Isolation of phenolic constituents and characterization of antioxidant markers from sunflower (Helianthus annuus) seed extract

Article abstract of DOI:10.1016/j.phytol.2013.03.013

A new compound, benzyl alcohol β-d-apiofuranosyl-(1→6)-β-d- (4-O-caffeoyl) glucopyranoside (1), was isolated from the seed of sunflower (Helianthus annuus), together with eight known phenolic compounds: caffeic acid (2), methyl caffeoate (3), chlorogenic acid (4), 4-O-caffeoylquinic acid (5), 3-O-caffeoylquinic acid (6), methyl chlorogenate (7), 3,5-di-O-caffeoylquinic acid (8), and eriodictyol 5-O-β-d-glucoside (9). Their structures were elucidated on the basis of spectroscopic methods and chemical evidence. The antioxidative effect of the phenolic constituents from the sunflower seeds was also evaluated based on the oxygen-radical absorbance capacity (ORAC), and the fraction containing caffeic acid derivatives showed a high antioxidant potency.

Full text of DOI:10.1016/j.phytol.2013.03.013

Phytochemistry Letters 6 (2013) 302–305
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Isolation of phenolic constituents and characterization of antioxidant markers
from sunflower (Helianthus annuus) seed extract
Yoshiaki Amakura *, Morio Yoshimura, Saori Yamakami, Takashi Yoshida
Department of Pharmacognosy, College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
A new compound, benzyl alcohol
b
-
D
-apiofuranosyl-(1!6)-
b-D-(4-O-caffeoyl) glucopyranoside (1), was
Received 26 December 2012
Received in revised form 14 March 2013
Accepted 18 March 2013
Available online 4 April 2013
isolated from the seed of sunflower (Helianthus annuus), together with eight known phenolic
compounds: caffeic acid (2), methyl caffeoate (3), chlorogenic acid (4), 4-O-caffeoylquinic acid (5), 3-O-
caffeoylquinic acid (6), methyl chlorogenate (7), 3,5-di-O-caffeoylquinic acid (8), and eriodictyol 5-O-
b-
D-glucoside (9). Their structures were elucidated on the basis of spectroscopic methods and chemical
evidence. The antioxidative effect of the phenolic constituents from the sunflower seeds was also
evaluated based on the oxygen-radical absorbance capacity (ORAC), and the fraction containing caffeic
acid derivatives showed a high antioxidant potency.
Keywords:
Sunflower seed
Helianthus annuus
Compositae
ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Phenolic constituent
Antioxidant
1
. Introduction
absorbance capacity (ORAC) assay (Ou et al., 2001; Huang et al.,
2002).
Sunflower (Helianthus annuus L.) is an annual plant native to
North America, and one of the most important oilseed crops, being
the second largest oilseed crop as a global source of vegetable oil
2. Results and discussion
(
Canella and Sodini, 1977). Sunflower seeds are widely used in the
A homogenate of sunflower seeds in 80% ethanol (EtOH) was
food and nutraceutical industries because of their high oil and
protein contents and other valuable bioactive components. The
most abundant fatty acids in sunflower oil are linoleic acid (ca.
extracted with n-hexane and ethyl acetate (EtOAc) to give the
respective n-hexane, EtOAc, and water extracts. The antioxidative
activity of each extract was evaluated by ORAC (Fig. 1A). The EtOAc
extract, which exhibited significant antioxidant activity, was
repeatedly chromatographed over MCI-GEL CHP-20P and/or
YMC GEL ODS-AQ with aqueous methanol (MeOH) in a stepwise
gradient mode to afford a new compound (1), together with caffeic
acid (2), methyl caffeoate (3) (Fujioka et al., 1999), chlorogenic acid
6
5
5%), oleic acid (ca. 25%), and palmitic and stearic acids (each ca.
%) (Canella et al., 1982; Economides, 1998; Pereira et al., 2003).
Sunflower oil also contains high levels of tocopherols and
phytosterols (Rashid et al., 2009). On the other hand, extracts
with high antioxidant activity may also be obtained from
sunflower seed shells and kernels (De Leonardis et al., 2003). In
Japan, natural antioxidant ‘‘sunflower seed extract’’ is defined as an
ethanol or hot water extract from the seeds of sunflower, and this
additive is characterized as an antioxidant containing isochloro-
genic and chlorogenic acids (Notice No. 210, 1996). Sunflower
seeds are thus suggested to be rich in polyphenols. This paper
describes the isolation and structural characterization of phenolics
including a new glycoside from the seed of sunflower. The
antioxidant activities of fractions partitioned with solvent and the
isolated phenolics were also estimated by an oxygen radical
(
1
4) (Iwai et al., 2004), methyl chlorogenate (7) (Deyama et al.,
987; Ge et al., 2007), isochlorogenic acid (3,5-di-O-caffeoylquinic
acid (8)) (Dini et al., 2006), and eriodictyol 5-O- -glucoside (9)
Gujer et al., 1986). Similar chromatographic separation of the
b
-D
(
water extract gave 4, 4-O-caffeoylquinic acid (5), and 3-O-
caffeoylquinic acid (6) (Iwai et al., 2004). The known compounds
2–9 were identified, respectively, by direct comparison with
authentic specimens and by comparison of their spectral data with
those reported in the literature.
Compound 1 was isolated as a brown amorphous powder. Its
molecular formula was assigned as C27
H
H
32
O
13 from its HR-ESI-MS
13-H: 563.1765) and
ꢀ
(
m/z 563.1741 [MꢀH] ; calcd. for C27
32
O
1
3
13
*
Corresponding author. Tel.: +81 89 925 7111; fax: +81 89 926 7162.
C-NMR (27 C signals) spectra. The UV spectrum showed
absorption maxima at 207, 248sh, 289, and 330 nm, respectively.
E-mail address: amakura@cc.matsuyama-u.ac.jp (Y. Amakura).
1
874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2013.03.013

Y. Amakura et al. / Phytochemistry Letters 6 (2013) 302–305
303
n-Hexane
(A)
EtOAc
H2O
1
(B)
2
3
4
5
6
7
8
9
Epigallocatechin gallate
(
EGCG)
Ascorbic acid
0
10000
20000
30000
40000
(
µmol TE/g)
Fig. 1. ORAC values of each fraction (A) and isolated compounds 1–9 (B).
1
13
The H- and
C-NMR spectra of
1
exhibited the signals
solvent for the sunflower seed extracts (Fig. 1A). As a result, the
EtOAc fraction exhibited a marked activity which is considered to
be responsible for 4 and 8 as the main component in the extract
(Fig. 3). The antioxidant activity of isolated compounds 1–9 was
also evaluated (Fig. 1B). All of them showed potent antioxidative
characteristic of trans-caffeoyl and benzyl moieties, as follows.
The H NMR spectrum exhibited AB-type proton signals due to a
benzylic methylene group at
and multiplets due to a phenyl group at
those assignable to a caffeoyl group [
d, J = 8.5 Hz, H-5 ), 6.95 (dd, J = 2, 8.5 Hz, H-6 ), 7.05 (d, J = 2 Hz, H-
), and 7.68 (dd, J = 16 Hz, H-7 )]. These aromatic units were also
supported by 16 carbon signals due to the caffeoyl [
1
d 4.68 and 4.91 (each d, J = 12 Hz, H-7)
d
7.25–7.43 (5H) as well as
0
d
6.28 (d, J = 16 Hz, H-8 ), 6.77
activity with ORAC values of ca. 10,000 mmol TE/g or above. It is
0
0
(
2
particularly notable that the potency of 2 was about three times
more potent than that of epigallocatechin gallate (EGCG), a typical
tea catechin. These results suggest that the antioxidant activity of
0
0
d
0
114.8, 115.2,
0
1
16.5, 123.1, 127.7, 146.8, 147.6, 149.8, 168.4 (C-1 -9 )] and benzyl
group [d 71.9, 128.8, 129.3 (4C), 138.9 (C-1–7)] in the C NMR
1
3
spectrum. The presence of two sugar units in 1 was shown by two
anomeric proton signals at 4.40 (d, J = 8 Hz, Glc H-1) and 4.95 (d,
J = 2 Hz, Api H-1) and others assigned by the H– H shift
Table 1
1
13
4
H-(500 MHz) and C-NMR(126 MHz) data of compound 1 measured in MeOH-d .
d
1
1
Position
d
C
d
H
(J in Hz)
correlation spectrum (Table 1). The sugar residues were presumed
to be hexose and branched pentose, as revealed by the C NMR
spectrum exhibiting 11 aliphatic carbon signals which included a
1
2
3
4
5
6
7
138.9
129.3
129.3
128.8
129.3
129.3
71.9
–
1
3
a
7.25-7.43
7.25-7.43
7.25-7.43
7.25-7.43
7.25-7.43
a
a
a
a
quaternary
resonances (
the sugar units in 1 were confirmed by acid hydrolysis followed by
RP-HPLC analysis for derivatives prepared by reaction with
(
d
80.6, Api C-3) and three methylene carbon
d
68.6, 65.7 and 75.1). -Glucose and -apiose as
D
D
4.68 (d, J = 12), 4.91 (d, J = 12)
L-
0
0
0
0
0
0
0
0
0
1
127.7
115.2
146.8
149.8
116.5
123.1
147.6
114.8
168.4
103.2
75.3
–
cysteine methyl ester and O-tolyl isothyocyanate according to the
previously reported method (Tanaka et al., 2007). The linking
position of each unit was determined by cross-peaks among
2
7.05 (d, J = 2)
3
–
4
–
5
6.77 (d, J = 8.5)
6.95 (dd, J = 2, 8.5)
7.68 (d, J = 16)
6.28 (d, J = 16)
–
glucose H-1 (
d
4.40)/C-7 (
168.4) of the caffeoyl group, and apiose H-1 (
glucose C-6 ( 68.6) in HMBC (Fig. 2). -Glycosidic linkage in the
d
71.9) of the benzyl group, glucose H-4 (
d
6
0
4
.87)/C-9 (
d
d
4.95)/
7
8
9
d
b
glucose core was evidenced by large coupling constants (J = 8 Hz).
Glucose (Glc)-1
4.40 (d, J = 8)
The configuration at the anomeric center of the apiose was also
2
3.35 (dd, J = 8, 9.5)
3.60 (t, J = 9.5
1
3
determined as
data for 1 with those for
et al., 1989), and the JH-1
reported data of -apiofuranoside (Liu et al., 2010). Therefore, 1
b
on the basis of the comparison of the C-NMR
- and -apiofuranoside (Kitagawa
H-2 coupling constant consistent with
3
75.8
a
b
-D
4
72.8
4.87 (t, J = 9.5)
3.66 (m)
5
74.9
,
6
68.6
3.53 (dd, J = 6.5, 11), 3.73 (dd, J =2.5, 11)
4.95 (d, J = 2)
b
-D
Apiose (Api)-1
111.1
78.1
was established as benzyl alcohol
O-caffeoyl) glucopyranoside.
b
-D-apiofranosyl-(1!6)-
b-D-(4-
2
3
4
5
3.89 (d, J = 2)
80.6
–
To clarify the relationship between the polarity of the
ingredients and antioxidative activity, the ORAC values were
estimated for each fraction obtained by partitioning with organic
65.7
3.53 (d, J = 9.5), 3.74 (d, J = 9.5)
3.56 (2H, s)
75.1
a
Overlapped signals.

3
04
Y. Amakura et al. / Phytochemistry Letters 6 (2013) 302–305
Fig. 2. Structures of compounds 1–9 and selected HMBC correlations of 1.
sunflower seed extract is largely attributed to these caffeic acid
derivatives.
1240 (Kyoto, Japan). Electrospray ionization (ESI)-MS and high-
resolution (HR) ESI-MS spectra were obtained using a micrOTOF-Q
(
Bruker Daltonics, Billerica, MA, USA) mass spectrometer with
1
13
3
. Experimental
acetonitrile as the solvent. H- and C-NMR spectra were recorded
on a Bruker AVANCE500 instrument (Bruker BioSpin, Billerica, MA,
1
13
3.1. General
USA) (500 MHz for H and 126 MHz for C) and chemical shifts are
given in ppm values relative to those of the solvents [methanol-d
3.30; 49.0)] on a tetramethylsilane scale. The standard pulse
4
Optical rotations were measured with a JASCO P-1020 digital
(
d
H
d
C
polarimeter. UV spectra were recorded on a Shimadzu UVmini-
sequences programmed for the instrument (AVANCE 500) were
used for each 2D measurement (COSY, HSQC, and HMBC). JCH was
set at 8 Hz in HMBC. Column chromatography was carried out with
Diaion HP-20, MCI-gel CHP-20P (Mitsubishi Chemical Co., Tokyo,
Japan), and YMC gel ODS (YMC Co. Ltd., Kyoto, Japan), respectively.
Normal-phase (NP) HPLC conducted on a YMC-Pack SIL A-003
(
YMC Co., Ltd.) column (4.6 mm i.d. ꢁ 150 mm) developed with n-
hexane-MeOH-tetrahydrofuran-formic acid (55:33:11:1) contain-
ing oxalic acid (450 mg/L) (flow rate: 1.5 mL/min; 280 nm UV
detection). Reversed-phase (RP) HPLC conditions were as follows:
[
Condition 1] column, L-column ODS (5
m
m, 150 mm ꢁ 2.1 mm
i.d.) (Chemicals Evaluation and Research Institute, Tokyo, Japan);
mobile phase, solvent A was 5% acetic acid and solvent B was
acetonitrile (0–30 min, 0–50% B in A; 30–35 min, 50–85% B in A;
3
5–40 min, 85–85% B in A); injection volume, 2
temperature, 40 8C; flow-rate, 0.3 mL/min; detection, 200–
00 nm. [Condition 2] column, COSMOSIL Cholester Waters
m, 150 ꢁ 2.0 mm i.d.) (Nacalai Tesque, Kyoto, Japan); mobile
phase, 10 mM H PO -10 mM KH PO -MeOH (37.5:37.5:25); col-
umn temperature, 40 8C; flow-rate, 0.2 mL/min; detection,
80 nm. The microplate reader was an Infinite F200 microplate
reader (TECAN, M a¨ nnedorf, Switzerland).
mL; column
4
(5 m
3
4
2
4
2
3.2. Samples and reagents
Fig. 3. RP-HPLC profiles of sunflower seed extracts. (a) 50% EtOH extract; (b) 50%
EtOH extract-n-hexane fraction; (c) 50%EtOH extract-EtOAc fraction; (d) 50%EtOH
Sunflower seeds (kernels of H. annuus) were obtained from
Nagaoka Perfumery Co., Ltd. (Osaka, Japan). N-Methylmorpholine-
extract-H
2
O fraction. 4, chlorogenic acid; 8, 3,5-di-O-caffeoylquinic acid. HPLC
0
conditions are described in condition 1 of ‘‘Experimental’’.
N-oxide, 2,2 -azobis-(2-amidinopropane) dihydrochloride (AAPH),

Y. Amakura et al. / Phytochemistry Letters 6 (2013) 302–305
305
L
-cysteine methyl ester hydrochloride, and O-tolyl isothiocyanate
to the microplate, and, after shaking for 15 s, the fluorescence was
recorded every minute for 90 min at excitation and emission
wavelengths of 485 and 528 nm, respectively. The area under the
curve (AUC) was calculated, and the net AUC was calculated by
subtracting the AUC of the blank (phosphate buffer only) from
that of each sample. ORAC values are expressed as trolox
were purchased from Wako Pure Chemical Industries (Osaka,
Japan). Fluorescein sodium salt and 6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox) were obtained from
Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of
analytical grade.
equivalents (
mmol TE/g) using the calibration curve generated
3.3. Extraction and isolation
in each assay.
The sunflower seeds (800 g) were homogenized in 50% EtOH
Acknowledgments
[
2
EtOH-H O (1:1)] (6 L) and the homogenate was filtered. The
filtrate was concentrated and extracted with n-hexane (2 L) and
EtOAc (5 L) to give the respective n-hexane (41.6 g), EtOAc (1.0 g),
and water (45.1 g) extracts. The EtOAc extract (1.0 g) was
We thank Ms. Kazusa Ouchi and Ayako Yoshimura of
Matsuyama University for their technical assistance. This work
was supported by a Health Sciences Research Grant from the
Ministry of Health, Labour and Welfare, Japan.
chromatographed over MCI-GEL CHP-20P with MeOH-H
0:100!10:90!20:80!30:70!40:60!100:0) in stepwise gradi-
ent mode. The fractions showing similar HPLC patterns (condition
) were combined and further purified by column chromatography
2
O
(
1
References
over Sephadex LH-20 with EtOH and/or YMC GEL ODS-AQ with
Amakura, Y., Yoshimura, M., Sugimoto, N., Yamazaki, T., Yoshida, T., 2009. Marker
constituents of the natural antioxidant eucalyptus leaf extract for the evalua-
tion of food additives. Biosci. Biotechnol. Biochem. 73, 1060–1065.
Canella, M., Sodini, G., 1977. Extraction of gossypol and oligosaccharides from
oilseed meals. J. Food Sci. 42, 1218–1219.
aqueous MeOH to afford caffeic acid (2) (2.6 mg), methyl caffeoate
(
(
3) (2.3 mg), chlorogenic acid (4) (15.0 mg), methyl chlorogenate
2.3 mg) (7), 3,5-di-O-caffeoylquinic acid (8) (20 mg), eriodictyol
5
-O-
b
-D-glucoside (9) (3.6 mg), and benzyl alcohol
b-D-apiofra-
Canella, M., Bernardi, A., Casalaina, A., Sodini, G., 1982. Albumin and globulin
components of different sunflower cultivars. Riv. Ital. Delle Soct. Grasse 59,
nosyl-(1!6)-
b
-D-(4-O-caffeoyl) glucopyranoside (1) (3.6 mg). The
water extract (2 g) was similarly separated by column chroma-
tography over MCI-GEL CHP-20P with aqueous MeOH to give
chlorogenic acid (4) (15 mg), 3-O-caffeoylquinic acid (6) (2.0 mg),
and 4-O-caffeoylquinic acid (5) (2.5 mg). These compounds were
identified by direct comparison with authentic specimens or by
comparison of their spectral data with those reported in the
literature. The physical data of the new compound 1 are as follows.
377–382.
De Leonardis, A., Macciola, V., Di Rocco, A., 2003. Oxidative stabilization of cold-
pressed sunflower oil using phenolic compound of the same seed. J. Sci. Food
Agric. 83, 523–528.
Deyama, T., Ikawa, T., Kitagawa, S., Nishibe, S., 1987. The constituents of Eucommia
ulmoides OLIV.V. Isolation of dihydroxydehydrodiconiferyl alcohol isomers and
phenolic compounds. Chem. Pharm. Bull. 35, 1785–1789.
Dini, I., Tenore, G.C., Dini, A., 2006. New polyphenol derivative in Ipomoea batatas
tubers and its antioxidant activity. J. Agric. Food Chem. 54, 8733–8737.
Economides, S., 1998. The nutritive value of sunflower meal and its effect on
replacing cereal straw in the diets of lactating ewes and foats. Livestock Prod.
Sci. 55, 89–97.
Fujioka, T., Furumi, K., Fujii, H., Okabe, H., Mihashi, K., Nakano, Y., Matsunaga, H.,
Katano, M., Mori, M., 1999. Antiproliferative constituents from Umbelliferae
plants. V. A new furanocoumarin and falcarindiol furanocoumarin ethers from
the root of Angelica japonica. Chem. Pharm. Bull. 47, 96–100.
3
.3.1. Benzyl alcohol
glucopyranoside (1)
A light brown amorphous powder. UV
07 (4.22), 248sh (3.89), 289 (4.00), 330 (4.12). [a]
D
b
-
D
-apiofranosyl-(1!6)-
b-D-(4-O-caffeoyl)
l
max (MeOH) nm (log
e
):
-548 (c 0.5,
) and C NMR (126 MHz,
) data provided in Table 1. HR-ESI-MS m/z: 563.1741 ([M–
13-H: 583.1770).
23
2
1
13
MeOH). H NMR (500 MHz, MeOH-d
MeOH-d
H] , Calcd. for C27
4
Ge, F., Ke, C., Tang, W., Yang, X., Tang, C., Qin, G., Xu, R., Li, T., Chen, X., Zuo, J., Ye, Y.,
2007. Isolation of chlorogenic acids and their derivatives from Stemona japonica
by preparative HPLC and evaluation of their anti-AIV (H5N1) activity in vitro.
Phytochem. Anal. 18, 213–218.
4
ꢀ
H
32
O
Gujer, R., Magnolato, D., Self, R., 1986. Glucosylated flavonoids and other phenolic
compounds from sorghum. Phytochemistry 25, 1431–1436.
3.4. Determination of sugar configuration
Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J.A., Deemer, E.K., 2002. Devw-
lopment and validation of oxygen radical absorbance capacity assay for lipo-
The sugar configuration was determined using a previously
philic antioxidants using randomly methylate
b-cyclodextrin as the solubility
described method (Tanaka et al., 2007). Compound 1 (1.0 mg) was
hydrolyzed by heating in 0.5 M HCl (0.2 mL) and neutralized with
Amberlite IRA400. After evaporation, the residue was dissolved in
enhancer. J. Agric. Food Chem. 50, 1815–1821.
Iwai, K., Kishimoto, N., Kakino, Y., Mochida, K., Fujita, T., 2004. In vitro antioxidative
effects and tyrosinase inhibitory activities of seven hydroxycinnamoyl deriva-
tives in green coffee beans. J. Agric. Food Chem. 52, 4893–4898.
Kitagawa, I., Sakagami, M., Hashiuchi, F., Zhou, J.L., Yoshikawa, M., Ren, J., 1989.
Apioglycyrrizin and araboglycyrrhizin, two new sweet oleanene-type triter-
pene oligoglycosides from the root of Glycyrrhiza inflate. Chem. Pharm. Bull. 37,
pyridine (0.2 mL) containing
(
(
L
-cysteine methyl ester hydrochloride
1.0 mg) and heated at 60 8C for 1 h. O-Tolyl isothyocyanate
1.0 mg) in pyridine (0.2 mL) was then added to the mixture and
551–553.
heated at 60 8C for 1 h. The reaction mixture was directly analyzed
by RP-HPLC (condition 2). The peaks coincided with those of
Liu, C.M., Li, B., Scen, Y.H., Zhang, W.D., 2010. Heterocyclic compounds and aromatic
diglycosides from Bretschneidera sinensis. J. Nat. Prod. 73, 1582–1585.
Notice No.210, 1996. List of Existing Food Additives. Ministry of Health and Welfare,
Japan.
Ou, B., Hampsch-Woodill, M., Prior, R.L., 2001. Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein as the
fluorescent probe. J. Agric. Food Chem. 49, 4619–4626.
derivatives of authentic samples,
from apiin (7-O-[ -apio-
-furanosyl-(1!2)-
syl] apigenin) (Yoshida et al., 1989).
D
-glucose and
D
-apiose, obtained
b
-
D
D
b-D-glucopyrano-
Pereira, M.G., Mudge, S.M., Latchford, J., 2003. Vegetable iol spills on salt marsh
sediments; comparison between sunflower and linseed oils. Marine Environ.
Res. 56, 367–385.
3.5. Antioxidant assay
Antioxidant activity was estimated by the ORAC method
Amakura et al., 2009; Yoshimura et al., 2011). Briefly, the ORAC
assay was performed in 75 mM phosphate buffer (pH 7.4) with a
final reaction volume of 200 L. Trolox (20 L) and fluorescein
120 L; 70 nM, final concentration) solutions were pipetted into
Rashid, U., Anwar, F., Arif, M., 2009. Optimization of base catalytic methanolysis of
suflower (Helianthus annuus) seed oil for biodiesel production by using response
surface methodology. Ind. Eng. Chem. Res. 48, 1719–1726.
Tanaka, T., Nakashima, T., Ueda, T., Tomii, K., Kouno, I., 2007. Facile discrimination of
aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 55, 899–901.
(
m
m
1
Yoshida, T., Jin, Z.X., Okuda, T., 1989. Taxifolin apioside and davuriciin M , a
(
m
hydrolysable tannin from Rosa davurica. Phytochemistry 28, 2177–2181.
Yoshimura, M., Amakura, Y., Yoshida, T., 2011. Polyphenolic compounds in clove
and pimento and their antioxidative activities. Biosci. Biotechnol. Biochem. 75,
each well of a 96-well microplate. The mixture was pre-
incubated in a microplate reader for 15 min at 37 8C. A solution
2207–2212.
of AAPH (60
mL: final concentration, 12 mM) was added rapidly