Jpn . J. Crop Sci. 70: 379-386. (in Japanese with an

Suda, 1., S. Furuta, Y . Nishiba, T. Noda, O. Yamakawa, K. Sugita and K. Matsugano, 1998 . Superoxide scavenging of sweet potato juice. Kyushu Agri c. Res. 60: 36. (in Japanese) Suda, I., H . Ogata, N. Mizuki, Y . Takahata and Y. Nishiba, 1999 . Measurement of radical sca venging activities of colored agricultural products and foods by DPPH/spectrophotometric assay . Kyushu Agric . Res. 61 : 32 . (in Japanese) Watanabe, M., A. Sato, R. Ohsawa and J. Terao, 1995 . Antioxidative activity of buckwheat seed extracts and its rapid estimate for evaluation of bre eding materials. Nippon Shokuhin Kogyo Gakkaishi 42 : 649-655. (in Japanese with an English summary) Watanabe, M., Y. Ohshi ta and T. Tsushida, 1997 . Antioxidant compounds from buckwheat (Fagopyrum esculentum Moench) hulls. J. Agri. Food Chern. 45 : 1039-1044. Watanabe, M ., 1998 . Catechins as antioxidants from buckwheat (Fagopyrum eseulentum Moench) groats. J. Agri. Food Chern . 46 : 839845 . Zielinski, H. and H. Kozlowska, 2000. Antio xidant activi ty and tota l phenolics in selected cereal grai ns and their different morphological fractions . J. Agri. Food Chern . 48 : 2008 -2016.