New monoterpene glycosides from sunflower seeds and their protective effects against H2O2-induced myocardial cell injury

Article abstract of DOI:10.1016/j.foodchem.2015.04.079

Three new monoterpene glycosides (1-3) and eleven known compounds (4-14) were isolated from seeds of Helianthus annuus L. (sunflower). Their structures were determined by spectroscopic and chemical methods. All the compounds were isolated from sunflower seeds for the first time. Protective effects of compounds 1-14 against H₂O₂-induced H9c2 cardiomyocyte injury were evaluated, and compounds 1 and 2 showed some cell-protective effects. No significant DPPH radical scavenging activity was observed for compounds 1-14.

Full text of DOI:10.1016/j.foodchem.2015.04.079

Food Chemistry 187 (2015) 385–390
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
New monoterpene glycosides from sunflower seeds and their protective
effects against H O -induced myocardial cell injury
2
2
Yonghe Fei , Jianping Zhao , Yanli Liu , Xiaoran Li , Qiongming Xu , Taoyun Wang a,c,
⇑
,
a
b
a
a
a,
⇑
a,b
a
Ikhlas A. Khan , Shilin Yang
a
College of Pharmaceutical Science, Soochow University, Suzhou 215123, China
National Center for Natural Products Research, and Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi,
b
MS 38677, United States
c
College of Chemical, Biological and Material Engineering, Suzhou Science and Technology University, Suzhou 215009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new monoterpene glycosides (1–3) and eleven known compounds (4–14) were isolated from seeds
of Helianthus annuus L. (sunflower). Their structures were determined by spectroscopic and chemical
methods. All the compounds were isolated from sunflower seeds for the first time. Protective effects of
compounds 1–14 against H O -induced H9c2 cardiomyocyte injury were evaluated, and compounds 1
2 2
and 2 showed some cell-protective effects. No significant DPPH radical scavenging activity was observed
for compounds 1–14.
Received 15 November 2014
Received in revised form 13 March 2015
Accepted 18 April 2015
Available online 24 April 2015
Keywords:
Sunflower seed
Ó 2015 Elsevier Ltd. All rights reserved.
Helianthus annuus L.
Monoterpene glycosides
Cardiomyocytes oxidative injury
DPPH radical scavenging activity
1
. Introduction
(British Pharmacopoeia, 2005), copper (USDA, 2012), zinc (USDA,
2
012), crude fiber (Skrbic & Filipcev, 2008), sesquiterpenes
Helianthus annuus L. (sunflower) is an important oilseed crop
Sackston, 1981). Due to its great capability of adaptation to differ-
(Macias et al., 2002), diterpenes (Suo et al., 2007), triterpenes
(Ukiya et al., 2007), and phenols (Zili c´ et al., 2010), are used to pre-
ˇ
(
ent climatic and soil conditions (Villamide & San Juan, 1998), the
crop is cultivated worldwide, and the total production of seeds
was 44.7 million metric tons in 2013 according to the Food and
Agriculture Organization (FAO) of the United Nations. Along with
rape seed, soybean, peanut, and palm oil, sunflower oil is one of
the most important vegetable oils and is widely used in the food
and nutraceutical industries (Schmidt, 2013).
Sunflower seeds have excellent nutritional properties. The qual-
ity of its edible oil ranks it as one of the best vegetable oils among
the cultivated plant oils. Up to 90% of the fatty acids in conven-
tional sunflower oils are typically unsaturated fatty acids, namely
oleic and linoleic, palmitic, stearic (British Pharmacopoeia, 2005),
and minor amounts of myristic, myristoleic, palmitoleic, arachidic,
behenic and other fatty acids account for the remaining 10%
pare breads, biscuits and snack foods. Kernels of sunflower seeds
are eaten raw or roasted as a rich source of protein and vitamins
B, D, E and K. Other studies have shown its benefits in the reduc-
tion of cardio-vascular diseases (Zhu, Liu, Xia, & Ma, 2003). In addi-
tion to being used as food, sunflower seeds were reported to be
used for medicinal purpose. The Indians of North America use
the seeds for the treatments of cold, cough, and the ailments
related to throat and lung (Putt, 1978). In Venezuela, the flowers
and seeds are used in folk medicine for treating cancer (Hartwell,
1982).
Sunflower seeds are a rich and renewable natural resource with
high economic value. The seeds are primarily utilized for produc-
tion of vegetable oil. However, the pressed cake (or the residue
resulting from oil extraction), a by-product from the oil production,
is underutilized (Weisz, Carle, & Kammerer, 2013). In order to fully
utilize this rich resource, more investigations on the chemical con-
stituents in sunflower seeds are needed. In addition to proteins,
other chemicals such as phenolic compounds, organic acids, phos-
pholipids, tocopherols, and phytosterols have been found in the
seeds (Amakura, Yoshimura, Yamakami, & Yoshida, 2013; Baydar
(Skoric, Jocic, Sakac, & Lecic, 2008). Sunflower seeds, containing
high amounts of proteins and significant contents of tocopherols
⇑
Corresponding authors at: College of Pharmaceutical Science, Soochow Univer-
sity, Suzhou 215123, China.
E-mail addresses: xuqiongming@suda.edu.cn (Q. Xu), wangtaoyun@usts.edu.cn
(
T. Wang).
http://dx.doi.org/10.1016/j.foodchem.2015.04.079
308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
0

3
86
Y. Fei et al. / Food Chemistry 187 (2015) 385–390
&
Erbas, 2005; Martinez-Force, Alvarez-Ortega, Cantisan, & Garces,
from Jiangxi Bencaotiangong Technology Co., Ltd. (Nanchang,
China). All of the other chemicals and reagents were of analytical
grade and purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China).
1
2
998; Rashid, Anwar, & Arif, 2009; Weisz, Kammerer, & Carle,
009). Recent studies reported more than twenty compounds iden-
tified from the seeds, and some of the compounds were found for
the first time from Helianthus genus (Amakura et al., 2013; Fei,
Chen, Li, Xu, & Yang, 2014). In this paper, we report the isolation
and structure identification of three new monoterpene glycosides
from sunflower seeds, together with eleven other compounds. In
addition, the results about the protective effects of these com-
2.2. Plant materials
The sunflower seeds (H. annuus L.) were purchased from a farm-
ers’ market in Qiqihar city (Heilongjiang province, China) in
September 2011 and authenticated by Prof. Xiaoran Li
pounds against cardiomyocytes injury induced by H
as their DPPH radical scavenging activities are included.
2 2
O , as well
(
Department of Pharmacognosy, College of Pharmaceutical
Science, Soochow University) using morphological identification.
A voucher specimen (No. 10-09-08-01) was deposited in the
herbarium of the College of Pharmaceutical Science at Soochow
University.
2
. Materials and methods
.1. Materials and chemicals
Melting points of the isolates were determined using the XT5
2
micro-melting-point system (Beijing Optical Instrument Factory,
Beijing, China). Specific optical rotation values were determined
using a 241 polarimeter (Perkin–Elmer Inc., Waltham, MA, USA).
IR spectra were recorded on a 983 G spectrometer (Perkin–Elmer
Inc., Waltham, MA, USA). H NMR, C NMR, and 2D NMR spectra
were obtained from an Inova 500 spectrometer (Varian Inc., Palo
2.3. Extraction and isolation
Sun-dried seeds (20 kg) were crushed into fine powder and
extracted twice in MeOH (200 L). The solvent was removed under
reduced pressure. The resulting residue (150 g) was dissolved in
distilled water and fractionated successively with petroleum ether,
chloroform, and n-butyl alcohol. The chloroform fraction (34 g)
was dissolved in chloroform, loaded onto a silica gel column, and
1
13
5 5
Alto, CA, USA) in C D N using tetramethylsilane (TMS) as the inter-
nal standard. HR-ESI-MS spectra were determined on a Q-TOF2
spectrometer (Micromass Corp., London, UK). EI-MS was deter-
mined on a Micromass Zabspec spectrometer (Micromass Corp.,
London, UK). High performance liquid chromatography (HPLC)
analysis and purification were performed with an Agilent Zorbax
eluted with CHCl
under reduced pressure. The CHCl
was subjected to separation on a MPLC/ODS column and eluted
with MeOH–H O (40:60, 60:40, 80:20, and 90:10; 1000 mL each)
3
–MeOH (80:20, 60:40, 40:60, 0:100; 4.0 L each)
3
–MeOH (60:40) eluate (6.2 g)
2
SB-C18 semipreparative HPLC column (250 ꢀ 9.4 mm i.d., 5
l
m,
at 20 mL/min to provide five fractions. Fraction 1 (196 mg) was
separated by Sephadex LH-20 gel column chromatography
(100 cm ꢀ 3 cm i.d.) eluting with MeOH to give compounds 6
Agilent Corp. Palo Alto, CA, USA) on a Shimadzu HPLC system com-
posed of a LC-20AT pump with an SPD-20A detector (Shimadzu
Corp., Kyoto, Japan), the flow rate was 2 mL/min, and the wave-
length for detection was 203 nm. Medium pressure liquid chro-
matography (MPLC) purification was performed on a Büchi Flash
Chromatography system composed of a C-650 pump with a flash
column (460 mm ꢀ 26 mm i.d., Büchi Corp., Flawil, Switzerland).
GC analysis was conducted on a GC-14C (Shimadzu Corp., Kyoto,
Japan) instrument with a flame ionization detector and the analyt-
ical conditions were as follows: DB-5 column (i.d. 0.25 mm, length
(43 mg) and 10 (32 mg). Compounds 7 (31 mg, t
R
13.27 min), 8
37.15 min) were obtained
from fraction 2 (163 mg) after separating by semi-preparative
RP-HPLC eluting with MeOH–H (67:33) at 2.0 mL/min.
Compounds 4 (15 mg, t 37.14 min), 3 (13 mg, t 14.61 min), 2
(15 mg, t 16.00 min), 5 (15 mg, t 26.61 min), and 1 (9.2 mg, t
32.70 min) were obtained from fraction 3 (120 mg) after the purifi-
cation using semi-preparative RP-HPLC with MeOH–H O (56:44)
for elution. Fraction 4 (90 mg), which was subjected to separation
by semi-preparative RP-HPLC and eluted with MeOH–H O (86:14),
yielded compounds 11 (32 mg, t 12.42 min) and 12 (19 mg, t
15.62 min). Compounds 13 (51 mg, t 28.16 min) and 14 (13 mg,
37.43 min) were obtained from fraction 5 (102 mg) after purifi-
R R
(7.2 mg, t 29.35 min) and 9 (6.4 mg, t
2
O
R
R
R
R
R
2
3
0 m; Suzhou Huitong Chromatography Technology Co., Ltd.,
Suzhou, China), column temperature at 210 °C; injector tempera-
ture at 270 °C; and detector temperature at 300 °C. The samples
2
R
R
(
1
lL) were injected manually into the column.
R
Silica gel (200–300 mesh) for column chromatography and pre-
t
R
coated silica gel TLC plates were purchased from Qingdao Marine
Chemical Factory. ODS for MPLC was purchased from Merck
KGaA (Darmstadt, Germany). Sephadex LH-20 for column chro-
matography was purchased from GE Healthcare Corp. (Beijing,
China). Compounds on TLC were colored by spraying 10% sulfuric
acid alcohol solution and heating. Vitamin E was purchased from
J&K Scientific Ltd. (Beijing, China). 3-(4,5-Dimethyl-2-thiazolyl)-
cation by using semi-preparative RP-HPLC with MeOH–H
(89:11) as the eluent.
2
O
2.3.1. (+)-Campholenol-10-O-b-
D-glucopyranoside (1)
(4S)-2,2,3-trimethyl-3-cyclopentene-1-ethanol-10-O-b-
D
-glu-
2
5
1
copyranoside. white powder; [
a]
D
ꢁ36 (c = 0.015, MeOH);
H
1
3
2
,5-diphenyl-2H-tetrazolium bromide (MTT), 1,1-diphenyl-2-
NMR (500 MHz, MeOD) and C NMR (125 MHz, MeOD) spectro-
picrylhydrazyl (DPPH) and phosphate buffered saline (PBS) were
purchased from Sigma–Aldrich Co. (St. Louis, MO, USA).
Dulbecco’s modified Eagle’s medium (DMEM), mitochondrial
dehydrogenase and fetal bovine serum (FBS) were purchased from
Gibco™ (Grand Island, NY, USA). Rat H9c2 cardiomyocytes were
obtained from the Cell Bank of the Chinese Academy of Sciences
scopic data are shown in Table 1; HR-ESI-MS (negative ion mode)
ꢁ
m/z 315.1813 ([MꢁH] , calcd for C16
28 6
H O ; 315.1808).
2.3.2. (+)-Campholenol-10-O-b-
glucopyranoside (2)
D-apiofuranosyl-(1 ? 6)-b-D-
(
Shanghai, China). 96-Well plates were obtained from Corning
Costar (Corning Costar, Cambridge, MA, USA). CD OD and CDCl
were obtained from Merck (Darmstadt, Germany). Methanol
MeOH) for HPLC analysis and purification was HPLC grade and
(4S)-2,2,3-trimethyl-3-cyclopentene-1-ethanol-10-O-b-D-apio-
25
3
3
furanosyl-(1 ? 6)-b-
(c = 0.016, MeOH); H NMR (500 MHz, MeOD) and
D
1
-glucopyranoside. white powder; [
a
]
D
ꢁ55
13
C NMR
(
(125 MHz, MeOD) spectroscopic data are shown in Table 1;
ꢁ
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China). Standard (+)-campholenol and (ꢁ)-myrtenol were obtained
HR-ESI-MS (negative ion mode) m/z 447.2236 ([MꢁH] , calcd for
21 36
C H O10; 447.2230).

Y. Fei et al. / Food Chemistry 187 (2015) 385–390
387
Table 1
NMR spectroscopic data of 1, 2 and 3 (in MeOD).
1
2
3
d
C
d
H
d
C
d
H
d
C
d
H
1
2
3
148.1
121.4
35.0
148.1
121.4
35.0
43.1
144.8
119.8
2.22 m
5.57 m
5.21 s
2.29 m
5.21 s
2.30 m
1.85 m
1.86 m
1
.84 m
4
5
6
7
46.7
46.4
11.4
24.8
1.85 m
46.7
46.4
11.4
24.8
30.8
40.8
37.5
31.1
2.28 m
2.08 m
1.59 s
0.98 s
1.59 s
0.99 s
1.20 m
2.42 m
8
9
18.7
29.9
0.77 s
1.54 m
18.7
29.9
0.78 s
1.54 m
25.2
20.2
1.30 s
0.87 s
1
.84 m
3.53 m
.97 m
1.84 m
3.53 m
3.94 m
4.23 d (8.0)
3.16 m
3.33 m
3.26 m
3.39 m
3.59 m
1
0
69.3
69.3
71.5
4.00 dd (2.0, 12.0)
4.18 dd (1.5, 12.0)
4.27 d (7.5)
3.18 m
3.32 m
3.28 m
3
0
a
Glc-1
103.1
73.7
76.7
70.2
76.5
61.3
4.25 d (8.0 )
3.17 m
3.35 m
3.28 m
3.27 m
103.2
73.7
76.6
70.3
75.4
67.2
102.0
73.7
76.6
70.6
75.1
66.6
0
2
0
3
0
4
0
5
3.38 m
3.60 m
0
6
3.66 m
3
.85 m
3.97 m
4.99 d (3.0)
3.89 d (3.0)
3.98 m
4.96 d (5.0)
4.01 d (5.0)
00
Apio-1
109.5
76.6
79.2
73.6
109.8
81.1
80.9
74.5
0
0
0
0
0
0
2
3
4
3.76 d (12.0)
3.82 d (12.0)
3.99 d (12.0)
3.65 d (12.5)
3.73 d (12.5)
3
.95 d (12.0)
3.57 d (12.0)
.66 d (12.0)
00
5
64.2
62.8
3
a
Data in parentheses are J values (in Hz).
2
.3.3. (ꢁ)-Myrtenol-10-O-
glucopyranoside (3)
1R)-6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-methanol-10-O-
-apiofuranosyl-(1 ? 6)-b- -glucopyranoside. white powder;
a
-
D
-apiofuranosyl-(1 ? 6)-b-
D
-
2.5. Assay for testing the protective effects against myocardial cell
injury induced by H
2 2
O
(
D
a
-
D
Rat H9c2 cardiomyocytes were seeded into 96-well flat micro-
2
5
+12 (c = 0.013, MeOH); 1H NMR (500 MHz, MeOD) and
13
5
[
a
]
D
C
titer plates at a density of 1 ꢀ 10 per well and cultured in
NMR (125 MHz, MeOD) spectroscopic data are shown in Table 1;
DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and
ꢁ
HR-ESI-MS (negative ion mode) m/z 445.2079 ([MꢁH] , calcd for
100
lg/mL of streptomycin at 37 °C in a humidified atmosphere
C
21
H
34
O10; 445.2074).
of 5% CO
2
, and allowed to adhere for 24 h before the tested com-
pounds (compounds 1–14) or positive control (vitamin E) were
2.4. Acid hydrolysis of compounds 1–3
introduced. The tested compounds with five different concentra-
tions (12.5–200
lM) were added and following 12 h of incubation,
The acid hydrolysis was used to identify the type and configura-
200 M H was added to each well for 24 h (Konorev, Kennedy,
l
2 2
O
tion of sugar moiety and aglycone (Gao, Zhang, Liu, Cai, & Yang,
011). A solution of each monoterpene glycoside (5 mg) was dis-
solved in 2 N HCl (2 mL) and heated at 90 °C for 2 h. After cooling,
& Kalyanaraman, 1999). The cells in the control groups were trea-
ted with the same volume of PBS. Cell viability was evaluated by
MTT assay, based on the reduction of MTT by the mitochondrial
dehydrogenase of intact cells to a purple formazan product.
Absorbance was read on an ELISA plate reader (Biorad iMark™
Microplate Reader, Bio-Rad Laboratories, Inc., Hercules, CA, USA)
at 540 nm to measure the quantity of formazan. The percentage
cell viability was calculated as a ratio of optical density (OD) value
of sample to the OD value of a control (Li et al., 2014). All experi-
ments were repeated three times.
2
the reaction mixture was neutralized with silver carbonate
(
10 mg), and evaporated to dryness under N
2
overnight. The reac-
O, and both lay-
tion mixture was extracted with chloroform and H
2
ers were concentrated to dryness. The chloroform extract was
purified by Sephadex LH-20 gel column chromatography eluting
with CHCl –MeOH, (1:1). The purified compound (aglycone) was
3
then analyzed to determine its chemical structure by comparing
its retention time with that of the standard in the GC analysis
and by comparing the specific optical rotation value with that of
the standard. The residue from the aqueous layer was dissolved
in anhydrous pyridine (1 mL) followed by the addition of 2 mg of
2.6. DPPH radical scavenging capacity assay
DPPH free radical scavenging activity of compounds 1–14 were
L
-cysteine methyl ester hydrochloride (0.2 mL). After heating at
0 °C for 2 h, the solvent was evaporated under N , and 0.2 mL of
trimethylsilyl) imidazole dissolved in H O was added. Then the
measured in triplicate following a previously described method
(Hatano, Kagawa, Yasuhara, & Okuda, 1988). Briefly, a solution of
6
(
2
2
0.5 mM methylated DPPH was prepared. An aliquot (100
tested compound at final concentrations (5–200 M) in methanol
was mixed with 100 L of methylated DPPH radical solution and
lL) of
mixture was heated at 60 °C for another 2 h and the dried reactant
was partitioned with n-hexane and water (0.1 mL, each). The
organic layer was used for GC analysis, and the following sugar
units in compounds 1–3 were identified by comparison with
authentic samples:
1
l
l
incubated in the dark for 30 min. Vitamin E served as a positive con-
trol. A freshly prepared DPPH solution exhibited a deep purple color
with an absorption maximum at 517 nm. The DPPH free radical
scavenging rate in percent (I %) was calculated in following way:
D
-glucose (t
R
11.49 min),
L-glucose (t
R
1.10 min), -apiose (t
D
R
9.15 min), -apiose (t
L
R
9.41 min).

3
88
Y. Fei et al. / Food Chemistry 187 (2015) 385–390
À
Â
À
Á
ÃÁ
I% ¼ 100 ꢀ 1 ꢁ Asample ꢁ Ablank =AControl
data and comparisons with the reference values and authentic
samples.
Compound 1 was isolated as white powder. The molecular for-
AControl is the absorbance of the control reaction (containing all
reagents except the test compound), Asample is the absorbance of
the test compound, and Ablank is the absorbance of a blank sample.
mula of 1 was determined as C16
28
H O
1
6
on the basis of its HR-ESI-
ꢁ
MS (m/z 315.1813 ([MꢁH] )). The H NMR spectrum showed sin-
glet resonances of three methyl groups at d 1.59 (3H, s, Me-6),
0.98 (3H, s, Me-7), 0.77 (3H, s, Me-8), an olefinic proton signal at
d 5.21 (1H, s, H-2) and signals of an oxymethylene group at d
3.97 (1H, m, H-10), 3.53 (1H, m, H-10). The NMR spectroscopic data
of compound 1 were similar to those of (+)-campholenol (Chapuis
& Brauchli, 1992), except for the presence of additional signals at dC
103.5, 76.7, 76.5, 73.7, 70.2 and 61.3, corresponding to one glu-
copyranosyl group. The assignment of the aglycone structure as
2
.7. Statistical analysis
The results were expressed as the mean ± SD. Differences were
evaluated with one way ANOVA. The post hoc test was done with
the student’s Dunnett test. P values less than 0.05 were considered
statistically significant. Statistical calculations were performed
using the SPSS 16.0 for Windows software package.
(
+)-campholenol was further confirmed by the GC analysis of the
acid-hydrolysis product of 1 (Zhou, Huang, Song, & Wang, 2012),
in which the GC retention time of the aglycone compound from
3
. Results and discussion
1
was same as that of the standard (+)-campholenol.
3
.1. Phytochemical investigation
Furthermore, the aglycone compound from 1 showed almost the
2
5
same specific optical rotation value of [
a
]
D
+20.4 (c = 0.005,
+18.0) (Chapuis &
Brauchli, 1992). The sugar residue obtained from the acid hydroly-
sis of 1 was identified as -glucose by the GC analysis. The anome-
ric proton signal at d 4.25 (1H, d, J = 8.0 Hz, H-1 of glucose) showed
the correlation to the carbon at d 103.5 (C-1 of glucose) in the
The MeOH extract of dried seeds of Helianthus annuus L. was
2
5
3 D
CHCl ) as that of (+)-campholenol ([a]
separated by successively partitioning with petroleum ether, chlo-
roform, n-butyl alcohol, and by repeated silica gel and ODS column
chromatography to afford three new monoterpene glycosides (1–
D
3
) (Fig. 1), as well as eleven known compounds, which were iden-
tified as (ꢁ)-myrtenol-10-O-b- -glucopyranoside (4) (Kawahara,
Fujii, Ida, & Akita, 2006), (ꢁ)-myrtenol-10-O-b- -apiofuranosyl-
1 ? 6)-b- -glucopyranoside (5) (Kikuchi et al., 2012), 1,4-butano-
C
D
HSQC spectrum (Table 1). The glucopyranosyl group was located
at C-10 of the aglycone, which was revealed by the HMBC correla-
tion between d
D
(
D
H C
4.25 (H-1 of glucose) and d 69.3 (C-10 of agly-
lide (6) (Yu & Yang, 2005), succinic acid (7) (Cui et al., 1995), erucic
acid (8) (Ji et al., 2007), hexadecanoic-2,3-dihydroxypropyl ester
cone) (Fig. 1). The b configuration of glucopyranosyl group was
3
confirmed on the basis of the large
Thus the structure of compound 1 was elucidated as (+) camp-
holenol-10-O-b- -glucopyranoside.
Compound 2 was isolated as white powder. The molecular
JH-1,H-2 coupling constant.
(
9) (Wang, Zhu, Sheng, Chen, & Yang, 2011), 9-octadeconic-2,3-di-
hydroxypropylester (10) (Zhu, Zhu, Zhu, Wen, & Liu, 2011),
,12,13-trihydroxy-10,15-octadecadienoic acid (11) (Li, Kuang,
D
9
Okada, & Okuyama, 2003), 9,12,13-trihydroxy-10,15-octadeca-
dienoic acid methyl ester (12) (Suemune, Harabe, & Sakai, 1988),
formula of 2 was determined to be C21
H
36
O
10 on the basis of its
ꢁ
1
HR-ESI-MS (m/z 447.2236 ([MꢁH] )). The H NMR spectrum of 2
9
,12,13-trihydroxy-10-octadecaenoic acid (13) (Li et al., 2003),
and 9,12,13-trihydroxy-10-octadecaenoic acid methyl ester (14)
Suemune, Harabe, & Sakai, 1988), based on their spectroscopic
displayed two anomeric signals at d
H
4.23 (1H, d, J = 8.0 Hz) and
4
.99 (1H, d, J = 3.0 Hz) suggesting it had two sugar moieties.
(
1
3
Based on the C NMR and COSY spectra of 2, the signals at d
C
6
4.2, 70.3, 76.6, 79.2, and 109.5 were attributed to a terminal
C
b-apiofuranose moiety, and the signals at d 67.2, 70.3, 73.7,
7
5.4, 76.6, and 103.1 were assigned to a b-glucopyranose moiety.
C
Compound 2 showed the same signals at d 148.1, 121.4, 69.3,
4
6.7, 46.4, 35.0, 29.9, 24.8, 18.7, and 11.4 as compound 1 did, sug-
gesting that it had the same monoterpene aglycone moiety. The
assignment of the aglycone structure as (+)-campholenol was fur-
ther confirmed by the GC analysis of the acid-hydrolysis product of
2
5
2
, as well as the specific optical rotation of [
CHCl ) of the aglycone compound (Chapuis & Brauchli, 1992). In
the HMBC spectrum of 2 (Fig. 1), the correlation between H-1 of
the glucopyranosyl moiety (d 4.23) and C-10 (d 69.3)
of the monoterpene moiety and the correlation between H-1 of
the apiofuranosyl moiety (d 4.99) and C-6 of the glucopyranosyl
moiety (d 67.2) were observed, revealing the connections
of the three moieties. Thus the structure of compound 2 was elu-
D
a] +19.6 (c = 0.004,
3
H
C
H
C
cidated as (+)-campholenol-10-O-b-D-apiofuranosyl-(1 ? 6)-b-D-
glucopyranoside.
Compound 3 was isolated as white powder. The molecular for-
mula of compound 3 was determined to be C21
H
34
O
10 on the basis
ꢁ
1
of its HR-ESI-MS (m/z 445.2079, ([MꢁH] )). The H NMR spectrum
of 3 showed two methyl singlet signals at d 1.30 (3H, s, Me-8) and
0
.87 (3H, s, Me-9), one olefinic proton signal at d 5.57 (1H, m, H-3),
one oxymethylene signals at d 4.00 (1H, dd, J = 2.0, 12.0 Hz, H-10)
1
13
and 4.18 (1H, dd, J = 1.5, 12.0 Hz, H-10) (Table 1). The H and
C
NMR spectra indicated that 3 is a myrtenol derivative by compar-
ing its spectroscopic data with previously published data
1
(
Yoshikawa et al., 1997). The H NMR spectrum of 3 displayed
Fig. 1. Structures of compounds 1–3 and their key HMBC and NOESY correlations.
H
two anomeric signals at d 4.27 (1H, d, J = 7.5 Hz) and 4.96 (1H,

Y. Fei et al. / Food Chemistry 187 (2015) 385–390
389
Table 2
Protective effects of monoterpene glycosides from sunflower on H
2 2
O -induced H9c2 cell injury.
Compounds
Viabilities of H9c2 cardiomyocyte (%)
12.5
0
lM
lM
25
l
M
50
lM
100
l
M
200 lM
*
*
1
2
3
4
5
6
7
8
9
65.72 ± 2.52
64.58 ± 1.86
64.28 ± 2.33
68.43 ± 3.20
67.16 ± 2.67
63.32 ± 1.81
68.94 ± 3.73
70.03 ± 5.16
69.36 ± 4.39
65.63 ± 2.97
69.96 ± 3.84
63.48 ± 3.76
67.26 ± 3.51
69.84 ± 3.51
63.56 ± 2.38
67.23 ± 1.69
64.14 ± 1.65
67.41 ± 4.70
66.17 ± 2.62
63.08 ± 4.39
64.59 ± 2.60
67.74 ± 3.70
67.36 ± 3.87
63.92 ± 5.58
64.03 ± 3.40
67.18 ± 2.58
66.50 ± 2.74
65.19 ± 2.63
67.87 ± 2.58
65.19 ± 2.03
66.18 ± 3.72
65.83 ± 2.55
65.52 ± 2.58
70.35 ± 5.02
64.74 ± 3.84
68.10 ± 4.39
68.02 ± 6.81
63.60 ± 4.14
69.81 ± 5.53
67.34 ± 3.08
68.93 ± 4.43
68.15 ± 5.32
70.08 ± 1.96
69.14 ± 3.45
67.28 ± 1.72
68.52 ± 3.03
65.22 ± 2.41
62.93 ± 3.37
66.48 ± 2.77
68.55 ± 2.48
62.97 ± 4.06
65.41 ± 3.20
66.75 ± 3.12
63.36 ± 1.28
68.09 ± 2.76
65.71 ± 3.12
64.36 ± 4.52
63.35 ± 2.93
67.35 ± 3.02
68.52 ± 2.46
73.14 ± 2.55
67.36 ± 2.60
66.62 ± 4.39
66.02 ± 3.34
63.02 ± 1.83
65.31 ± 2.49
63.72 ± 4.06
71.21 ± 4.74
66.42 ± 3.37
66.86 ± 3.25
70.55 ± 3.31
66.93 ± 3.81
68.37 ± 3.64
69.49 ± 4.70
75.80 ± 1.83
75.29 ± 3.38
72.57 ± 2.86
63.75 ± 3.56
69.95 ± 3.60
65.72 ± 3.61
67.75 ± 2.94
69.06 ± 4.37
66.95 ± 2.32
69.76 ± 2.94
63.95 ± 2.72
66.73 ± 3.79
64.77 ± 4.18
69.09 ± 5.53
68.31 ± 4.01
81.68 ± 1.66
*
1
1
1
1
1
0
1
2
3
4
a
*
**
Vitamin E
a
*
*
Positive control.
p < 0.05.
p < 0.01 vs. 0 lM group.
*
Table 3
Maximal DPPH radical.
3.2. Protective effects of the compounds against H
myocardial cell injury
2
O
2
-induced
Compounds
Scavenging rate (200 lM)
The intracellular damage induced by reactive oxidative species
ROS) could lead to various diseases such as cardiovascular dis-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1.26 ± 0.22%
6.67 ± 2.32%
5.68 ± 2.65%
0
(
eases (Fearon & Faux, 2009). It is believed that oxidative stress
plays a pivotal role in the pathogenesis of cardiomyocytes ischemic
diseases. Studies showed that antioxidants could mitigate oxida-
tive-stress-mediated cellular injury (Yousuf et al., 2009).
Hydrogen peroxide can easily cross cellular membranes, triggering
the chain reactions resulting in the damage of cells (Valko et al.,
1.92 ± 0.71%
4.72 ± 1.41%
1.52 ± 1.74%
5.23 ± 0.81%
4.28 ± 1.41%
5.17 ± 3.72%
3.85 ± 3.84%
2.79 ± 1.01%
3.23 ± 2.41%
4.83 ± 1.41%
93.23 ± 0.72%
0
1
2
3
4
2
2 2
007). Therefore, H O is usually employed in the models for inves-
tigating H9c2 cardiomyocyte injury (Hescheler et al., 1991;
Yasuoka et al., 2004). In this study, compounds 1–14 were investi-
gated for their protective effects against H
cell injury. Compounds 1 and 2 increased the viability of H9c2
induced by H with a dose-dependent manner at the concentra-
tion range from 12.5 to 200 M (Table 2). Vitamin E can incorpo-
2 2
O -induced myocardial
Vitamin E
2 2
O
l
rate into the membrane lipids of endothelial cells to protect
against membrane lipid peroxidation by quenching cytotoxic ROS
(Fujimoto, Mizoi, Yoshimoto, & Suzuki, 1984; Yamamoto et al.,
1983). We speculated that the active compounds isolated from
sunflower might play a similar antioxidant role in the prevention
of peroxidative damage. Therefore, the radical scavenging capacity
of the isolates were evaluated in the present study. Compounds 1
and 2 might play a similar role according to the results. Further
studies are warranted to understand their potential mechanism
and to reveal their other bio-activities.
d, J = 5.0 Hz) suggesting it possessed two sugar units. The GC anal-
ysis of the sugar residue obtained from the acid hydrolysis of 3
revealed that the two sugars were
The glucopyranosyl group was located at C-10 of the aglycone,
which was revealed by the HMBC correlation between d 4.27
H-1 of glucose) and d 71.5 (C-10 of aglycone). Similarly, the
apiofuranosyl unit was located at C-6 of the glucose, which was
revealed by the HMBC correlation between d 4.96 (H-1 of
apiofuranose) and d 66.6 (C-6 of glucose) (Fig. 1). The b-anomeric
D-glucose and D-apiofuranose.
H
(
C
H
C
configuration of the glucuronopyranosyl unit was determined on
the basis of the observation of the large JH-1, H-2 coupling constant,
3
3.3. DPPH radical scavenging capacity assay
and the
a-anomeric configuration of the apiofuranosyl unit was
indicated by NOESY correlations between d 4.96 (H-1 of apiofura-
nose) and d 3.65, 3.73 (H-5 of apiofuranose) (Fig. 1). The NMR data
of compound 3 were similar to those of compound 5, and the
main differences arose from the significant down-field shifts of
Compounds 1–14 were evaluated for antioxidant activity by
DPPH radical scavenging capacity assay. As shown in Table 3, the
maximal scavenging rates of compounds 1–14 were less than
20% at the concentration of 200 lM. The results suggested that
C-2 of
nose (+1.7 ppm) at d 80.9, due to a
replace of the b- -apiofuranosyl unit in compound 5. The specific
optical rotation value of the aglycone compound obtained by acid
D
-apiofuranose (+4.5 ppm) at d 81.1 and C-3 of
D
-apiofura-
compounds 1–14 had no significant DPPH radical scavenging
capacity.
a
-D
-apiofuranosyl unit in
D
4. Conclusion
25
hydrolysis of compound 3 was [
a
]
D
ꢁ43.2 (c = 0.006, CHCl
3
),
2
5
almost the same as that of (ꢁ)-myrtenol ([
a]
D
ꢁ47.5), indicating
Three new monoterpene glycosides (1–3) and eleven known
compounds were isolated and characterized from sunflower seeds.
To our knowledge, it is the first time report about the compounds
isolated from sunflower seeds. The protective effects of the isolated
that the aglycone of compound 3 was (ꢁ)-myrtenol (Kover,
Schottelius, & Hoffmann, 1991). Thus, compound 3 was identified
as (ꢁ)-myrtenol 10-O-a-D-apiofuranosyl-(1 ? 6)-b-D-glucopyranoside.

3
90
Y. Fei et al. / Food Chemistry 187 (2015) 385–390
compounds against H
investigated. Compounds 1 and 2 increased the viability of H9c2
induced by H with a dose-dependent manner at the concentra-
tion range from 12.5 to 200 M. The DPPH radical scavenging
capacity assay suggested that protective effects on H induced
2
O
2
-induced myocardial cell injury were
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