Novel oxidation products of cyanidin 3-O-glucoside with 2,2′-azobis-(2,4-dimethyl)valeronitrile and evaluation of anthocyanin content and its oxidation in black rice

Article abstract of DOI:10.1016/j.foodchem.2014.01.077

The radical oxidation mechanism of anthocyanin derivatives was investigated by the reaction of cyanidin 3-O-glucoside in the presence of radical initiator 2,2′-azobis-(2,4-dimethyl)valeronitrile (AMVN) in EtOH and aqueous CH ₃CN. Six different oxidation products were isolated, depending on the solvent employed. These products were identified using NMR spectroscopy and multistep derivatisation reactions. Of the products obtained, two novel oxidised anthocyanin derivatives were isolated from black rice under prolonged storage. A radical reaction mechanism is proposed on the basis of these reaction products. Quantification of oxidised anthocyanins in black rice is demonstrated as a method to verify freshness of the rice.

Full text of DOI:10.1016/j.foodchem.2014.01.077

Food Chemistry 155 (2014) 221–226
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Novel oxidation products of cyanidin 3-O-glucoside with 2,
20-azobis-(2,4-dimethyl)valeronitrile and evaluation of anthocyanin
content and its oxidation in black rice
⇑
Haruna Kamiya, Emiko Yanase , Shin-ichi Nakatsuka
Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 31 January 2014
The radical oxidation mechanism of anthocyanin derivatives was investigated by the reaction of cyanidin
3-O-glucoside in the presence of radical initiator 2,20-azobis-(2,4-dimethyl)valeronitrile (AMVN) in EtOH
and aqueous CH3CN. Six different oxidation products were isolated, depending on the solvent employed.
These products were identified using NMR spectroscopy and multistep derivatisation reactions. Of the
products obtained, two novel oxidised anthocyanin derivatives were isolated from black rice under
prolonged storage. A radical reaction mechanism is proposed on the basis of these reaction products.
Quantification of oxidised anthocyanins in black rice is demonstrated as a method to verify freshness
of the rice.
Keywords:
Anthocyanin
Cyanidin 3-O-glucoside
Radical oxidation
2,20-Azobis-(2,4-dimethyl)valeronitrile
(AMVN)
Ó 2014 Elsevier Ltd. All rights reserved.
Black rice
1. Introduction
they investigated the reaction of 1 (Fig. 1) with an alkylperoxyl rad-
ical species and reported the chemical structures of the resultant
The pigment anthocyanin is widely distributed in the plant king-
dom and is well known to be responsible for the red, blue, and violet
colours in flowers and fruits of some plants (Castañeda-Ovando,
Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal,
2009; Harborne, 1994; Harborne & Williams, 1995). Anthocyanins,
members of the flavonoid group of phytochemicals, are recognised
to play an important role in the enhancement of an array of bioactiv-
ities in animals, including improving vision (Jang, Zhou, Nakanishi, &
Sparrow, 2005; Matsumoto & Iida, 2007), antioxidative activities
(Castañeda-Ovando et al., 2009; Tsuda et al., 1994), and anticancer
activities (Hou, 2003; Katsube, Iwashita, Tsushida, Yamaki, & Kobori,
2003; Lule & Xia, 2005; Nichenametla, Taruscio, Barney, & Exon,
2006). Structurally, anthocyanins are the sugar (glucose)-containing
analogues of anthocyanidin aglycone, which are composed of a char-
acteristic flavylium (2-phenylchromenium) cation (Fig. 1). Numer-
ous anthocyanin derivatives are known with varying phenolic
substitution about the chromenylium (2-benzopyrylium) core and
the 2-phenyl moiety, in addition to differences in sugar combina-
tions (Harborne, 1994; Harborne & Williams, 1995).
products. In our continuing studies directed at natural product iso-
lation and characterisation, we noted the appearance of a new peak
that had been initially absent when a solution of cyanidin 3-O-gluco-
side 1 in MeOH was kept under daylight conditions for three days; in
contrast, this peak was not observed when the solution was kept in
the dark. Accordingly, we believed this peak to be the product of rad-
ical oxidation of 1. To investigate this hypothesis, we performed the
oxidation of 1, with a radical initiator in EtOH at 60 °C and analysed
the reaction mixture using HPLC. Three new peaks appeared in the
chromatogram with the concomitant decrease of 1; one of the peaks
was observed at the same retention time as the peak noted under
daylight conditions, indicating that the compound produced under
daylight conditions was formed by radical oxidation.
In this study, we investigated the reaction of 1 with the alkyl-
peroxyl radical 2,20-azobis-(2,4-dimethyl)valeronitrile (AMVN) in
detail and report the isolation of two novel oxidative products.
We propose an oxidation mechanism based on the identity of the
obtained products. Furthermore, we report the isolation of these
oxidative products from black rice that had been stored.
Several authors have reported the oxidation reactions of antho-
cyanins (Es-Safi et al., 2008; Jurd, 1964, 1972; Karrer & Widmer,
1927; Karrer et al., 1927; Tsuda, Ohshima, Kawakishi, & Osawa,
1996). Tsuda et al. first reported the radical oxidation of 1 in 1996;
2. Materials and methods
2.1. Instruments
⇑
NMR spectra were recorded on a JEOL ECA-500 or a JEOL ECA-
600 instrument (Tokyo, Japan), using tetramethylsilane (TMS) as
Corresponding author. Tel./fax: +81 58 293 2914.
E-mail address: e-yanase@gifu-u.ac.jp (E. Yanase).
http://dx.doi.org/10.1016/j.foodchem.2014.01.077
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

222
H. Kamiya et al. / Food Chemistry 155 (2014) 221–226
R
pressure and purified by HPLC (column: NB-ODS-9) using CH3CN/
3'
2'
OH
H2O (22:78 v/v) containing 0.5% TFA. Compound 2a (3.2 mg,
87.8%) was obtained: 1H NMR (600 MHz, CD3OD): d 6.22 (1H, d,
J = 2.0 Hz, H-5), 6.43 (1H, d, J = 2.0 Hz, H-7), 6.84 (1H, d,
J = 8.2 Hz, H-50), 7.20 (1H, dd, J = 2.0, 8.2 Hz, H-60), 7.27 (1H, d,
J = 2.0 Hz, H-20); 13C NMR (150 MHz, CD3OD): d 90.1 (C7), 99.6
(C5), 108.2 (C3), 108.7 (C9), 115.5 (C50), 118.0 (C20), 122.5 (C10),
123.4 (C60), 145.4 (C30), 148.3 (C40), 152.7 (C4), 157.1 (C8), 158.5
Cl
4'
5'
B
6'
8
HO
O
C
4
2
7
A
5
3 O
6"
6
4" OH
OH
1" O
HO
5"
R=OH Cyanidin 3-O-glucoside (1)
R=OCH3 Peonidin 3-O-glucoside (10)
2"
OH
OH
3"
Fig. 1. Representative anthocyanins; cyanidin 3-O-glucoside 1 and peonidin 3-O-
glucoside 10.
(C6), 161.1 (C2), 170.5 (C10); UV kmax (MeOH) nm (e): 247
(14,400), 334 (12,600).
an internal standard. Multiplicities are reported using the follow-
ing abbreviations: singlet (s), doublet (d), triplet (t), doublet of
doublets (dd), multiplet (m), broad (br). Coupling constants (J)
are reported in hertz (Hz). IR spectra were recorded on a Perkin
Elmer 2000 FT-IR spectrometer (Perkin Elmer Japan Co., Ltd.,
Kanagawa, Japan). UV–Vis spectra were recorded on a Hitachi
4000U UV–Vis spectrometer (Tokyo, Japan) using methanol as
the solvent. High-resolution electron impact mass spectrometry
(HREIMS) and high-resolution electrospray ionisation mass spec-
trometry (HRESIMS) data were obtained using a JEOL JMS-700/GI
mass spectrometer (Tokyo, Japan). High-performance liquid
chromatography (HPLC) analyses were carried out with a JASCO
PU-2089 intelligent pump equipped with a JASCO MD-2010 PDA
detector, and JASCO CO-2065 column oven (Tokyo, Japan). The
HPLC columns used for analytical and preparative HPLC were COS-
MOSIL 5C18-MS-II (4.6 mm ID ꢀ 150 mm; Nacalai Tesque Inc.,
Kyoto, Japan) and NB-ODS-9 (10 mm ID ꢀ 250 mm; Nagara Science
Co., Ltd., Gifu, Japan), respectively.
2.5. Methylation of 2a
A solution of diazomethane (excess) in Et2O was added to a
solution of 2a (10 mg) in MeOH (1 mL) and kept at room tempera-
ture for 1.5 h. The reaction mixture was concentrated under re-
duced pressure and then purified by preparative thin-layer
chromatography (p-TLC) with 1% MeOH in CH2Cl2 to give 2b
(8.0 mg, 65.0%). EIMS: m/z 372 [M]+; 1H NMR (600 MHz, CD3OD):
d 3.86 (3H, s, OMe), 3.88 (3H, s, OMe), 3.90 (6H, s, OMe), 3.92
(3H, s, OMe), 6.42 (1H, d, J = 1.7 Hz, H-5), 6.75 (1H, d, J = 1.7 Hz,
H-7), 7.03 (1H, d, J = 8.6 Hz, H-50), 7.37 (1H, dd, J = 2.1, 8.6 Hz,
H-60), 7.39 (1H, d, J = 2.1 Hz, H-20); IR mmax (CH2Cl2) cmꢁ1: 1512,
1606, 1727, 3054, 3058, 3686.
2.6. Catalytic reduction of 2b
A solution of compound 2b (8.0 mg) in EtOH (2 mL) was hydro-
genated over Pd/C catalyst (10 wt% loading) under hydrogen at
atmospheric pressure and under reflux for 1 h. After removal of
the catalyst by filtration, the filtrate was concentrated under re-
duced pressure, and the residue was purified by p-TLC (1% MeOH
in CH2Cl2) to give 4 (3.8 mg, 47.2%). HREIMS: m/z 374.1360 [M]+
(calcd. for C20H22O7, 374.1366); 1H NMR (500 MHz, CDCl3): d
3.24 (3H, s, OMe), 3.77 (3H, s, OMe), 3.81 (3H, s, OMe), 3.87 (3H,
s, OMe), 3.88 (3H, s, OMe), 4.41 (1H, d, J = 9.7 Hz, H-3), 5.93 (1H,
d, J = 9.7 Hz, H-2), 6.07 (1H, d, J = 2.3 Hz H-5), 6.20 (1H, d,
J = 2.3 Hz, H-7), 6.84 (1H, d, J = 9.2 Hz, H-50), 6.92–6.93 (2H, m, H-
20and H-60); 13C NMR (125 MHz, CDCl3): d 51.7 (C3 & OMe), 55.5
(OMe), 55.6 (OMe), 55.9 (OMe), 56.0 (OMe), 87.0 (C2), 88.5 (C7),
91.8 (C5), 105.0 (C9), 109.5 (C20), 110.6 (C50), 119.1 (C60), 129.2
(C10), 148.7 (C30 or 40), 148.9 (C30 or 40), 157.0 (C4), 162.3 (C8),
162.8 (C6), 170.7 (C10); IR mmax (CH2Cl2) cmꢁ1: 1608, 1737, 3055,
3061, 3686.
2.2. Materials
Cyanidin 3-O-glucoside 1 was isolated from a commercial black
bean extract and recrystallised using a HCl–MeOH mixture (Lee
et al., 2009; Valls, Millán, Martí, Borràs, & Arola, 2009). 2,20-Azo-
bis-(2,4-dimethyl)valeronitrile (AMVN) was purchased from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan). Black rice samples
were obtained from the Gifu Field Science Center at Gifu
University.
2.3. Reaction of Cyanidin 3-O-glucoside 1 with AMVN in EtOH
Compound 1 (50.0 mg) and AMVN (25.0 mg, 1 equiv.) were dis-
solved in 40 mL of EtOH, and the mixture was stirred at 60 °C for
4 h. The reaction mixture was purified by HPLC (column: NB-
ODS-9) with CH3CN/H2O (20:80 v/v) containing 0.5% trifluoroacetic
acid (TFA) to obtain compound 2 (13.5 mg, 28.2%), protocatechuic
acid ethyl ester (3b; 6.2 mg, 33.0%), and compound 2a (5.0 mg,
9.6%). 2-(3,4-Dihydroxyphenyl)-4,6-dihydroxybenzofuran-3-car-
boxylic acid glucose ester 2: 1H NMR (600 MHz, CD3OD): d 3.26
(1H, t, J = 8.9 Hz, H-200), 3.41 (1H, t, J = 8.9 Hz, H-400), 3.41–3.45
(1H, m, H-500), 3.45 (1H, t, J = 8.9 Hz, H-300), 3.75 (1H, dd, J = 4.5,
12.0 Hz, H-600), 3.87 (1H, dd, J = 2.4, 12.0 Hz, H-600), 5.81 (1H, d,
J = 8.2 Hz, H-100), 6.25 (1H, d, J = 2.1 Hz, H-5), 6.46 (1H, d,
J = 2.1 Hz, H-7), 6.87 (1H, d, J = 8.3 Hz, H-50), 7.27 (1H, dd, J = 2.0,
8.3 Hz, H-60), 7.33 (1H, d, J = 2.0 Hz, H-20); 13C NMR (150 MHz, CD3-
OD): d 61.9 (C600), 70.4 (C400), 73.4 (C200), 78.0 (C300), 78.5 (C500), 90.4
(C7), 96.5 (C100), 100.0 (C5), 107.1 (C3), 108.1 (C9), 115.6 (C50),
118.4 (C20), 122.1 (C10), 123.7 (C60), 145.3 (C30), 148.8 (C40), 152.4
(C4), 157.1 (C8), 158.7 (C6), 162.4 (C2), 167.8 (C10); UV kmax
2.7. LiAlH4 reduction of 4
A solution of compound 4 (3.8 mg) in Et2O (anhydrous, 1.5 mL)
was added to a solution of LiAlH4 (6.1 mg) in Et2O (anhydrous,
0.5 mL), and the mixture was stirred at room temperature for
10 min. The reaction was then quenched by the dropwise addition
of EtOAc and the mixture was extracted with 1 M HCl aq./CH2Cl2.
The organic layer was collected and concentrated under vacuum.
The residue was purified by p-TLC (40% EtOAc in hexane) to give
compound 5: HREIMS: m/z 346.1430 [M]+ (calcd. for C19H22O6,
346.1416); 1H NMR (600 MHz, CD3OD): d 3.37 (1H, dd, J = 5.5,
11.0 Hz, H-10), 3.49 (1H, dd, J = 5.5, 11.0 Hz, H-10), 3.65–3.72
(1H, m, H-3), 3.78 (3H, s, OMe), 3.81 (3H, s, OMe), 3.83 (3H, s,
OMe), 3.85 (3H, s, OMe), 5.77 (1H, d, J = 8.2 Hz, H-2), 6.12 (1H, d,
J = 1.8 Hz, H-5), 6.15 (1H, d, J = 1.8 Hz, H-7), 6.93 (1H, d,
J = 8.3 Hz, H-50), 6.96 (1H, dd, J = 1.4, 8.3 Hz, H-60), 7.03 (1H, brs,
H-20); 13C NMR (150 MHz, CDCl3): d 48.4 (C3), 55.8 (OMe), 56.0
(OMe), 56.4 (OMe), 61.9 (C10), 88.7 (C2), 89.7 (C7), 92.3 (C5),
108.1 (C9), 111.5 (C20), 112.3 (C50), 120.2 (C60), 131.3 (C10), 149.8
(C30 or 40), 149.9 (C30 or 40), 158.5 (C4), 162.9 (C8), 163.4 (C6); IR
mmax (CH2Cl2) cmꢁ1: 1501, 1519, 1602, 3054, 3686.
(MeOH) nm (e): 253 (15,100), 347 (9400).
2.4. Acid hydrolysis of 2
A solution of 2 (5.6 mg) in 2% TFA aq. (1 mL) was stirred at 80 °C
for 1 h. After cooling, the solution was concentrated under reduced

H. Kamiya et al. / Food Chemistry 155 (2014) 221–226
223
2.8. Reaction of 1 with AMVN in aqueous CH3CN
systems in the presented order: 0.5% TFA/H2O; 10% CH3CN/0.5%
TFA/H2O; 15% CH3CN/0.5% TFA/H2O; 20% CH3CN/0.5% TFA/H2O;
30% CH3CN/0.5% TFA/H2O; 0.5% TFA/MeOH. Fraction 2 (38.7 mg)
was further purified by reversed-phase HPLC (15% CH3CN/
0.5%TFA/H2O) to yield 1. Fraction 3 (10.1 mg) was further purified
by reverse-phase HPLC twice (18% CH3CN/0.5% TFA/H2O and 11.5%
CH3CN/0.5% TFA/H2O) to yield 7 and 10. Fraction 4 (22.3 mg) was
further purified by reversed-phase HPLC (15% CH3CN/0.5% TFA/
H2O) to yield 2. The 1H NMR spectra obtained for the isolated
compounds were in agreement with previously reported data. As
a result of quantitative analysis by HPLC, the amount of each com-
pound in black rice per 10 g sample was as follows: 1, 14 mg; 2,
0.07 mg; 7, 0.1 mg; 10, 0.9 mg.
Compound 1 (50.0 mg) and AMVN (25.0 mg, 1 equiv.) were dis-
solved in 20% CH3CN aq. (40 mL), and the mixture was stirred at
60 °C for 6 h. The reaction mixture was purified by HPLC (column:
NB-ODS-9) using CH3CN/H2O (15:85 v/v) containing 0.5% TFA.
Compound 6 (5.4 mg, 15%), protocatechuic acid (3a; 5.3 mg,
34.0%), and 7 (6.8 mg, 13.6%) were obtained. 2-O-(3,4-Dihydroxy-
benzoyl)-2,4,6-trihydroxyphenylacetic acid glucose ester 7: 1H
NMR (600 MHz, CD3OD): d 3.27–3.40 (4H, m, H-200 ꢂ 500), 3.55
(1H, d, J = 16.3 Hz, H-7), 3.61 (1H, d, J = 16.3 Hz, H-7), 3.64 (1H,
brd, H-600), 3.81 (1H, brd, J = 12.3 Hz, H-600), 5.42 (1H, d, J = 8.3 Hz,
H-100), 6.17 (1H, d, J = 2.0 Hz, H-5), 6.27 (1H, d, J = 2.0 Hz, H-3),
6.87 (1H, d, J = 8.3 Hz, H-50), 7.56–7.58 (2H, m, H-20, 60); 13C NMR
(150 MHz, Acetone-d6): d 29.6 (C7), 62.2 (C600), 70.8 (C400), 73.5
(C200), 77.4 (C300), 78.0 (C500), 95.5 (C100), 100.8 (C3), 102.2 (C5),
106.2 (C1), 115.9 (C50), 117.5 (C20), 121.5 (C10), 124.3 (C60), 145.5
(C30), 151.5 (C40), 152.2 (C6), 157.6 (C2), 158.1 (C4), 165.1 (C70),
170.7 (C8).
2.13. Quantitative analysis
Black rice samples (20 grain, approx. 500 mg) were extracted
overnight with 2 mL of 50% MeOH aq. with 1% TFA and then fil-
tered. The residue and filtrate were collected. The residue was
dried at 105 °C for 5 h and weighed. The filtrate was concentrated
under reduced pressure and weighed. This extract was dissolved in
0.2 mL of 50% MeOH aq. in 1% TFA and was used for the quantita-
tive analysis of 1 and 10 by HPLC (15% CH3CN/0.5% TFA/H2O). After
the quantitative analysis of 1 and 10, this sample solution was re-
dissolved in 1 mL of 2% TFA aq. after concentration and extracted
three times with EtOAc. The organic layer was concentrated and
dissolved in 0.2 mL of 50% MeOH aq. with 1% TFA. This solution
was used for the quantitative analysis of 2 and 7 by HPLC (15% CH3-
CN/0.5% TFA/H2O).
2.9. Acid hydrolysis of 7
A solution of 7 (6.8 mg) in 2% TFA aq. (1 mL) was stirred at 80 °C
for 1 h. After cooling, the solution was concentrated under reduced
pressure and purified by HPLC (column: NB-ODS-9) using CH3CN/
H2O (15:85 v/v) containing 0.5% TFA to obtain 7a (4.4 mg, 98%).
The 1H NMR data of 7a were identical to that described in the lit-
erature (Ryu, Park, & Ho, 1998).
2.10. Methylation of 7a
A solution of diazomethane (excess) in Et2O was added to a
solution of 7a (3.5 mg) in MeOH (0.5 mL) and kept at room temper-
ature for 1.5 h. The reaction mixture was concentrated under re-
duced pressure and then purified by p-TLC (2% MeOH in CH2Cl2)
to give 7b (2.6 mg, 61.4%). HRESIMS: m/z 391.1383 [M+H]+ (calcd.
for C20H23O8, 391.1393); 1H NMR (600 MHz, CD3OD): d 3.54 (2H, s,
H-7), 3.56 (3H, s, COOMe), 3.80 (3H, s, OMe), 3.84 (3H, s, OMe), 3.90
(3H, s, OMe), 3.94 (3H, s, OMe), 6.42 (1H, d, J = 2.4 Hz, H-5), 6.49
(1H, d, J = 2.4 Hz, H-3), 7.08 (1H, d, J = 8.9 Hz, H-50), 7.64 (1H, d,
J = 2.0 Hz, H-20), 7.80 (1H, dd, J = 2.0, 8.9 Hz, H-60); 13C NMR
(150 MHz, CD3OD): d 29.8 (C7), 52.4 (COOMe), 56.0 (OMe), 56.5
(OMe), 56.6 (OMe), 97.2 (C5), 100.8 (C3), 109.6 (C1), 112.0 (C50),
113.6 (C20), 122.5 (C10), 125.6 (C60), 150.2 (C30), 152.0 (C2), 155.4
(C40), 160.3 (C6), 161.5 (C4), 165.8 (C70), 173.7 (C8).
3. Results and discussion
3.1. Radical oxidation of 1 in EtOH
Tsuda et al. previously described the radical oxidation of 1 with
the radical initiator AMVN in 1996 (Tsuda et al., 1996) and re-
ported the isolation of a 2,3-benzofuran derivative after reaction
in aqueous CH3CN; the derivative is formed by elimination of the
B-ring from 1, yielding protocatechuic acid (3,4-dihydroxybenzoic
acid). We conducted the same reaction under the reported condi-
tions and analysed the reaction mixture by HPLC. A similar HPLC
chromatographic pattern was observed; however, the peaks dif-
fered from those observed from the analogous radical oxidation
in EtOH (Fig. 2).
The reaction products were purified using preparative HPLC
(Fig. 2A, P1–P3), and the structures were determined using NMR
techniques and mass spectrometry (MS). We found that the peak
labelled P2 was due to ethyl protocatechuate 3b, which originated
from the cleavage of the B-ring of 1. The 1H NMR spectrum of the
compound corresponding to P1 indicated that the C-ring moiety of
the unknown anthocyanin was altered by the reaction and that the
A- and B-rings, as well as the glucoside unit remained intact.
To confirm the identity of P1, a pentamethoxy derivative was
produced by permethylation using diazomethane, followed by acid
hydrolysis to remove the glucoside unit (Fig. 3). The 1H NMR and IR
spectra of the transformed pentamethoxy P1 derivative indicated
the presence of four phenolic hydroxyl groups and an ester moiety.
Upon catalytic hydrogenation of the permethylated P1 derivative,
the appearance of two additional proton signals was observed
(compound 4); furthermore, alcohol 5 was generated upon reduc-
tion of 4 with LiAlH4. From this information, P1 was identified as 2-
(3,4-dihydroxyphenyl)-4,6-dihydroxybenzofuran-3-carboxylic
acid glucose ester (2) based on its reactivity as described above
(Fig. 3). HRESIMS data showed that the molecular formula of 2
2.11. LiAlH4 reduction of 7b
A solution of 7b (1.8 mg) in Et2O (anhydrous, 0.1 mL) was added
to a solution of LiAlH4 (2 mg) in Et2O (anhydrous, 0.1 mL), and the
mixture was stirred at room temperature for 20 min. The reaction
was then quenched with EtOAc, and the mixture was extracted
with EtOAc/1 M HCl aq. The organic layer was collected and con-
centrated under vacuum. The residue was purified by p-TLC (55%
EtOAc in hexane) to give 8 (0.9 mg) and 9 (0.9 mg). The chemical
structures of 8 and 9 were identified using 1H NMR.
2.12. Isolation of 1, 2, 7, and 10 from black rice
Samples of black rice (10 g) were extracted overnight using
20 mL of 50% MeOH aq. with 1% TFA and then filtered. The sample
was extracted twice. The filtrate was washed with chloroform after
concentration under reduced pressure. The aqueous layer was con-
centrated under reduced pressure and loaded onto a Diaion
HP20SS (Mitsubishi Chemical Corporation) column packed with
0.5% TFA aq. The column was eluted with the following solvent
was
C21H20O12 (calcd. for C21H20O12Na, 487.0852; found,

224
H. Kamiya et al. / Food Chemistry 155 (2014) 221–226
2a, i.e., the aglycone of 2, which was confirmed through 1H NMR
spectral analysis after hydrolysis of 2.
Compound 2c, in which the glucoside unit of 2 is substituted
with a methyl residue, has been previously isolated from black rice
(Oryza sativa L.) and bran (Han, Ryu, & Kang, 2004). To provide fur-
ther confirmation for the structure of 2, 2a was methylated in 2%
H2SO4/MeOH. Compound 2c was obtained and analysed using 1H
NMR, which was identical to that previously published.
The yields of 2, 2a, and 3b from the oxidation of 1 in EtOH were
28%, 10%, and 32%, respectively.
3.2. Radical oxidation of 1 in aqueous CH3CN
Compound 1 was treated with AMVN in aqueous CH3CN using
conditions modified from a previous report (Tsuda et al., 1996),
and the solution was analysed using HPLC. Three new peaks were
formed with an accompanying decrease of 1 (Fig. 2B, P4–P6). The
reaction products were purified using preparative HPLC and the
structures were determined using NMR and MS techniques. The re-
sults showed that the compounds corresponding to the peaks P4
and P5 were 6 and protocatechuic acid 3b, respectively, as previ-
ously reported by Tsuda et al. (1996).
The NMR data obtained indicated that P6 was a novel com-
pound containing the A-, B-rings, and glucoside unit from 1. HRE-
SIMS data showed that the molecular formula of P6 was C21H22O13
(C21H22O13Na: calcd. for C21H22O13Na, 505.0958; found, 505.0949).
The chemical structure of P6 was determined using derivatisation
reactions, i.e., acid hydrolysis, methylation, and reduction (Fig. 3).
The results allowed us to identify P6 as 2-O-(3,4-dihydroxyben-
zoyl)-2,4,6-trihydroxyphenylacetic acid glucose ester 7, generated
by the cleavage of the C2–C3 bond in compound 1. Compound
7a, the acid hydrolysate of 7, was previously isolated from the pet-
als of Papaver rhoeas and the fruit of Jaboticaba (Myrciaria caulifl-
ora); the spectral data for 7a were in agreement with published
data (Hillenbrand, Zapp, & Becker, 2004; Reynertson et al., 2006).
The yields of 6, 3a, and 7 from the oxidation of 1 in aqueous
CH3CN were 15%, 34%, and 14%, respectively.
Fig. 2. HPLC chromatogram of products from the reaction of
1 with AMVN
conducted in (A) EtOH, and (B) CH3CN/H2O. The reversed-phase HPLC system was
operated under the following conditions: column, COSMOSIL 5C18-MS-II, 4.6 mm
ID ꢀ 150 mm; solvent, 20% CH3CN in a 0.5% aqueous TFA solution; flow rate, 1 mL/
min; temp., 35 °C; detector, UV (254 nm).
3.3. Proposed radical oxidation mechanism of 1
Our experiments indicated that the radical oxidation of 1 with
AMVN produced different products in the two solvent systems
examined; i.e., 2, 2a, and 3b were produced in EtOH, and 3a, 6,
and 7 were produced in aqueous CH3CN (Fig. 4). To explain these
results, we propose mechanisms for the formation of these com-
pounds in each solvent, as shown in Fig. 5.
487.0880), which is in good agreement with the structure obtained
from the 1H NMR spectrum. The 1H NMR spectrum of P3 was very
similar to that of 2, except for the absence of a characteristic glyco-
side peak at approximately 3–4 ppm, suggesting that P3 was in fact
3'OR
OMe
OMe
OMe
OMe
7
2'
6
8
9
H
RO
MeO
MeO
LiAlH
4
H
O
O 2
3
O2
4' OR
H
2
5
6'
10 OR'5'
3
4
H
OMe
Pd / C
H
OR
MeO
OMe
OH
O
O
5
4
R=H, R'=Glc 2
R=R'=H 2a
R=R'=Me 2b
Hydrolysis
Methylation
R=H, R'=Me 2c
OMe
OMe
OH
OMe
3'
2'
4O' H
OMe
HO
3
CH N
7'
LiAlH
4
2
2
5'
4
2 O
HO
O
MeO
6'
8
O
1
O
5
O
7
O
6
MeO
OH
8 OR'
OH
OMe
OMe
OH
OMe
R'=Glu 7
R'=H 7a
7b
9
Hydrolysis
Fig. 3. Transformation of 2 and 7.

H. Kamiya et al. / Food Chemistry 155 (2014) 221–226
225
OH
OH
OH
OH
HO
O
O
+
EtO
OR
O
OH
AMVN
EtOH
OH
3b: 32%
2: R=Glu 28%
2a: R=H 10%
OH
+
HO
O
AMVN
MeCN / H O
OH
OGlc
OH
OH
OH
HO
O
O
H
OGlc
2
OH
O
1
HO
+
+
HO
O
O
OH
O
OH
OGlc
7:14%
6: 15%
3a: 34%
Fig. 4. Chemical structures of the radical oxidation products of 1.
It is known that anthocyanins exist in different chemical forms
in solution (Castañeda-Ovando et al., 2009). An equilibrium exists
between the flavylium ion I, anhydrobase II, and carbinol pseudo-
base III because of reversible hydration at the 2-position; thus, all
three anthocyanin forms can coexist in solution. Therefore, we sup-
pose that AMVN oxidation occurs on each of the three forms of
anthocyanin 1.
In the AMVN-mediated oxidation of III in aqueous CH3CN, the
species undergoing oxidation is chalcone IV, a ring-opened species
existing under aqueous conditions. The position in chalcone IV cor-
responding to the 4-position of 1 is oxidised, resulting in formation
of 3a and 6. In addition, oxidation of tautomeric species II occurs at
the position corresponding to the 3-position in 1, resulting in for-
mation of intermediate product V. In the presence of water, V is
subsequently transformed to 7 by hydration at the 2-position
and successive ring opening.
reaction does not occur. Therefore, because of the equilibrium
reaction described above, III is converted to I or II. Oxidation of
II in EtOH with AMVN generates V by oxidation at the 3-position,
in a manner similar to that in aqueous CH3CN, and is then con-
verted to VI by the nucleophilic attack of EtOH. Compound VI is
converted to 2 by a rearrangement reaction accompanied by ring
contraction. In EtOH, because the oxidation product derived from
III is not produced, compound 2 and its aglycone 2a are the main
oxidation products.
3.4. Isolation of 1 and its oxidised derivatives from black rice
Black rice (Oryza Sativa L. indica) is a variety of rice with dark
purple colouration primarily cultivated in Asia. Anthocyanins are
thought to play an important antioxidative role in black rice during
its storage; thus, it is likely that black rice contains an anthocyanin
radical oxidant as above. Early studies have shown compound 1 as
a major anthocyanin component in black rice (Abdel-Aal, Young, &
Rabalski, 2006; Kim et al., 2008; Ryu et al., 1998). Accordingly, we
In the AMVN-mediated oxidation of 1 in EtOH, III can be formed
by the nucleophilic attack of EtOH; however, oxidation to a com-
pound analogous to 6 does not proceed because the C-ring opening
OH
OH
EtO
O
OH
R
OH
3b
OH
H
O
O
H
O
OH
OR
O
OH
in EtOH
HO
O
O
O
OH
ROH
O
O O
O
OGlc
OGlc
OGlc
OH
O
O
OH
O
OH
OH
OH
O
OGlc
OH
OH
O
V
VI
O
II
2
in H O
2
OH
OH
HO
OH
O
+
O
HO
O
OGlc
OH
OGlc
7
OH
OH
I
OH
HO
O
ROH
3a
OH
H
O
HO
OH
OH
H
O
O
OH
OGlc
HO
RO
in H O
2
HO
O
O
OH
OGlc
OH
6
OGlc
O
OH
III
O
IV
Fig. 5. Hypothetical oxidation mechanism for 1.

226
H. Kamiya et al. / Food Chemistry 155 (2014) 221–226
Table 1
Indeed, the amount of compound 7 increased during prolonged
storage periods. This result suggests the possibility that the
amount of 7 detected is a suitable criterion for the determination
of the freshness of black rice.
The amounts of 1, 7, and 10 in black rice at different storage periods.
Storage period
Content (mg/g)a
Ratiob
1
10
7
1
10
7
d
d
20 daysc
0 years
1 years
2 years
3 years
8.25
1.70
1.74
0.97
1.29
0.44
0.07
0.07
0.03
0.05
–
9.5
9.4
9.1
8.5
8.4
0.5
0.4
0.4
0.3
0.3
–
Acknowledgments
0.01
0.03
0.05
0.08
0.03
0.2
0.4
We thank Dr. Yoshihiro Miwa, professor emeritus at Gifu
University, for providing the black rice samples. This work was
partially supported by a C19 Kiyomi Yoshizaki Research Grant.
0.5
a
b
c
Total weight of samples were calculated from residue and extract weights.
Content ratio of compound 1, 10, and 7 in sample.
20 days before harvest.
References
d
Not detected.
Abdel-Aal, E. S. M., Young, J. C., & Rabalski, I. (2006). Anthocyanin composition in
black blue, pink, purple, and red cereal grains. Journal of Agricultural and Food
Chemistry, 54, 4696–4704.
Castañeda-Ovando, A., Pacheco-Hernández, M. L., Páez-Hernández, M. E., Rodríguez,
J. A., & Galán-Vidal, C. A. (2009). Chemical studies of anthocyanins: A review.
Food Chemistry, 113, 859–871.
investigated the isolation of 1 and its oxidised derivatives from a
black rice sample subjected to long-term storage and performed
a quantitative analysis of the isolated materials.
Es-Safi, N. E., Meudec, E., Bouchut, C., Fulcrand, H., Ducrot, P. H., Herbette, G., et al.
(2008). New compounds obtained by evolution and oxidation of malvidin 3-O-
glucoside in ethanolic medium. Journal of Agricultural and Food Chemistry, 56,
4584–4591.
Han, S. J., Ryu, S. N., & Kang, S. S. (2004). A new 2-arylbenzofuran with antioxidant
activity from the black colored rice (Oryza satuca L.) bran. Chemical and
Pharmaceutical Bulletin, 52, 1365–1366.
Harborne, J. B. (1994). The flavonoids: Advances in research since 1986. Chapman &
Hall.
Harborne, J. B., & Williams, C. A. (1995). Anthocyanins and other flavonoids. Natural
Product Reports, 639–657.
Hillenbrand, M., Zapp, J., & Becker, H. (2004). Depsides from the petals of Papaver
rhoeas. Planta Medica, 70, 378–380.
Hou, D.-X. (2003). Potential mechanisms of cancer chemoprevention by
anthocyanins. Current Molecular Medicine, 3, 149–159.
Jang, Y. P., Zhou, J., Nakanishi, K., & Sparrow, J. R. (2005). Anthocyanins protect
against A2E photooxidation and membrane permeabilization in retinal pigment
epithelial cells. Photochemistry and Photobiology, 81, 529–536.
Jurd, L. (1964). Anthocyanins and related compounds. III. Oxidation of substituted
flavylium salts to 2-phenylbenzofurans. Journal of Organic Chemistry, 29,
2602–2605.
Jurd, L. (1972). Recent progress in the chemistry of flavylium salts. Recent Advances
in Phytochemistry, 5, 135–164.
Karrer, P., & Widmer, R. (1927). Plant coloring matters. V. Colors of primrose.
Helvetica Chimica Acta, 10, 758–763.
Karrer, P., Widmer, R., Helfenstein, A., Hurliman, W., Nievergelt, O., & Monsasrrate-
Thomas, P. (1927). Plant coloring matters. IV. Anthocyans and anthocyanidins.
Helvetica Chimica Acta, 10, 729–757.
Katsube, N., Iwashita, K., Tsushida, T., Yamaki, K., & Kobori, M. (2003). Induction of
apoptosis in cancer cells by bilberry (Vaccinium myrtillus) and the anthocyanins.
Journal of Agricultural and Food Chemistry, 51, 68–75.
A black rice sample that had been stored for one year in the dark
at room temperature was extracted with 50% MeOH aq. with 1%
TFA overnight, and the extract was purified by a Diaion HP20SS
(Mitsubishi Chemical Co.) column and reversed-phase HPLC. Com-
pounds 1 (as the major anthocyanin) and peonidin 3-O-glucoside
(10; minor) were obtained. The amounts of 1 and 10 in black rice
were 0.19% and 0.007%, respectively. In addition, HPLC analysis
of this extract revealed the presence of two peaks having the same
retention time as those of 2 and 7, which were further purified
using preparative HPLC. The 1H NMR spectra of the isolated com-
pounds were in good agreement with the corresponding com-
pounds obtained by the laboratory oxidation of 1.
Quantitative analyses of 2 and 7 were performed on black rice
samples at varying storage periods, i.e., 20 days before harvest, just
after harvest and after 1–3 years of storage; the results are shown
in Table 1. Because growth conditions and water content differed
with each sample, data are shown as the ratio of each component
relative to the total dry weight. Compound 2 was not detected for
all conditions examined due to its low concentration. In contrast, 7
was detected after harvest, although it could not be detected
before harvest. The data suggested that 7 increased with concom-
itant decrease in anthocyanin compounds (1, 10) with prolonged
storage.
Kim, M. K., Kim, H., Koh, K., Kim, H. S., Lee, Y. S., & Kim, Y. H. (2008). Identification
and quantification of anthocyanin pigments in colored rice. Nutrition Research
and Practice, 2, 46–49.
4. Conclusions
Lee, J. H., Kang, N. S., Shin, S. O., Shin, S. H., Lim, S. G., Suh, D. Y., et al. (2009).
Chemical studies of anthocyanins: A review. Food Chemistry, 112, 226–231.
Lule, S. U., & Xia, W. (2005). Food phenolics, pros and cons: A review. Food Reviews
International, 21, 367–388.
Matsumoto, H., & Iida, H. (2007). The improvement of refractive function by oral
intake of blackcurrant anthocyanins and its action mechanism. Japanese
Pharmacology & Therapeutics, 35, 447–455.
Nichenametla, S. N., Taruscio, T. G., Barney, D. L., & Exon, J. H. (2006). A review of the
effects and mechanisms of polyphenolics in cancer. Critical Reviews in Food
Science and Nutrition, 46, 161–183.
Reynertson, K. A., Wallace, A. M., Adachi, S., Gil, R. R., Yang, H., Basil, M. J., et al.
(2006). Bioactive depsides and anthocyanins from jaboticaba (Myrciaria
cauliflora). Journal of Natural Products, 69, 1228–1230.
In conclusion, two novel oxidation products, compounds 2 (2-
(3,4-dihydroxyphenyl)-4,6-dihydroxybenzofuran-3-carboxylic
acid glucose ester) and 7 (2-O-(3,4-dihydroxybenzoyl)-2,4,6-tri-
hydroxyphenylacetic acid glucose ester), were isolated from the
AMVN-mediated oxidation reaction of 1, with the discovery that
different oxidation products were generated in different solvents.
Based on these products, a radical reaction mechanism is proposed
(cf. Fig. 5). Under the oxidation conditions of the proposed mecha-
nism, 1 was transformed to the corresponding oxidised products
with a total yield of around 70%, as shown in Fig. 5. This result indi-
cates that our proposed oxidative mechanism is a preferred route
for the radical degradation of anthocyanin. Investigations are cur-
rently underway for complete identification of oxidation products
of other anthocyanin derivatives (e.g., delphinidin glycoside) under
similar oxidative conditions, in order to provide further insights
into this radical oxidation route. In addition, the presence of these
oxidised compounds in black rice after prolonged periods of stor-
age under dark conditions indicates a high probability of this rad-
ical reaction occurring in food products by a similar mechanism.
Ryu, S. N., Park, S. Z., & Ho, C. T. (1998). High performance liquid chromatographic
determination of anthocyanin pigments in some varieties of black rice. Journal
of Food and Drug Analysis, 6, 729–736.
Tsuda, T., Ohshima, K., Kawakishi, S., & Osawa, T. (1996). Oxidation products of
cyanicin 3-O-b-
Tsuda, T., Watanabe, M., Ohshima, K., Norinobu, S., Choi, S. W., Kawakishi, S., et al.
(1994). Antioxidative activity of the anthocyanin pigments cyanidin 3-O-b-
D-glucoside with a free radical initiator. Lipids, 31, 1259–1263.
D-
glucoside and cyanidin. Journal of Agricultural and Food Chemistry, 42,
2407–2410.
Valls, J., Millán, S., Martí, M. P., Borràs, E., & Arola, L. (2009). Advanced separation
methods of food anthocyanins, isoflavones and flavanols. Journal of
Chromatography A, 1216, 7143–7172.