Food Chemistry 119 (2010) 1300–1306

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Antioxidative properties and flavonoid composition of Chenopodium quinoa

seeds cultivated in Japan
Yuko Hirose a,*, Tomoyuki Fujita b, Toshiyuki Ishii c, Naoya Ueno c
a
Faculty of Education and Human Sciences, University of Yamanashi, Takeda 4-4-37, Kofu, Yamanashi 400-8510, Japan
b
Graduate School of Agriculture, Shinshu University, Minami-minowa 8304, Kami-ina, Nagano 399-4598, Japan
c
Yamanashi Prefectural Agritechnology Center, Shimoimai 1100, Kai, Yamanashi 400-0104, Japan

a r t i c l e i n f o a b s t r a c t

Article history: To evaluate the nutritional advantages of quinoa seeds (Chenopodium quinoa Willd.) cultivated in Japan,
Received 27 February 2009 antioxidative properties and flavonoid composition were determined and compared to corresponding
Received in revised form 31 August 2009 data for conventionally-used cereals and pseudo-cereals, including quinoa seeds from South America.
Accepted 2 September 2009
The antioxidant activities of these grains against DPPH radicals were strongly associated with the total
phenolic content of the tested samples. The crude extracts of quinoa seeds cultivated in Japan exhibited
higher antioxidative effects than those from South America and other cereals, excluding buckwheat. Four
Keywords:
flavonol glycosides were isolated and identified from the Japanese quinoa seeds, and the chemical com-
Chenopodium quinoa Willd.
Flavonol glycoside
position of the flavonoids – quercetin and kaempferol 3-O-(200 ,600 -di-O-a-rhamnopyranosyl)-b-galacto-
Quercetin pyranosides (1 and 4), quercetin 3-O-(200 ,600 -di-O-a-rhamnopyranosyl)-b-glucopyranoside (2), and
Kaempferol quercetin 3-O-(200 -O-b-apiofuranosyl-600 -O-a-rhamnopyranosyl)-b-galactopyranoside (3) – was evaluated
Acidic hydrolysis through quantitative determination. Trioside 2 was isolated for the first time from quinoa seeds. These
Total phenolic content glycosides were not detected in extracts from any of the tested grains except quinoa. The aglycone quer-
DPPH cetin content of the Japanese quinoa seeds is higher than in the seeds from South America and buck-
Functional foodstuff wheat. The amounts of quercetin and kaempferol formed via acidic hydrolysis in quinoa are much
higher than those of conventionally-used edible plants. The quinoa seeds cultivated in Japan are the most
effective functional foodstuff – in terms of being a source of antioxidative and bioactive flavonoids –
among cereals and pseudo-cereals.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction (NASA) (Schlick & Bubenheim, 1993) and has been noted as a
new foodstuff in the world. There has been growing interest in a
Quinoa (Chenopodium quinoa Willd.) is a grain crop of Andean number of countries, especially in Europe, in initiating introduction
origin and the family Chenopodiacea; it is a pseudo-cereal used and research work. Nevertheless, little cultivation has occurred in
principally in the same manner as wheat and rice. Until recently, Japan.
its cultivation was restricted to subsistence farming in some re- Beyond their basic nutritional function of supplying nutrients,
gions of South America (Bhargava, Shukla, & Ohri, 2006). Although foods also have health-promoting and/or disease-preventing prop-
a lesser-known plant, there has been increasing interest in quinoa erties. Polyphenols have beneficial effects on health and are ubiqui-
due to its perceived superior nutritional quality compared to other tous in plant foods. Recent studies have identified flavonoid
grains. Quinoa seeds contain carbohydrates (77.6%), protein conjugates in quinoa seeds harvested in South America: kaempferol
(12.9%), a balanced amino acid spectrum of high lysine and methi- and quercetin oligomeric glycosides (Dini, Tenore, & Dini, 2004;
onine contents, lipids (6.5%), and is rich in dietary fibre (Ando et al., Zhu et al., 2001). Flavonoids, one of the typical polyphenols in veg-
2002). Quinoa seeds are also rich in mineral nutrients (3.0%), and etable, fruits, and tea, can prevent degenerative diseases such as
the K, Ca, Mg, P, and Fe contents are much higher than those of coronary heart disease (Arts & Hollman, 2005), atherosclerosis
conventional cereals (Konishi, Hirano, Tsuboi, & Wada, 2004). In (Kurosawa et al., 2005), cancers (Rice-Evans & Packer, 1998), diabe-
the last few decades, quinoa has been evaluated as a food with tes, and Alzheimer’s disease (Youdim, Shukitt-Hale, & Joseph,
excellent nutritional characteristics by the National Research 2004), through antioxidative action and/or the modulation of sev-
Council and the National Aeronautics and Space Administration eral protein functions. It is important to determine the amount
and composition of flavonoids in edible parts of vegetables, fruits,
* Corresponding author. Tel.: +81 55 220 8182; fax: +81 55 220 8791. and teas. Murota and Terao (2003) reported that flavonoid bioactiv-
E-mail address: [email protected] (Y. Hirose). ity can be attributed to aglycone structures, not sugar moieties.

0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.09.008
 Y. Hirose et al. / Food Chemistry 119 (2010) 1300–1306 1301

Therefore, qualitative and quantitative analyses of aglycone moie- formed on a JASCO 980 series pumping system with a Shimadzu
ties from flavonoid glycosides provide important information on SPD-10A UV–VIS detector.
their bioavailability. No paper has explained the quantitative rela-
tionships between these compounds in quinoa seeds.
In Japan, the traditional Japanese diet has recently given way to 2.3. Preparation of crude extract solutions from quinoa and other
European and American styles; this has led to an increase in life- grains
style diseases such as cancer, coronary heart disease, and diabetes.
In response, there has been a stronger demand for foods that are Grain samples (100 mg) were milled to a powder, using a coffee
healthier and/or have added value. This means that people not only grinder as needed, and extracted with MeOH:H2O (2:1 v/v; 5 ml)
want food that contains fundamental nutrients, but also functional for 60 min at 50 °C. The crude extracts were filtered through a
ingredients that are sufficiently effective at maintaining health. 0.45-lm membrane filter and made up to 10 ml in a volumetric
In the course of our screening program for functional foodstuffs flask with MeOH:H2O (2:1 v/v).
in Japan, we cultivated quinoa and isolated antioxidative ingredi-
ents from the quinoa seeds. No investigation of phenolic com-
2.4. Total phenolic content of crude extract solutions from quinoa and
pounds from quinoa seeds grown in Japan has been reported
other grains
prior to our own work, although there has been a recent article
published on the antioxidant potency of various extracts and frac-
The total phenolic contents of crude extract solutions were
tions of quinoa seeds produced in Japan (Nsimba, Kikuzaki, & Koni-
determined according to Folin–Ciocalteu’s method (Singleton &
shi, 2008).
Rossi, 1965). Three millilitres of water and 1 ml of crude grain ex-
The aim of this study is to evaluate the nutritional advantages of
tract solution were mixed with 1 ml of 5-fold diluted Folin–Ciocal-
quinoa seeds cultivated in Japan. The usefulness of quinoa as a
teu’s reagent and 1 ml of 10% sodium carbonate. The mixture was
foodstuff was evaluated by structural and quantitative determina-
allowed to stand for 1 h at room temperature and the absorbance
tion of flavonol glycosides, the content of aglycones formed via
was measured at 760 nm against a blank. The total phenolic con-
acidic hydrolysis, and radical-scavenging activity; these were com-
tent in the extract is expressed as mg equivalents of gallic acid/
pared to corresponding data for conventional cereals and pseudo-
100 g fresh weight (FW).
cereals.

2.5. Scavenging ability by crude extract solutions from quinoa and

2. Materials and methods other grains on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals

2.1. Plant materials The extract solution of a grain (2 ml) was mixed with MeOH
solution containing DPPH radicals (0.15 mM, 2 ml). The mixture
Quinoa (C. quinoa Willd.) was supplied by Yamanashi Prefec- was shaken vigorously and left to stand for 30 min in the dark be-
tural Agritechnology Center; it was cultivated in five farms in fore the absorbance was measured at 517 nm against a blank. The
Yamanashi, Japan over the period 2005–2008. The quinoa ecotype scavenging activity was calculated using the following equation:
tested in this experiment was the sea-level NL-6 variety. Samples DPPH radical-scavenging ability (%) = [(A  B)/A]  100.
1, 2, 4, and 6 were harvested during summer, while samples 3 A and B are the control and sample absorbances at 517 nm of
and 5 were harvested during winter. After harvest, the seeds were the reaction mixture, respectively. The scavenging ability (%) of
dried in a room and slightly polished by a rice-cleaning machine. Trolox was plotted against the concentration. The antioxidant
Amaranthus seeds (Amaranthus spp.) of the new Asteka variety activity of the grain is defined as the concentration (lmol) of Trol-
were provided by the National Agriculture and Food Research ox with the antioxidant equivalent to 100 g of fresh weight (FW).
Organization (NARO). The other cereal and pseudo-cereal crops
were purchased at a local market in Kofu City (Yamanashi, Japan).
Cereals: brown rice, proso millet, foxtail millet, Japanese barnyard 2.6. Isolation of flavonol glycosides from quinoa seeds harvested in
millet, wholewheat flour, barley (pressed grain), oatmeal, ryeflour, Japan
Job’s tears, and cornflour. Pseudo-cereals: four samples of quinoa
seeds produced in South America (three in Bolivia and one in Peru), The quinoa seeds (1.0 kg), harvested from Yamanashi in 2005,
two samples of buckwheat flour, buckwheat seed, and two samples were powdered and extracted with MeOH:H2O (10 L, 2:1 v/v).
of amaranthus. These varieties were unknown. The extracts were evaporated in vacuo and partitioned between
EtOAc and H2O. The water layer was evaporated under reduced
pressure for the removal of EtOAc and absorbed on an ODS column
2.2. General methods (20  250 mm). After washing with water, the column was eluted
with MeOH:H2O (35:65 v/v) and 100% MeOH to give fractions 1
Quercetin, morin and 1,1-diphenyl-2-picrylhydrazyl (DPPH) (6.17 g), and 2 (11.52 g). Fraction 1 was loaded repeatedly onto a
were purchased from Sigma–Aldrich (St. Louis, MO); kaempferol preparative ODS column (20  250 mm, YMC-Pack ODS-A) using
and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid 1% aq. AcOH:MeOH (7:3 and 6:4 v/v) as the mobile phase, to yield
(Trolox) were purchased from Extrasynthese (Genay, France); Fo- compounds 1 (556 mg), 2 (113 mg), 3 (388 mg), and 4 (302 mg).
lin–Ciocalteu’s reagent, L-ascorbic acid, and rutin trihydrate were
purchased from Kanto Chemicals Co. (Tokyo, Japan). All other
chemicals used were of analytical reagent grade. 2.7. Quercetin 3-O-(200 ,600 -di-O-a-rhamnopyranosyl)-b-
UV spectra were recorded on a UV-1700 Shimadzu (Kyoto, Ja- galactopyranoside (1)
pan) spectrophotometer. NMR spectra were run on a Bruker
AVANCE-400 spectrometer (1H at 400 MHz; 13C at 100 MHz) with Yellow amorphous powder. FAB-MS (neg.) m/z: 755 [MH],
TMS as the internal standard. The MS data were recorded on a Jeol 301 [MHtriose moiety]. UV (MeOH) kmax nm (loge): 356
JMS 700 mass spectrometer. Elemental analysis was performed (4.27), 256 (4.36). Found: C, 47.22; H, 5.55%. Calcd. for
using a Flash EA 1112 CHNS analyser. HPLC separations were per- C33H40O204.5 H2O: C, 47.31; H, 5.90%.
1302 Y. Hirose et al. / Food Chemistry 119 (2010) 1300–1306

2.8. Quercetin 3-O-(200 ,600 -di-O-a-rhamnopyranosyl)-b- antiviral activities. These activities may be partially related to their
glucopyranoside (2) antioxidant activity, especially their free radical-scavenging ability.
DPPH is a relatively stable free radical used extensively in evaluat-
Yellow amorphous powder. FAB-MS (neg.) m/z: 755 [MH], ing the antioxidant activity of natural products; antioxidant activ-
301 [MHtriose moiety]. UV (MeOH) kmax nm (loge): 354 ity is largely attributable to the amount of phenolic compounds.
(4.25), 256 (4.35). Found: C, 47.28; H, 5.75%. Calcd. for Fifteen samples of quinoa seeds cultivated in Japan, with differ-
C33H40O204.5 H2O: C, 47.31; H, 5.90%. ences in terms of the harvest season and farms, were extracted
with MeOH:H2O (2:1 v/v); the crude extracts were used to deter-
2.9. Quercetin 3-O-(200 -O-b-apiofuranosyl-600 -O-a-rhamnopyranosyl)- mine their DPPH radical-scavenging abilities. The range in scav-
b-galactopyranoside (3) enging ability was from 502 ± 6 to 950 ± 20 lmol equivalent of
Trolox/100 g FW.
Yellow amorphous powder. FAB-MS (neg.) m/z: 741 [MH],
301 [MHtriose moiety]. UV (MeOH) kmax nm (loge): 357
3.2. Antioxidative properties of quinoa seeds compared with other
(4.25), 256 (4.33). Found: C, 47.52; H, 5.34%. Calcd. for
grains
C32H38O203.5 H2O: C, 47.71; H, 5.63%.

To evaluate the antioxidative activity of quinoa seeds cultivated

2.10. Kaempferol 3-O-(200 ,600 -di-O-a-rhamnopyranosyl)-b-
in Japan, the DPPH radical-scavenging ability and total phenolic
galactopyranoside (4)
content were compared to corresponding data for 10 conventional
cereals – namely, brown rice, proso millet, foxtail millet, Japanese
Yellow amorphous powder. FAB-MS (neg.) m/z: 739 [MH],
barnyard millet, wholewheat flour, barley (pressed grain), oatmeal,
285 [MHtriose moiety]. UV (MeOH) kmax nm (loge): 349
ryeflour, Job’s tears, and cornflour – and three pseudo-cereals –
(4.21), 266 (4.29). Found: C, 48.14; H, 5.65%. Calcd. for
namely, four samples of quinoa seeds produced in South America,
C33H40O194.5 H2O: C, 48.24; H, 6.01%.
three samples of buckwheat, and three samples of amaranthus. As
demonstrated in Fig. 1, there is a good correlation between the to-
2.11. Quantitative analysis of flavonol glycosides in quinoa and other
tal phenolic contents and the DPPH radical-scavenging abilities of
grains by HPLC
the tested grains. The antioxidative effect of quinoa seeds from Ja-
pan was higher than those from South America and other grains
According to Section 2.3, the tested grains were extracted with
excluding buckwheat. The results suggest that quinoa seeds culti-
MeOH:H2O (2:1 v/v). The crude extracts were filtered and evapo-
vated in Japan have phenolic compounds responsible for their high
rated to dryness under reduced pressure and suspended in water.
antioxidant property.
The extract solution was passed through InertSep C18 (GL Science),
eluted with 70% MeOH and made up to 5 ml in a volumetric flask. The
flavonol glycosides in the eluate were analysed with HPLC. The HPLC 3.3. Isolation and structural determination of four flavonol glycosides
conditions were as follows: column, ODS (4.6  250 mm, Inertsil from quinoa seeds
ODS-3, GL Science); mobile phase, A (H2O:MeCN:AcOH 90:8:2 v/v/
v) and B (H2O:MeCN:AcOH 80:18:2 v/v/v); flow rate, 1 ml/min; tem- Samples of the quinoa seeds cultivated in Japan were milled and
perature, 40 °C; detection, UV at 370 nm. The applied elution condi- extracted with several solvents such as hexane, EtOAc, EtOH,
tions were: 0–20 min, 25% B isocratic; 20–40 min, linear gradient MeOH, and MeOH:H2O (2:1 v/v). The crude extract with MeOH:
from 25% to 50% B; 40–60 min, linear gradient from 50% to 100% B. H2O (2:1 v/v) exhibited the highest antioxidant potential and phe-
nolic content among the solvent extracts (data not shown). The
2.12. Acid hydrolysis of glycosides in crude extract solution from MeOH:H2O extract of whole flour (1.0 kg) from the quinoa seeds
quinoa and other grains was partitioned by solvent extraction and solid phase extraction.
The repeated preparative HPLC of the crude glycosidic fraction
One millilitre of extract solution, according to Section 2.11, was afforded four flavonol glycosides 1–4. The structures of 1–4 were
transferred into a 25-ml test tube; 1.6 M HCl aq. soln. (2.0 ml),
morin in MeOH (7.2 lg/1.0 ml) as an internal standard, and ascor-
bic acid in MeOH (1.6 mg/500 ll) as an antioxidant were then
1400
added and refluxed for 1 h in boiling water. 6 M NaOH aq. soln.
DPPH Radical-Scavenging Activity

(270 ll) was added and the resulting solution was filtered through 1200
0.45-lm PTFE filters and adjusted with 10 ml of MeOH in a volu-
(Trolox µmol E/100 g FW)

metric flask. The HPLC conditions for aglycone analysis were as fol- 1000
lows: solvent, 0.01 M H3PO4:EtOH (62.5:37.5 v/v); column, ODS
800
(6  150 mm, A-312, YMC); flow rate, 1.0 ml/min; temperature,
40 °C; detection, UV at 370 nm. 600

2.13. Statistics 400

200
The data was processed using Microsoft Office Excel 2002 and is
presented as the mean value ± SD of three determinations. 0
0 100 20 0 30 0 400
Total Phenolic Content
3. Results and discussion ( Gallic acid mg E/100 g FW )

3.1. Antioxidative effect of quinoa seeds cultivated in Japan Fig. 1. Correlation between total phenolic content and DPPH radical-scavenging
activity. Values are means (n = 3). d: quinoa seeds cultivated in Japan, 4: quinoa
seeds produced in South America, s: cereals (brown rice, proso millet, foxtail
Functional foods possess several important biological proper- millet, Japanese barnyard millet, wholewheat flour, barley (pressed grain), oatmeal,
ties, such as antithrombotic, anti-inflammatory, anticancer, and ryeflour, Job’s tears and cornflour), j: buckwheat (flour and seeds), N: amaranthus.
 Y. Hirose et al. / Food Chemistry 119 (2010) 1300–1306 1303

determined by spectroscopic methods including UV, MS and NMR, than that of 1 (3.3 Hz). The large coupling constant (JH100 –
and identified through direct comparison of their physical proper- H200 = 7.7 Hz) indicated a b-anomeric configuration for glucose.
ties with those previously reported. The 1H and 13C NMR spectral These facts supported the fact that the b-galactopyranose moiety
data of 1–4 were assigned based on DEPT, HSQC, and HMBC mea- in 1 was replaced with the b-glucopyranose in 2. In the HMBC
surements (Tables 1 and 2). The compounds 1, 3, and 4 had been spectra of 2, the cross-peak were observed between a carbon at
previously reported as flavonol glycosides from quinoa seeds C-3 (dC 134.5) and an anomeric proton at H-100 (dY 5.59) in glucose;
grown in South America and identified as quercetin 3-O-(200 ,600 - between C-200 (dC 80.1) and H-1000 (dY 5.22) in rhamnose; and be-
di-O-a-rhamnopyranosyl)-b-galactopyranoside (1) (Dini et al., tween C-600 (dC 68.3) and H-10000 (dY 4.50) in another rhamnose.
2004), quercetin 3-O-(200 -O-b-apiofuranosyl-600 -O-a-rhamnopyr- These indicate an O-glycosidic substitution at C-3 and a glycosidic
anosyl)-b-galactopyranoside (3) (Zhu et al., 2001), and kaempferol linkage at C-2 and C-6 of glucose with two rhamnopyranosyl units,
3-O-(200 ,600 -di-O-a-rhamnopyranosyl)-b-galactopyranoside (4) respectively. From these results, the structure of 2 was established
(Dini et al., 2004), which has been named mauritianin (Nishibe as quercetin 3-O-(200 ,600 -di-O-a-rhamnopyranosyl)-b-glucopyrano-
et al., 1996). side (2). Compound 2 was isolated for the first time from quinoa
The mass spectrum of 2 in negative ion mode showed an seeds, though it was isolated from aerial parts of Chenopodium al-
[MH] ion at m/z 755. A C33H40O20 molecular formula was de- bum, another species within this genus (Chludil, Corbino, & Lei-
duced from the MS and NMR data. The 1H and 13C NMR spectra cach, 2008). The structures of 1–4 are depicted in Fig. 2.
of 2 were very similar to those of 1, except for the small shifts in
the saccharide moiety corresponding to the b-galactose of 1. The 3.4. Quantitative analysis of flavonol glycosides in quinoa seeds and
proton signals for the saccharide moiety of 2, except at H-600 a, were other grains by HPLC
shifted 0.07–0.49 ppm upfield against those of 1; the carbon sig-
nals, except at C-100 , were 1.0–3.3 ppm downfield. The coupling The content of flavonol glycosides was determined on six sam-
constant between H-400 and 500 of 2 (JH400 –H500 = 9.4 Hz) was larger ples of quinoa seeds cultivated in Japan (with differences in harvest

Table 1
1
H NMR spectral data for the flavonol glycosides (400 MHz, CD3OD).

1 2 3 4
Aglycone
H-6 6.17 (d, 2.1) 6.18 (d, 2.1) 6.17 (d, 2.0) 6.18 (d, 2.0)
H-8 6.37 (d, 2.1) 6.37 (d, 2.1) 6.37 (d, 2.0) 6.38 (d, 2.0)
H-20 7.69 (d, 2.0) 7.59 (d, 2.2) 7.72 (d, 2.1) 8.06 (d, 8.9)
H-30 6.89 (d, 8.9)
H-50 6.87 (d, 8.5) 6.87 (d, 8.3) 6.86 (d, 8.5) 6.89 (d, 8.9)
H-60 7.55 (dd, 8.5, 2.0) 7.61 (dd, 8.3, 2.2) 7.61 (dd, 8.5, 2.1) 8.06 (d, 8.9)
3-Glucosyl
H-1 5.59 (d, 7.7)
H-2 3.64 (dd, 9.4, 7.7)
H-3 3.55 (t, 9.4)
H-4 3.34 (t, 9.4)
H-5 3.33 (ddd, 9.4, 5.6, 1.5)
H-6a 3.82 (dd, 11.4, 1.5)
H-6b 3.40 (dd, 11.4, 5.6)
3-Galactosyl
H-1 5.69 (d, 7.8) 5.43 (d, 7.7) 5.61 (d, 7.8)
H-2 3.95 (dd, 9.4, 7.8) 3.95 (dd, 9.6, 7.7) 3.93 (dd, 9.5, 7.8)
H-3 3.73 (dd, 9.4, 3.3) 3.71 (dd, 9.6, 4.6) 3.70 (dd, 9.5, 3.7)
H-4 3.81 (t-like, 3.3) 3.80 (t-like, 4.6) 3.77 (t-like, 3.7)
H-5 3.67 (m) 3.65 (m) 3.64 (m)
H-6a 3.75 (ddd, 11.0, 3.3) 3.72 (dd, 10.0, 5.0) 3.72 (dd, 10.1, 5.8)
H-6b 3.47 (dd, 11.0, 6.4) 3.42 (dd, 10.0, 6.6) 3.44 (dd, 10.1, 6.6)
200 -Rhamnosyl
H-1 5.21 (d, 1.4) 5.22 (d, 1.4) 5.21 (d, 1.4)
H-2 3.99 (dd, 3.3, 1.4) 4.00 (dd, 3.3, 1.4) 4.00 (dd, 3.4, 1.4)
H-3 3.78 (dd, 9.5, 3.3) 3.80 (dd, 9.7, 3.3) 3.79 (dd, 9.6, 3.4)
H-4 3.33 (t, 9.5) 3.26 (t, 9.7) 3.34 (t, 9.6)
H-5 4.04 (dd, 9.5, 6.2) 4.08 (dd, 9.7, 6.2) 4.06 (dd, 9.6, 6.2)
H-6 (CH3) 0.94 (d, 6.2) 1.00 (d, 6.2) 0.97 (d, 6.2)
200 -Apiofuranosyl
H-1 5.46 (d, 1.4)
H-2 4.06 (d, 1.4)
H-4a 4.05 (d, 9.6)
H-4b 3.71 (d, 9.6)
H-5a 3.75 (d, 11.6)
H-5b 3.65 (d, 11.6)
600 -Rhamnosyl
H-1 4.55 (d, 1.7) 4.50 (d, 1.4) 4.53 (d, 1.4) 4.52 (d, 1.4)
H-2 3.59 (dd, 3.3, 1.7) 3.59 (dd, 3.4, 1.4) 3.58 (dd, 3.3, 1.4) 3.57 (dd, 3.3, 1.4)
H-3 3.51 (dd, 9.5, 3.3) 3.47 (dd, 9.5, 3.4) 3.51(dd, 9.5, 3.3) 3.50 (dd, 9.6, 3.3)
H-4 3.27 (t, 9.5) 3.23 (t, 9.5) 3.27 (t, 9.5) 3.27 (t, 9.6)
H-5 3.54 (dd, 9.5, 6.2) 3.43 (dd, 9.5, 6.2) 3.53 (dd, 9.5, 6.2) 3.52 (dd, 9.6, 6.1)
H-6 (CH3) 1.18 (d, 6.2) 1.08 (d, 6.2) 1.18 (d, 6.2) 1.18 (d, 6.1)

The values in the parenthesis indicate multiplicity and coupling constants (J Hz).
1304 Y. Hirose et al. / Food Chemistry 119 (2010) 1300–1306

Table 2
13
C NMR spectral data for the flavonol glycosides (100 MHz, CD3OD).
R1
1 2 3 4
OH
Aglycone
C-2 158.4 159.0 158.4 158.7
HO O
C-3 134.6 134.5 135.0 134.5
C-4 179.3 179.3 179.4 179.5
C-5 163.2 163.2 163.1 163.2 OR2
C-6
C-7
99.7
165.6
99.8
165.7
99.8
165.7
99.8
165.7
OH O
C-8 94.6 94.7 94.6 94.7
C-9 158.4 158.5 158.4 158.5
R1 R2
C-10 105.9 105.9 105.8 105.9
C-10 123.3 123.5 123.2 123.1 1: OH α-rhamnopyranosyl-
2,6-di-O-α
C-20 117.4 117.4 117.4 132.3 β-galactopyranosyl
C-30 145.9 145.9 145.9 116.2
2: OH 2,6-di-O-α-rhamnopyranosyl-
C-40 149.7 149.6 149.7 161.3
C-50 116.1 116.2 116.2 116.2 β-glucopyranosyl
C-60 123.0 123.6 123.2 132.3 3: OH 2-O-β-apiofuranosyl-6-O-α-
3-Glucosyl rhamnopyranosyl-β-galactopyranosyl
C-1 100.5 4: H 2,6-di-O-α-rhamnopyranosyl-
C-2 80.1
C-3 79.0
β-galactopyranosyl
C-4 71.9
C-5 77.1 Fig. 2. Structures of flavonol glycosides isolated from quinoa seeds.
C-6 68.3
3-Galactosyl
C-1 101.1 101.6 100.9
C-2 77.4 76.7 77.6
C-3 75.7 75.3 75.8
C-4 70.9 70.7 70.7
C-5 75.3 75.2 75.3
C-6 66.9 67.0 67.1
200 -Rhamnosyl
C-1 102.6 102.7 102.7
C-2 72.4 72.4 72.5
C-3 72.3 72.3 72.3
C-4 74.1 74.1 74.1
C-5 69.9 70.0 69.9
C-6 17.4 17.6 17.6
200 -Apiofuranosyl
C-1 110.7
C-2 78.1
C-3 80.9
C-4 75.5
C-5 66.3
600 -Rhamnosyl
C-1 101.8 102.3 101.8 101.9
C-2 72.1 72.2 72.1 72.1
C-3 72.3 72.3 72.3 72.4
C-4 73.9 73.9 73.9 73.9
C-5 69.7 69.8 69.7 69.7
C-6 18.0 17.9 18.0 18.0

season and farms) in comparison to four quinoa seeds grown in Bo-

livia and Peru. Along with the quinoa seeds, 10 grains of cereals,
three amaranthus samples, and three buckwheat samples were
prepared with MeOH:H2O extraction followed with SPE pretreat-
ment. The MeOH:H2O extracts of quinoa seeds and other grains Fig. 3. HPLC profiles (370 nm) for flavonol glycosides in quinoa extracts. (A) Quinoa
were evaluated by HPLC analysis of 1–4. Typical HPLC chromato- seeds produced in Bolivia and (B) in Japan.

grams of quinoa seed extracts are shown in Fig. 3. The sample of

A was produced in Bolivia and B in Japan.
Compounds 1, 2, 3, and 4 were eluted at 31.5, 33.3, 36.8, and These molecular formulas containing water crystals were deter-
42.7 min, respectively. The recovery with extraction was examined mined based upon elemental analysis results; the flavonol contents
using quercetin 40 -O-glucopyranoside as the internal standard. are expressed as mg anhydrous/100 g of fresh weight (FW). Com-
Quercetin monoglucoside (7.08 lg), isolated from onions, was pounds 1, 2, 3, and 4 were detected in extracts from all of the
added to every extraction and eluted at 65.2 min under these HPLC tested quinoa seeds but were not in the extracts from the other
conditions. The recovery of quercetin monoglucoside was in the grains of cereals and pseudo-cereals. Table 3 shows the flavonol
range 90–99% and is considered to be sufficient for quantitative contents in the quinoa seed.
determination of the flavonols. Compounds 1–4 were isolated from The composition of flavonol glycosides was somewhat different
quinoa seeds as pure chemicals to give linear calibration curves. depending upon the place of production. In the quinoa seeds from
 Y. Hirose et al. / Food Chemistry 119 (2010) 1300–1306 1305

Table 3
Content of flavonol glycosides and their derivative aglycones by acidic hydrolysis from quinoa seeds.

Sample no. Place of Harvesting Content of flavonol Total content of 1–4 Content of aglycones formed
production season glycosides (mg/100 g FW) (mg/100 g FW) via acidic hydrolysis (mg/100 g FW)
1 2 3 4 Quercetin Kaempferol
1 Japan Summer 83.8 ± 0.8 14.0 ± 0.2 47.2 ± 0.3 41.1 ± 0.4 186.1 67.4 ± 0.9 18.4 ± 0.3
2 Japan Summer 69.0 ± 0.5 9.8 ± 0.2 42.5 ± 1.1 34.6 ± 0.8 155.9 55.0 ± 0.3 15.9 ± 0.5
3 Japan Winter 79.5 ± 1.5 15.0 ± 0.3 46.7 ± 0.3 48.8 ± 1.9 190.0 64.9 ± 0.6 20.3 ± 0.7
4 Japan Summer 87.3 ± 2.5 11.2 ± 0.2 51.7 ± 1.2 42.6 ± 1.2 192.8 68.0 ± 2.1 18.0 ± 0.8
5 Japan Winter 51.5 ± 0.4 10.4 ± 0.2 34.4 ± 0.2 33.8 ± 0.2 130.1 45.3 ± 0.9 15.0 ± 0.5
6 Japan Summer 83.9 ± 1.9 9.9 ± 0.3 48.9 ± 1.4 39.3 ± 0.7 182.0 61.0 ± 1.8 16.7 ± 0.2
7 Bolivia – 53.3 ± 0.8 6.9 ± 0.0 36.9 ± 0.3 78.7 ± 1.6 175.8 42.9 ± 1.2 36.6 ± 0.6
8 Bolivia – 24.3 ± 0.7 3.3 ± 0.2 21.5 ± 0.6 113.3 ± 2.9 162.4 22.5 ± 0.7 52.1 ± 0.6
9 Bolivia – 45.4 ± 1.3 5.8 ± 0.2 28.5 ± 0.8 113.3 ± 3.0 193.0 34.7 ± 0.9 50.1 ± 0.9
10 Peru – 42.0 ± 0.5 6.0 ± 0.2 30.1 ± 0.2 93.0 ± 0.3 171.1 34.7 ± 1.2 41.2 ± 1.7

Values expressed as a mean of three determinations ± standard deviation. –: harvest season is unknown.

Japan, the major compound was quercetin glycoside 1. The total 3.5. Acidic hydrolysis of flavonol glycosides in extracts from quinoa
content of quercetin glycosides, namely 1, 2, and 3, was much seeds and other grains
higher than that of kaempferol glycoside 4, while the content of
4 in the seeds from South America was higher than that of querce- Flavonoids are mostly present as glycosides in plants. During
tin glycosides 1–3. In order for comparisons based on mole equiv- intestinal absorption, these glycosides are mostly hydrolysed to
alents, the flavonol glycoside contents were re-expressed as lmol/ their aglycones, followed by conjugation to glucuronides and/or
100 g of FW and are shown graphically in Fig. 4. For the samples sulphates (Murota & Terao, 2003); the bioactivity is attributed to
from Japan, the total content of 1, 2, and 3 was 3.5-fold higher than aglycone structures and not sugar moieties (Bors, Heller, Michel,
that of 4 in samples 1, 2, 4, and 6, which were harvested in sum- & Saran, 1990). Since quercetin and kaempferol are beneficial to
mer, whereas that of 1, 2, and 3 was 2.8-fold higher than 4 in sam- human health, a better understanding of the levels of the two agly-
ples 3 and 5, which were harvested in winter. Since this was cones is needed. In order to determine the content of quercetin and
observed in all tested quinoa seeds from Japan (data for other kaempferol, including their glycosidic form, the crude extracts of
tested seeds is not shown), it was deduced that the sunlight is all tested grain samples were hydrolysed with hydrochloric acid
responsible for the accumulation of quercetin glycosides. Accord- and injected after neutralisation into the HPLC system. The recov-
ing to numerous reports on flavonoid antioxidant activity, querce- ery of morin as an internal standard with hydrolysis was in the
tin glycosides are more active than the corresponding kaempferol range 92–102% and sufficient for quantitative determination of
derivatives. In this study, the crude extracts from the quinoa seeds the two aglycones. After hydrolysis of the extracts from quinoa
cultivated in Japan showed stronger antioxidant activity than those seeds with acid, none of the four flavonol glycosides were detected
from South America; quantitative analysis supports the assump- in the reactant. The aglycones quercetin and kaempferol were
tion that the antioxidant properties of quinoa seeds are correlated measured at up to 2.1 and 2.2 lg, respectively. Quercetin was de-
with the amount of quercetin glycosides. As it has been reported tected in quinoa and buckwheat, whereas kaempferol was found
that some flavonoids accumulate in stressed plants and are related in quinoa. These aglycones were not detected in tested samples
to chemical defence strategies (Chludil et al., 2008), the difference of 10 cereals and amaranthus. The amount of quercetin and
in glycoside concentration between the quinoa seeds from Japan kaempferol formed via hydrolysis from quinoa seeds is shown in
and South America may be explained by differences in genetic Table 3. The content of quercetin in buckwheat flour was approx-
background rather than the environmental conditions. imately 20 mg/100 g FW. The amount of quercetin in quinoa seeds
was much higher than in buckwheat, which was abundant in rutin.
For easy comparison of the amount of the two aglycones formed
by hydrolysis with the flavonol glycosides, the amounts were re-
compound 4 expressed as lmol/100 g of FW and are shown graphically in
250
compound 3 Fig. 5. The amount of quercetin in samples 1–6, which were from
Flavonol glycoside (μmol/100 g FW )

compound 2 Japan, was 150 ± 3 to 225 ± 7 lmol/100 g FW, which is about three
200 compound 1 times higher than that of kaempferol (52.3 ± 1.8 to 71.0 ± 2.5 lmol/
100 g FW). The amount of quercetin in samples 7–10, which were
150 grown in South America, was 74.6 ± 2 to 142 ± 4 lmol/100 g FW,
which is lower than that of kaempferol (128 ± 2 to 182 ± 2 lmol/
100 g FW). The amounts of quercetin in quinoa seeds from Japan
100 were higher than those from South America, whereas the amounts
of kaempferol in the seeds from Japan were much lower than those
50 from South America. These results coincided with the quantitative
analysis of the flavonol glycosides. On average, the total contents of
1, 2, and 3 reached 89.2% of the amount of aglycone quercetin and
0
the contents of 4 reached 87.5% of the amount of aglycone kaempf-
1 2 3 4 5 6 7 8 9 10 erol. Furthermore, Sakakibara, Honda, Nakagawa, Ashida, and
Sample Kanazawa (2003) deduced that quantification by hydrolysis may
Fig. 4. Flavonol glycosides contents of quinoa seeds. 1–6: quinoa cultivated in
produce a loss of content due to the decomposition and polymeri-
Japan; 7–9: quinoa produced in Bolivia; 10: quinoa produced in Peru. Values are sation of flavonols. The results showed that all derivatives related
means (n = 3). to quercetin and kaempferol, except for the four glycosides, could
1306 Y. Hirose et al. / Food Chemistry 119 (2010) 1300–1306

250 quercetin database, several examples of plants rich in quercetin are onions
kaempferol (21.4 mg/100 g), green tea leaves (dry, 662 mg/100 g), capers
(234 mg/100 g), lovage leaves (170 mg/100 g), buckwheat
200
Flavonol (μmol/100 g FW)

(23.1 mg/100 g), etc. The quinoa seeds cultivated in Japan are the
most effective functional foodstuff in terms of being a source of
150 antioxidative and bioactive flavonoids among cereals, pseudo-
cereals, and regularly-consumed plant foods. A study on the effects
on bioactivity by dosing mice with quinoa seeds is currently in
100 progress.

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