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Comparison of Flavonoids in Bran of Four Classes of Wheat'YUN FENG2 ' 3 and C. E. MC DONALD2

ABSTRACT Cereal Chem. 66(6):516-518Flavonoids from bran of hard red spring, hard red winter, durum, ultraviolet spectra, and fluorescent color to those of Len, a variety ofand white wheats were extracted with diluted NaOH solution (pH 11). hard red spring wheat in which the flavonoid was earlier purified andThey were purified by chromatography on Amberlite XAD-2 resin and identified as 6,8-di-C-glycosylapigenins. Finally, after partly purifying ona Sephadex G-15 column followed by paper chromatography with two the Amberlite XAD-2 column, the flavonoid contents of nine wheatdifferent solvents. The properties of the hard red winter, durum, and varieties were measured by ultraviolet spectrometry at 405 nm.white wheat flavonoids were identical in chromatography R, values,

Flavonoids are plant pigments widely distributed in nature.A mixture of two di-C-glycosylflavones wa s isolated from branof hard red spring (HRS) wheat (Len variety) and identified as6-C-pentosyl-8-C-hexosyl- and 6-C-hexosyl-8-C-pentosylapi-genins by ultraviolet (UV) spectroscopy, mass spectroscopy, and'3C-nuclear magnetic resonance spectroscopy (Feng et al 1988).In this paper, the work wa s extended to the characterization offlavonoids in bran of other wheat classes using the di-C-glycosylapigenins fraction identified in HRS wheat bran (Len)as a standard. Also the quantity of flavonoid in varieties ofdifferent wheat classes was measured by UV spectroscopy.

MATERIALS AND METHODSWheat SamplesBran samples from four classes of wheat were used in thisstudy. Varieties of three wheat classes grown at Hettinger, ND,in 1986 were Len, Stoa, an d Alex (HRS class); Roughrider andAgassiz (hard red winter [HRW] class); and Rugby, Lloyd, andVic (durum class). The white wheat used in this study was agift of unknown variety. Th e bran samples were cleaned andground by the methods used before (Feng et al 1988).Isolation an d Characterization of Bran Flavonoid FractionTh e extraction and purification procedures for bran flavonoidsfrom four classes of wheat were the same as those used previously(Feng et al 1988) except for omission of the paper chromatographicstep with tert-butanol/acetic acid/water and the thin-layerchromatography step.Characterization of FlavonoidsThe purified flavonoid fraction from three classes of wheat(HRW, durum, and white) wa s characterized by directlycomparing chromatography R1 values, UV spectra, andfluorescent color with those of the purified flavonoid from Lenvariety HRS wheat. Paper (Whatman 3MM) and polyamide thin-layer plates (Alltech Associates, Deerfield, IL) were used asadsorbents. The solvents, 15% acetic acid, ethyl acetate/formicacid/water (66:14:20, v/v), and t-BuOH/acetic acid/H 2 0 (3:1:1,v/v) were used for paper chromatography an d H20/methanol/butanone (4:3:3, v/v) wa s used for polyamide thin-layerchromatography. The flavonoids from the four classes of wheatwere cochromatographed ascendingly each time.Th e reagents and procedures used for obtaining UV spectra

'Published with the approval of the Director of the Agricultural Experiment Stationas Journal Series 1778.2Graduate student and professor, respectively, Department of Cereal Science and

Food Technology, North Dakota State University, Fargo 58105.3Present Address: 310 Hawkeye Court, Iowa City, IA 52240.This paper was prepared for electronic processing.@ 1989 American Association of Cereal Chemists, Inc.516 CEREAL CHEMISTRY

of flavonoids were those described by Mabry et al 1970. TheUV spectra were determined on flavonoid solution and thenonflavonoid phenol fraction by scanning from 225-525 nm ata speed of 1,200 nm/ min on a Beckman DU-7 spectrophotometerusing methanol as a blank.The fluorescent color of the wheat flavonoid fraction with andwithout ammonia treatment was also determined. The dry paperchromatograms were first viewed under UV light (366 nm) alone;the fluorescent color was noted, and then the paper wa s exposedto NH 3 for 2 sec and the color again recorded.Quantative Analysis of Flavonoids in BranFor a flavonoid standard curve, 10-mg samples of purifiedflavonoids from Le n bran (Feng et al 1988) were dissolved in100 ml of spectroscopic methanol, and from this 10-100 ,ug/mlsolutions were prepared. Using a l-ml absorption cell, on e dropof NaOMe (Mabry et al 1970) wa s added to 0.5 ml of flavonoidsolution, and light absorption wa s determined at 405 nm usingmethanol as a blank. The standard curve of light absorption versusflavonoid concentration (,um/ml) is shown in Figure 1.For the determination, wheat bran (2 g) wa s extracted 12 hrtwice with 80 ml of water, pH 11 (NaOH). The combined extractswere acidified to pH 5 with acetic acid and centrifuged to removethe precipitate that was formed. The supernatant was appliedto a 2.8 X 6 cm column of Amberlite XAD-2 (Eastman KodakCo., Rochester, NY). The column was washed with 300 ml ofwater and then 10 ml of methanol. The flavonoid fraction wa sthen eluted with 50 ml of methanol. The light absorption at 405nm by the flavonoid was determined after adding on e drop ofNaOMe to 0.5 ml of solution. The quantity of flavonoid in eachsample wa s calculated from the standard curve (Fig. 1). Thenonflavonoid phenols that are also adsorbed and eluted fromthe Amberlite XAD-2 column were separated from the flavonoidfraction by paper chromatography with ethyl acetate/ formic acid/

ECLO0c00.I-0.0

1.81.61.41.21.00.80.60.40.20.0 I I -I I I I a I I A

0 10 20 30 40 50 60 70 80 90 100/,g/mlFig. 1. Standard curve for bran flavonoids.

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in Len bran flavonoids, reported to be a mixture of 6-C-pentosyl-8-C-hexosyl- and 6-C-hoxosyl-8-C-pentosylapigenin (Feng et al1988).UV spectra in methanolic reagents. In Figure 2 the UV spectrain methanol (solid line, column 1) an d five methanolic shiftreagents (column 1, dotted line, and both lines in columns 2 an d3) for flavonoid fractions from the other HRW, durum, and whiteclasses of wheat were almost identical to those of purifiedflavonoids from the Len (HRS) bran. Flavonoids are identifiedby the presence or absence of spectra shifts that occur in thepresence of these shift reagents; this is explained in detail in the

first paper (Feng et al 1988). Th e spectra were also identical tothose of apigenin (Mabry et al 1970). The flavonoids in eachclass apparently have an apigenin aglycone as found for branof Len wheat by UV spectra and 1 C-NMR (Feng et al 1988).The fluorescent colors oj bran flavonoids. Th e appearance ofa flavonoid under UV light with and without ammonia (NH 3)treatment can be used to differentiate types of flavonoids. Th efluorescent color is related to the flavonoid structure and canbe used as a guide in the preliminary identification of flavonoids(Mabry et al 1970). For all four wheat classes the color was darkpurple, which turned to yellow green on exposure of the flavonoidspot to NH 3 gas. This indicates that the flavonoid in all fourclasses of wheat is a flavone with 5-OH and 4'-OH (Mabry etal 1970), as found in apigenin.Thus, R

1 values, UV spectra, an d the color under UV lightwith or without NH 3 indicate that the flavonoids in HRW, durum,and white wheat are probably 6,8- diglycosylapigenins as foundin the bran of Len variety HRS wheat (Feng et al 1988).

Quantitative Analysis for FlavonidsThe flavonoids in bran give an absorption peak at 396 nmwhen alkaline NaOMe is added to a methanol solution of theflavonoid (Fig. 3). This absorption peak was used to measurethe quantity of flavonoid. The wavelength of 405 nm was chosento decrease interference from nonflavonoids, which have a peaktailing off into the 396 nm range (Fig. 4). The results of quantitativeanalysis for flavonoids in wheat are given in Table I. Varietiesin the same wheat class were found to vary in the level of wheatflavonoids. However, varieties of durum and white wheatcontained higher average amounts of flavonoids than the averagefor varieties of HRS and HRW wheat. However, the results inTable I show only the relative amounts of flavonoids in wheatbran. On repeated extractions of the bran sample with pH 11water (NaOH), each extraction continued to extract moreflavonoids. Th e rate of extraction, however, decreased after two12-hr extractions, as used here, because 60% of the flavonoidextracted was extracted in the first two of the seven extractions.

ACKNOWLEDGMENTSWe thank Gordon Rubenthaler of USDA at Pullman, WA, for thegift of a sample of bran from white wheat.

LITERATURE CITEDFENG, Y., McDONALD, C. E., and VICK, B. A. 1988. C-Glycosylflavones from hard red spring wheat bran. Cereal Chem 65:452.MABRY, T. J., MARKHAM, K. R., and THOMAS, M. B. 1970. Pages12-13, 35-40, 81 in: The Systematic Identification of Flavonoids.Springer-Verlag: New York.

[Received February 27, 1989. Revision received May 25, 1989. Accepted June 21, 1989.]

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