Journal of Cereal Science 43 (2006) 108–119

www.elsevier.com/locate/jnlabr/yjcrs

Chemical structure of flavonoid compounds in wheat (Triticum aestivum L.)

flour that contribute to the yellow colour of Asian alkaline noodles
R.E. Asenstorfer a, Y. Wang b,c, D.J. Mares a,*
a
School of Agriculture and Wine, The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA 5064, Australia
b
Plant Breeding Institute, The University of Sydney, P.O. Box 219, Narrabri, NSW 2390, Australia
c
New Products Development, 286 Fison Ave. E, Eagle Farm, Qld 4009, Australia
Received 11 June 2005; revised 16 August 2005; accepted 1 September 2005

Abstract
Colour is an important criterion of quality in Asian noodles prepared from bread-wheat flour and ranges from white to cream for white salted
noodles to a bright yellow for alkaline noodles. A high-performance liquid chromatographic protocol for the analysis of naturally occurring
flavonoids and related phenolic compounds in wheat was developed to identify the compounds responsible for the yellow colour of alkaline
noodles. The separation system consisted of a C18 reversed-phase column and a gradient system of water/formic acid (99:1,v/v) (A) and
methanol/acetonitrile/formic acid (95:4:1,v/v/v) (B). A post column derivatisation technique was employed to identify the compounds that turned
yellow at alkaline pH. A comparison of the HPLC profiles of water, alkaline water and hydroxylamine soluble fractions revealed that the level of
compounds contributing to yellow alkaline noodle colour was highest in the hydroxylamine extract. Two flavonoid apigenin-C-diglycosides and
their Wessely–Moser isomers, together with their sinapic acid esters, were isolated and identified by absorbance spectroscopy, ES-MS
spectrometry and 13C NMR. These flavone-C-diglycosides were found to be the major components responsible for the alkali-induced change in
noodle colour and are relatively stable at ambient temperature. Furthermore the compounds are located only in the germ (embryoCscutellum
tissue) of wheat grains and hence their presence in flour must represent a redistribution during the milling process.
q 2005 Elsevier Ltd. All rights reserved.

Keywords: Flavone-C-glycosides; Yellow alkaline noodles

1. Introduction
Noodles are an important part of the diet in many countries
of eastern and south eastern Asia, accounting for approxi-
mately one third of Australia’s bread wheat exports and about
40% of wheat consumed in Asian countries (Hou and Kruk,
1998). These end-products are also becoming increasingly
popular in Western countries. Asian noodles made from wheat
may be divided into two general classes based on the
ingredients used: white salted noodles (WSN, made from
Abbreviations: ES-MS, electrospray mass spectrometry; HPLC, high-
performance liquid chromatography; HVPE, high-voltage paper electrophor-
esis; LC-MS, liquid chromatography mass spectrometry; MS/MS, daughter ion
mass spectrum; NMR, nuclear magnetic resonance; Rf, relative to front; Rm,
relative mobility; SPE, solid phase extraction; TLC, thin layer chromatography;
UV–vis, ultraviolet–visible; YAN, yellow alkaline noodle..
* Corresponding author. Tel.: C61 8 8303 7262; fax: C61 8 8303 7109.
E-mail address: daryl.mares@adelaide.edu.au (D.J. Mares).
0733-5210/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2005.09.001
flour, sodium chloride and water) and yellow alkaline noodles
(YAN, made from flour, alkaline salts such as sodium and
potassium carbonate and water). Yellow alkaline noodles
represent a major end use product of Australian hard wheat. In
addition to modifying the texture of the noodle, the addition of
alkaline salts results in a concomitant increase in the intensity
of the yellow colour.
Colour is a key quality trait (Mares and Campbell, 2001)
because of the visual impact at the point of sale. It provides
some indication of quality of the starting materials and in some
cases the age of the product. Asian customers prefer bright
yellow alkaline noodles that retain a stable colour for 24–48 h
after preparation and perceive red or dull grey colours as
undesirable. A preliminary study by Mares et al. (1997)
indicated that the colour of alkaline noodles was due partly to
xanthophylls and partly to compounds that could be extracted
with aqueous solvents such as water, hydroxylamine at neutral
pH and dilute alkali. These latter compounds, in contrast to the
xanthophylls, were colourless at neutral or acid pH but turned
yellow at higher pH. Alkaline noodles prepared from flour that
had been extracted with hydroxylamine solution, failed to turn
 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119
yellow and had reflectance curves that were similar to control
noodles made from flour and 2% NaCl solution. Whilst a
certain amount of yellow colour develops as a result of natural
pigments in wheat flour, noodle manufacturers often add
pigments such as tartrazine, sunset yellow and b-carotene
because there is insufficient natural pigmentation to meet
customer expectations.
Aqueous methanol, a solvent traditionally applied to the
analysis of phenolic compounds, is not an ideal solvent for this
sample matrix that is rich in alcohol soluble proteins and
polysaccharides. These proteins and polysaccharides precipi-
tate at the beginning of the gradient during HPLC, often
blocking the column, interfere with the analysis and
dramatically reduce the life of the column. Feng et al. (1988)
noted that bran flavonoids are more easily extracted by alkaline
solution than either methanol or aqueous methanol solutions
and therefore an alkaline aqueous extraction method, based on
the work of these authors was developed for routine analysis.
Identification of the chemical structure and the tissue of
origin of the compounds responsible for the yellow colour that
develops following the addition of alkaline solutions to flour
are important in manipulating the colour of YAN either by
modifying milling procedures or by genetic improvement. The
objectives of this work were to determine which of the
components in aqueous extracts prepared from various grain
tissues and wheat cultivars are involved in the development of
the yellow colour of alkaline noodles and to develop a routine
procedure for analysis of these compounds in wheat flour or
meal.
2. Methods
2.1. Wheat and flour preparation
Australian bread wheat cultivars Angus, Avocet, Canna,
Sunco, and Sunvale, and one durum cultivar Kamilaroi, were
grown in the field at Narrabri in northern NSW, Australia.
Grain was harvested at a moisture content of less than 12% and
either milled on a Quadrumat Senior Mill to produce flour
extractions in the range 66–72% or ground to yield wholemeal
using an Udy cyclone mill with a 0.1 mm particle size setting.
Germ tissue (embryoCscutellum) and de-embryonated
grains were obtained using two methods. In the first, grains
were soaked in cold water (4 8C) overnight and the germ
dissected from the grain with a scalpel. The tissues were
freeze-dried and ground with an Udy cyclone mill. In the
second, germ-free bran and flour were obtained by first
removing the germ from dry, intact grains using a scalpel,
then milling the de-embryonated grains in a Quadrumat Junior
Mill. The bran fraction was subsequently reduced to a fine
meal. This bran fraction was compared with bran derived from
whole grains milled with the Quadramat Junior mill.
2.2. Preparation of alkaline noodle sheets
Flour (10 g) and 3.5 ml of sodium carbonate solution (2%
w/v) were mixed in a small stainless steel bowl using a paddle
109
attached to an electric drill press. The mixture was pressed into
a ball and formed into a sheet by passing several times through
the rolls of a hand operated spaghetti machine. Noodle sheets
were immediately frozen and stored at K20 8C or held at
ambient temperature in a resealable plastic bag for 2, 6, 24 or
48 h before freezing. Noodle sheets were freeze-dried, reduced
to powder with a mortar and pestle, and extracted with
hydroxylamine (0.1 M, pH 7.0) for HPLC analysis. In a parallel
experiment, sheets were placed in boiling water for 3 min
immediately following preparation then stored and sampled as
described above.
2.3. Comparison of extractants and preparation of extracts
for HPLC analysis
Extracts for HPLC analysis were prepared according to the
procedure reported by Feng et al. (1988) with minor
modifications. Wholemeal (5 g) or flour (20 g) was extracted
with 200 ml of water (pH 7), alkaline water (pH 12, adjusted
with concentrated NaOH), or hydroxylamine (0.1 M, pH 7.2).
The mixtures were gently stirred at room temperature for 12 h,
the supernatant decanted and the remaining solid residue
extracted once more with 100 ml of the corresponding solvent
for 12 h. This second batch of extracts was centrifuged
(2100 g) for 20 min and the two supernatants pooled.
Extracts were adjusted to pH 5.2 with acetic acid to
precipitate proteins. The cloudy solution was left for 12 h at
room temperature then centrifuged (2100 g) for 20 min. The
precipitate was discarded and the supernatant concentrated to a
volume of approximately 20 ml by rotary evaporation under
reduced pressure at 40 8C. To precipitate arabinoxylans,
120 ml methanol was added to the concentrate. The mixture
was left overnight and then filtered. The filtrate was
concentrated to a volume of approximately 5 ml on a rotary
evaporator under reduced pressure at 40 8C. Methanol
precipitation was repeated and the final volume of the
concentrate adjusted to 25 ml with 60% methanol. Approxi-
mately 2 ml of the sample solution was filtered through a
0.45 mm cellulose-acetate disposable filter (Advantec MFC,
Pleasanton, California, USA) prior to injection onto the HPLC
column.
2.4. Isolation of major components responsible for the yellow
colour of YAN by HVPE
Flour (200 g) was extracted with 400 ml methanol for 12 h,
the mixture allowed to stand and the supernatant decanted and
filtered. The methanol was removed by rotary evaporation at
(50 8C, K100 kPa). The crude extract was fractionated by high
voltage paper electrophoresis (HVPE). Chromatography paper
(Chr 3; Whatman, Clifton, New Jersey, USA), wetted with
0.05 mol lK1 potassium tetraborate buffer (pH 9.4) was used to
separate samples according to the procedure outlined by Tate
(1968, 1981). A run time of 60 min with a potential of 1500 V
and a current of 150 mA was used. Relative mobilities were
compared with Orange G (1-phenylazo-2-naphthol-3,5-disul-
phonate; BDH Chemicals Ltd; Poole, England) and xylene
110 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119

cyanol FF (BDH Chemicals Ltd; Poole, England) as the anionic
standards and 2-deoxyadenosine as the neutral standard. After
running, the papers were dried in air. A yellow-coloured band
with mobility relative to Orange G (RmOG) of 0.59 was eluted
from the paper with water. The eluted fraction was further
purified using a C18-reverse phase cartridge (Sep-Pak classic;
Waters Corporation, Milford, Massachusetts, USA). The yellow
material was eluted from the C18 cartridge with methanol and
analysed by both HPLC and LC–MS.
2.5. Separation and identification of constituents by HPLC
Separation of phenolics and flavonoids was performed
using a Hewlett-Packard HPLC 1100 instrument consisting of
a quaternary pump, a solvent degasser, an autosampler, a
column compartment maintained at 30 8C, a photodiode-array
detector, and an isocratic pump for post-column derivatiza-
tion. A 250!4 mm analytical column (Merck, Darmstadt,
Germany) packed with spherical LiChrospher 100 RP-18
(5 mm) was employed, fitted with a 4!4 mm guard column
using the same packing material. A stepwise elution profile,
with linear gradients between isocratic stages was used. The
chromatographic protocol was: eluent A–HCOOH/H2O (1:99,
v/v), eluent B–HCOOH/CH3CN/MeOH (1:4:95, v/v/v);
elution program: 0–3 min, isocratic 10% B in A; 3–8 min,
gradient 10% B in A to 24% B in A; 8–11 min, isocratic 24%
B in A; 11–18 min, gradient 24% B in A to 34% B in A; 18–
28 min, gradient 34% B in A to 44% B in A; 28–35 min,

Fig. 1. RP-HPLC separations of flavonoids and related compounds in (a) water, (b) 0.1 M hydroxylamine (pH 7.2) and (c) alkaline water (pH 12) extracts of
wholemeal, detected at 340 nm.
 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119
gradient 44% B in A to 65% B in A; 35–40 min, gradient 65%
B in A to 95% B in A; 40–55 min, isocratic 95% B in A; 55–
60 min, gradient 95% B in A to 10% B in A; 60–70 min,
isocratic 10% B in A; flow rate—0.65 ml/min; detection—
(a)—UV 340 nm, (b)—UV 280 nm. In post column deriva-
tisation experiments, the flow rate of the isocratic pump was
set to 0.5 ml/min and shift reagent, 1 mol lK1 sodium
carbonate was pumped into the eluent prior to detection.
Naringin and ferulic acid (Sigma-Aldrich, St Louis, MO,
USA) were used as external standards. Flavone-glycoside
concentrations for all grain tissues are expressed as naringin
(Sigma-Aldrich) equivalents.
2.6. LC-mass spectrometry
Samples were loaded onto a HPLC column (Spherisorb
S5 ODS2, 250!1 mm, Waters Corporation) with a sample
injector (Rheodyne model 8125, Cotati, CA) fitted with a
5 ml loop. The separation was performed with solvent A
(5% formic acid) and solvent B (5% formic acid/80%
acetonitrile, v/v) by using a multi stage linear gradient
system, 10–35% of solvent B in 35 min, kept constant at
Fig. 2. Chromatograms of (a) aqueous, (b) hydroxylamine (pH 7.2) and (c) alkaline water extracts of flour after derivatisation measured at 436 nm.
111
35% for 5 min and then 35–95% solvent B in a further
20 min, at a flow rate of 25 ml/min delivered by a syringe
pump (140B Solvent Delivery System, Applied Biosystems).
The HPLC column was connected to a UV–vis diode array
detector (HP1100, Hewlett-Packard) monitoring at 280 and
340 nm, and then by mass spectrometry using an ionspray
ion source (API-300, PE Sciex, Thornhill, Ontario, Canada).
The mass spectrometer was operated in positive ion
mode and was scanned from m/z 250 to m/z 1000 in
1.88 s. Ion spray and orifice potentials were set at 5.5 kV
and 30 V, respectively. Curtain and nebuliser gases were
nitrogen and air, respectively. All mass spectral data were
processed using Bio-Multiview software (version 1.2b3, PE
Sciex).
2.7. Isolation for NMR
Wheat germ (3 kg) was extracted with 8 l 70% acetone for
16 h. The extract was filtered and acetone removed by
evaporation (50 8C, K100 kPa) until 2 l of the solution
remained. This was then washed once with 500 ml petroleum
spirit. The solution was then further concentrated to 1 l and
112 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119

place in the refrigerator for 12 h. After filtering, the extract was
loaded onto a C18 column (280 mm!300 mm diameter).
The column was washed with 30% methanol and then eluted
with 40% methanol. Fractions (10 ml) were collected and
tested by HPLC. The yields were 47.5 mg compound 1,
53.6 mg compound 2 and 20.3 mg for the sinapic acid adducts.
NMR experiments were performed on a 600 MHz (Varian)
instrument.
2.8. Sugar analysis
Approximately 20 mg of flavone-C-diglycoside was
oxidized at 110 8C for 6 h with 20 mg FeCl3 (Yasukawa
et al., 1982) in 1 ml water. After oxidation the sample was
passed through a sulfoxyethyl cellulose (Sigma-Aldrich)
cation exchange column to remove ferric ions and then
through a small C18 SPE column (Sep-Pak classic cartridge;
Waters Corporation, Milford, USA) to remove any phenolic
compounds present. The sugars were then analysed by TLC
(Yasukawa et al., 1982) using pyridine/ethyl acetate/acetic
acid/water (36/36/7/14, v/v/v/v) on a cellulose plate (CEL
300; Alltech associates, Deerfield, USA). The sugars were
reacted with p-anisidine (Sigma-Aldrich), after which
pentoses stain cherry red and the hexoses stain yellow.
TLC Rf values were: arabinose, 0.60; xylose, 0.68; lyxose,
0.72; glucose, 0.58 and galactose, 0.49. Galactose was
converted to lyxose during the oxidation process (Yasukawa
et al., 1982).
3. Results and discussion
3.1. Comparison of extraction methods
Extraction of flour samples followed by precipitation of
water-soluble proteins and polysaccharides with acetic acid and
methanol eliminated problems such as increasing pressure and
eventual blockage of the HPLC columns that reduced column
life and efficiency, but was time-consuming. Nevertheless, this
extraction method proved to be most effective, generating
reproducible results.
When compared to water, 0.1 M hydroxylamine (pH 7.2)
released additional compounds from wholemeal that were
more hydrophilic, whereas alkaline aqueous extracts contained
components that were more hydrophobic (Fig. 1). In aqueous
extracts, peaks 9 and 11 accounted for nearly all yellow colour
that developed following the post column treatment (Fig. 2(a)).
Similarly, peaks 9 and 11 accounted for most of the yellow
colour that developed at high pH in the alkaline aqueous
extracts (Fig. 2(c)). In contrast to water and hydroxylamine,
dilute alkali or alkaline water extracted large amounts of ferulic
acid (peak 12 in Fig. 2(c)). By comparison, hydroxylamine
extracts contained four additional alkali sensitive components
(peaks 1, 4, 5 and 7). However of these additional peaks, only
peak 7 contributed significantly to the yellow colour
(Fig. 2(b)). The intensity of peaks 9 and 11 was increased in
both hydroxylamine and alkaline water compared with to

2.9. Reaction product of 2,6-dimethoxy-1,4-benzoquinone and

hydroxylamine

2,6-Dimethoxy-1,4-benzoquinone-4-oxime was synthesised

from 2,6-dimethoxy-1,4-benzoquinone according to Bolker
and Kung (1969). ES-MS, m/z (relative intensity): 367
(2MHC, 0.07), 184.0 (MHC, 0.20), 170.0 (0.08), 167.2
(0.36), 166.2 (0.80), 152.0 (b.p.), 151.0 (0.70), 140.0 (0.09),
112.0 (0.17), 111.0 (0.26). HPLC retention time was 19.1 min.
Absorbances (aq.): lmaxZ300 nm (3Z16,000) with shoulder
at 395 nm (3Z1200) at pH 2.5 and lmaxZ353 nm (3Z25,300)
at pH 10.

2.10. Synthesis of a putative quinone-hydroxylamine adduct

(peak 7)

Approximately 10 mg of 2,6-dimethoxy-1,4-hydroxyben-
zene and 20 mg hydroxylamine hydrochloride were added to
2 ml of water and adjusted to pH 7. This was allowed to react
for 16 h at 45 8C. The solution was extracted with dichloro-
methane. The dichloromethane was carefully evaporated under
reduced pressure and the residue dissolved in methanol for
HPLC and LC-mass spectroscopy. For direct comparison, flour
(200 g) was extracted with 1 l 0.1 M hydroxylamine hydro-
chloride for 2 h. This was filtered and 100 ml of the filtrate
extracted with 3!100 ml dichloromethane. The dichloro- Fig. 3. UV spectra of peaks 9 and 11. Solid lines represent the spectra recorded
methane was evaporated and the residue dissolved in methanol without alkaline treatment, dotted lines represent spectra recorded after alkaline
for MS-analysis. treatment.
 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119 113

Table 1 column, facilitated the identification of the water-soluble

Major components in the germ and non-germ fractions of whole meal of compounds which turn yellow at high pH and which were
cultivar ‘Sunvale’ extracted using 0.1 M hydroxylamine (pH 7.2)
presumed to contribute to YAN colour. The RP-HPLC profiles
Peak no. Retention time (min) Fraction of the extracts from whole meal and flour were very similar
Non-germ Germ except that all compounds in the flour were present in lower
4 14.5 41.7 123.2 concentrations. While at least fifteen compounds became
5 18.9 72 222.3 yellow following post column derivatisation, most were
7 19.8 14.4 527 present in trace amounts. There were only two major and one
9 24.1 *nd 868
11 25.7 nd 1005.8
minor component in flour that were noteworthy. Peaks 9 and 11
had spectra (Fig. 3) with absorption maxima at 272 and 340 nm
The concentrations of the components were determined at 340 nm, and for peak 9, and at 272 and 334 nm for peak 11 and maxima of
expressed as naringin equivalents (mg/100 g). *nd, not detectable.
282 and 402 nm for both following the addition of alkali. These
water alone. spectra were similar to those reported for flavones and their
OH glycosides (Feng et al., 1988; Mabry et al., 1970). King (1962)
isolated two related flavone-C-glycosides from wheat germ
8
HO 7 O 2 that had apigenin (1) nuclei with highly hydroxylated glycosyl-
6 3 type side chains, one flavone-C-glycoside being the sinapic
5 4 1 acid ester of the other. In a subsequent study, Feng et al. (1988)
OH O identified apigenin-6-C-arabinoside-8-C-hexoside as the major
flavone-glycoside and 6-C-hexosyl-8-C-pentosylapigenin as a
3.2. Identification of major chemical constituents that turn minor flavone-glycoside of bran from red spring wheat. As
yellow at alkaline pHs these two compounds were major yellowing agents, for
simplicity an analytical wavelength of 340 nm was chosen
Liquid chromatography coupled with UV–vis detection in for subsequent HPLC analysis. The minor component, peak 7,
combination with a shift reagent, 3% NaOH added post was characterized for further study.

Fig. 4. Chromatograms of 0.1 M hydroxylamine extracts (pH 7.2) of (a) bran from de-embryonated grain, (b) bran from whole grain and (c) flour prepared from de-
embryonated grain. Detected at 436 nm, following post column reaction with alkali.
114 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119

565.4
(a)
55

50
511.2
45

40
Intensity, cps (x103)

35
529.2
30 427.2
379.2
25
391.2 409.4 547.2
20
481.2
15 499.2
349.0
10 493.2
337.0
325.2 457.4
5

250 300 350 400 450 500 550

m/z, amu

565.4
(b) 19
18
17
511.4
16
15
14
13
Intensity, cps (x103)

12
11 409.2
427.0 529.2
10
379.2
9
547.2
8 499.2
481.2
7 391.2
6
5
4 337.2
349.2 457.2
3
295.4
2
1

250 300 350 400 450 500 550

m/z, amu

Fig. 5. Mass spectra of (a) peak 9 and (b) peak 11.

3.3. Location of alkali-sensitive pigments in wheat grains
Peaks 7, 9 and 11 were present in the germ fraction (Table 1), at
concentrations that were 20–25-fold higher than levels in whole
meal. Peaks 9, 10 and 11 were absent from the non-germ portion
of the grain. The non-germ tissue appeared to be the source of
peak 4, which was yellow at high pH (O9), however it does not
normally appear in significant amounts in plain flour. These
results suggest that the major determinants of alkali induced
yellow colour in noodles were derived from the germ tissue.
Bran from de-embryonated grains contained only half the
concentration of alkali induced colour components compared
 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119 115

with bran from whole grains (Fig. 4). By contrast, the quantity Table 2
13
of peaks 4 and 7 was similar in both samples (Fig. 4(a) and (b)), C NMR (150 MHz) assignments: d 13C (ppm) of apigenin-C-diglycosides 2/3
and 4/5
indicating that these components are primarily located in the
seed coat/aleurone tissue. The flour fraction (predominantly Position Peak 9 (2/3) Peak 11 (4/5)
starchy endosperm) derived from de-embryonated dried grains Apigenin
contained only a trace of alkali sensitive components 4 182.4 182.4
(Fig. 4(c)). Since most of these appeared to be represented 2 164.3 164.5
7 161.3 162.1
by peak 4, it is assumed that this contribution can be attributed
40 161.3 161.8
to contamination of flour with bran, which inevitably occurs 5 158.4 158.0
during milling (Miskelly, 1996). The starchy endosperm of 9 153.6 155.3
wheat therefore, appears to contain little or none of peaks 9 and 20 129.6 129.1
11. As a consequence, the component of the desirable yellow 60 128.9 128.8
colour of alkaline noodles that arises as a direct result of 10 121.2 121.1
30 116.2 116.2
interaction with alkaline salts appears to be almost totally
50 116.2 116.2
dependent on the amount of germ/bran tissue introduced into 6 108.1 108.1
the flour during the milling process. 8 103.6 103.8
10 102.6 102.6
3 102.3 102.2
3.4. Identification of peaks 9 and 11
Glucose C-6 C-8

The observed parent ions of peaks 9 and 11 were identical 1 74.45 73.96
with a mass of m/z 565.2 [MHC] (Fig. 5). While the ionization 2 70.41 71.23
3 79.46 79.34
pattern is quite complex, losses of H2O (m/z 547.2), 2xH2O (m/
4 70.14 70.05
z 529.2), 3xH2O (m/z 511.2), 4xH2O (m/z 493.2) 5xH2O (m/z 5 81.3 82.0
475.2) 6xH2O (m/z 457.4) and the presence of an m/z 295 ion 6 61.14 60.98
are indicative of an apigenin-C-diglycoside (Chopin and Galactose C-6 C-8
Bouillant, 1975). The difference between the observed parent
mass (564) and apigenin molecular mass (270), indicated that a 1 74.03 74.36
2 68.56 69.69
hexose (180-H2O) and pentose (150-H2O), were attached to the 3 74.77 75.01
apigenin moiety. From the mass spectral data it is not possible 4 68.41 69.32
to decide whether these two compounds were Wessely–Moser 5 79.65 80.69
isomers or have isomeric sugars. 6 61.41 61.00
Arabinose C-8 C-6 C-8 C-6
OH 1 74.87 75.1 75.14 75.83
R2
2 70.23 73.86 73.33 73.97
HO O
3 76.93 76.51 76.92 76.56
4 69.21 73.37 70.56 73.92
R1 2 R1 = arabinose, R2 =glucose 5 68.67 68.6 70.26 70.15
3 R1 = glucose, R2 = arabinose
OH O 4 R1 = arabinose, R2 =galactose
5 R1 = galactose, R2 = arabinose 3.5. Sinapic acid and ferulic acid esters

Mass spectrometric analysis of peaks 9 and 11, enabled the

The 13C NMR data clearly indicate that the two compounds
are different apigenin-C-glycosides, each consisting of a
mixture of Wessely–Moser isomers (Table 2). The carbons
were assigned according to Besson et al. (1984, 1985), Wagner
et al. (1980); Yasukawa et al. (1986). Because of the large
number of sugar carbons, the assignment was more difficult
than expected. Our data were in general agreement with the
partial identification of apigenin-6C-arabinoside-8C-hexoside
from red wheat germ (Feng et al., 1988) and identification of
apigenin-6C-galactoside-8C-arabinoside by 13C NMR in wheat
seedlings (Wagner et al., 1980). Peak 9 was a mixture of
apigenin-6C-arabinoside-8C-glucoside (isoschaftoside, 2) and
apigenin-6C-glucoside-8C-arabinoside (schaftoside, 3) while
peak 11 was a mixture of apigenin-6C-arabinoside-8C-
galactoside (4) and apigenin-6C-galactoside-8C-arabinoside
(5). TLC confirmed the identity of the sugars present.
identification of six compounds (two major and four minor) or
isomers with mass (771.4 m/z) identical to the sinapic acid
adduct of the flavone-C-diglycoside described by King (1962).
The loss of a sinapoyl group was not observed, as acylated
anthocyanins do not lose the acyl group easily (Aritomi et al.,
1970). The ionisation pattern, m/z (relative intensity), 771
(b.p.), 735.4 (0.10), 705.4 (0.07) and 681.2 (0.12), shows only
loss of H2O (753.4 m/z), 2xH2O (735.4 m/z) while the MS/MS
data (Fig. 6) provided evidence for a loss of a third H2O (717.4
m/z). This indicates that one of the sugars and not the apigenin
moiety is esterified.
13
C NMR spectra were recorded using a mixture of
compounds/isomers, as it was not possible to isolate sufficient
quantity of each. The 13C NMR spectroscopy was consistent
with the presence of the sinapic acid ester (Table 3). Our 13C
116 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119

705.4
13

12
681.4
11 207.2

10
651.2 735.4
9 753.2
Intensity, cps (x103)

369.0
8

5 385.0 771.2
4

3 337.2 427.0 633.0

313.2 585.4 663.4
2
249.4 475.0 511.0 717.4
174.8
1

200 300 400 500 600 700

m/z, amu

Fig. 6. MS/MS spectrum of sinapic acid adduct.

NMR assignments for the sinapic ester were in general putative ferulic acid esters with mass m/z (relative intensity) of
agreement with Ternai and Markham (1976); Wagner et al. 741.4 (MHC, b.p.), 723.2 (MHC–H2O, 0.22), 705.2 (MHC–
(1980). In wheat leaves, Wagner et al. (1980) recorded three 2xH2O, 0.14), 675.2 (MHC–66, 0.09), 651.2 (MHC–90, 0.12),
gamma-carbons indicating a mixture of 2, 3 and 4-OH acyl- 535.2 (0.12). The patterns of ion loss and relative
glycoside isomers. In contrast, in our data only a single intensity were similar to the sinapic acid esters. These
gamma-carbon was observed suggesting the acyl group is compounds were not in sufficient concentration for identifi-
attached only to a single glycoside-hydroxyl. The two cation by NMR.
O-methyl carbons indicates that these methyl groups may Apigenin-C-diglycosides may exist with or without
interact with the rest of the molecule in two different ways, acylation with sinapic acid or ferulic acids. Esterification
possibly through the sinapic acyl group favouring a folded of the apigenin-C-diglycosides by sinapic ester increases the
conformation through a stacking interaction with the flavonoid molecule’s hydrophobicity and the affects where it partitions
nucleus (Alluis and Dangles, 1999). within the flour and its extractability in aqueous solvents.
Using this isolation method and LC-mass spectrometry, it Furthermore, the folded conformation of the aromatic acyl
was also possible to identify six (two major and four minor) group may significantly influence properties such as protein
binding (Alluis and Dangles, 1999). Thus, by breaking the
Table 3
13
C NMR (150 MHz) assignments: d13C (ppm) for the sinapic acid esters of a
ester-linkages, both alkaline water and hydroxylamine
mixture of apigenin-C-diglycosides 2/3 and 4/5 extract a greater amounts of apigenin-C-diglycosides.
Position d13C
C-gamma 165 3.6. Characterisation of peak 7
C3,C5 147.9!2
C-alpha 145.1 In the hydroxylamine extracts, peak 7 appeared to be the
C4 138.3 next most important contributor to the colour of alkaline
C1 124.1
noodles after peaks 9 and 11. This pigment is also colourless at
C1 124
C-beta 116.1 low pH (lmaxZ340 nm) and is yellow at high pH (lmaxZ
C-beta 116 400 nm). The mass spectrum of this pigment showed a peak
C2,6 106.1 with an m/z (relative intensity) of 367.2 (MHC, 0.21), 184.1
C2,6 105.4 (0.81), 156.1 (0.45), 154.0 (b.p.), 139.1 (0.42) and 124.0 (0.18).
O-Methyl 56.1
Initially it was proposed that this compound was an oxime
O-Methyl 55.9
formed by the reaction of hydroxylamine with 2,6-dimethoxy-
 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119 117

Table 4
Absorbance maxima of 2,6-dimethoxyquinone, 2,6-dimethoxy-1,4-hydroquinone, 2,6-dimethoxyquinone glycoside compared with the hydroxylamine/2,6-
dimethoxy-1,4-hydroquinone reaction product

Compound Charge lmax Extinction coefficient

2,6-Dimethoxy-p-quinone (a) Neutral 288, 395 8900, 380
2,6-Dimethoxy-1,4-hydroquinone (a) Neutral 284 3900
Dianion 210, 318 14,000, 4000
1-Hydroxy-2,6-dimethoxy-4-O-b-D-glucoside (b) Neutral 280 3300
Monoanion 250, 290
Hydroxylamine/2,6-dimethoxy-1,4-hydroquinone reaction product Neutral 340 16,000
Monoanion 400 17,000

Data was obtained from (a) Fitzpatrick and Steelink, 1972, (b) Ogawa et al., 1973.

(a) 125 (b) 125

Peak area (% of time 0 hr)

Peak area (% of time 0 hr)

100 100

75 75

50 50

25 25

0 0
0 10 20 30 40 50 0 10 20 30 40 50
Time (hours) Time (hours)

(c) 125 (d) 125

Peak area (% of time 0 hr)
Peak area (% of time 0 hr)

100 100

75 75

50 50

25 25

0 0
0 10 20 30 40 50 0 10 20 30 40 50

Time (hours) Time (hours)

(e) 125 (f) 125

Peak area (% of time 0 hr)
Peak area (% of time 0 hr)

100 100

75 75

50 50

25 25

0 0
0 10 20 30 40 50 0 10 20 30 40 50

Time (hours) Time (hours)

Fig. 7. Stability of peak 7 (solid line) compared with apigenin-C-diglycosides, peak 9 (dotted line) and peak 11 (dashed line) for the wheat cultivars Tasman (a),
Kamilaroi (b) Lark (c), Timgalen (d), Sunvale (e) and Sunvale after boiling (f).
118 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119

Table 5 cultivar used as a source of flour, between 85 and 100% of

Concentrations of apigenin-C-diglycosides, 2/3 and 4/5, in wholemeal and the initial levels were retained during 48 h storage of noodle
Quadrumat Senior mill flours of five Australian cultivars grown in 1995
(expressed as naringin equivalents, mg/100 g) sheets at ambient temperature (Fig. 7). This pattern was
duplicated in sheets that were boiled immediately following
Variety Wholemeal Mill Percentage grain flavonoids
preparation (Fig. 7(f)). By contrast, the content of peak 7
flour flour in mill flour
declined steadily during storage of raw and boiled noodle
Angus
sheets to between 25 and 40% of the initial value by 48 h.
31.7 10 31.5
57.5 16.3 28.3 The failure of boiling to prevent this decline indicates that
Avocet the breakdown of peak 7 was not an enzymatic process.
27.5 6.8 24.7
57.5 13 22.6
Canna 3.8. Cultivar variation in the flavonoid content of wholemeal
26.3 6.8 25.9 and flour
51.9 12.1 23.3
Sunco
36.4 6 16.5 Table 5 shows the variation in apigenin-C-diglycosides 2/
44.7 7.2 16.1 3 and 4/5 content for wholemeal and Quadrumat Senior mill
Sunvale flours of five Australian wheat cultivars grown in 1995. The
50.9 9.8 19.3 proportions of these compounds in the wholemeal flours of
53 11.9 22.5
Sunvale and Sunco appeared to be different from the other
bread wheat cultivars. This may be due to their pedigrees
since both are Cook backcross derivatives but this clearly
p-quinone, a product first isolated from fermented wheat germ requires further investigation. The two flavone-C-diglyco-
by Vuataz (1950) and Cosgrove et al. (1952). However, there sides are derived from germ tissue of wheat grain and
were significant differences in the absorbance spectra, mass differences observed in the extraction percentage (proportion
spectral daughter ion pattern and HPLC retention times
of total recovered in flour) may result from the morpho-
compared with peak 7. Nevertheless, it was possible to
logical properties of grain cultivars. These results highlight
synthesise a compound with the same retention time, UV/vis
the importance of both the milling process and
and mass spectrum as peak 7 by reacting 2,6-dimethoxy-1,4-
the differences in the milling properties of individual
hydroquinone with hydroxylamine (data not shown). This
cultivars in determining the final colour of yellow alkaline
compound has not been described previously. Its complete
noodles.
identification will be published separately. For the purpose of
this work it is sufficient to note that it is not identical with any
simple oxime of 2,6-dimethoxy-p-quinone.
De Jong et al. (1953) and Bouvier and Horvath (1987) 4. Conclusions
suggested that the precursor to 2,6-dimethoxy-p-benzoqui-
none was a glycoside and that the quinone was released There are four main compounds that contribute to the
during fermentation. However, this putative precursor has yellow colour generated in noodle sheets as a result of the use
never been isolated. Neither 2,6-dimethoxy-1,4-hydro- of alkaline salt solutions: apigenin-6C-arabinoside-8C-gluco-
quinone, the glycoside nor their ionised forms are expected side (isoschaftoside, 2) and apigenin-6C-glucoside-8C-arabi-
to contribute to the colour of YAN, but the oxidisation noside (schaftoside, 3), apigenin-6C-arabinoside-8C-
product, 2,6-dimethoxy-p-quinone, appears yellow (Table 4). galactoside (4) and apigenin-6C-galactoside-8C-arabinoside
It is currently not possible to estimate the relative (5). Each may occur either in the free form or acylated with
contribution of 2,6-dimethoxy-p-quinone to the YAN colour sinapic acid. These apigenin-C-diglycosides are initially
because the exact fate of 2,6-dimethoxy-1,4-hydroquinone is present in the wheat germ, and become incorporated into the
unknown. However, extinction coefficients suggest that at flour during the milling process.
high pH, the hydroxylamine/2,6-dimethoxy-1,4-hydro-
quinone reaction product has an absorbance that is 40-fold Acknowledgements
greater than 2,6-dimethoxy-p-quinone. Therefore the relative
contribution 2,6-dimethoxy-p-quinone to YAN colour is Financial support from the Grains Research and Develop-
significantly less than suggested by the hydroxylamine ment Corporation, including a postgraduate scholarship for
extraction data and it probably is only a minor contributor Yasmin Wang, and the Value Added CRC is gratefully
to YAN colour. acknowledged. David Leach and Grant Wiley, University of
Western Sydney, contributed helpful advice during the early
3.7. Stability of colour compounds in YAN stages of Y Wang’s PhD candidature. We would also like to
acknowledge Yoji Hayasaka, The Australian Wine Research
Peaks 9 and 11, the apigenin-C-diglycosides, appeared to Institute, for mass spectral and Philip Clements, The University
be relatively stable in YAN and, depending on the wheat of Adelaide, for NMR analyses.
 R.E. Asenstorfer et al. / Journal of Cereal Science 43 (2006) 108–119
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