Isolation, characterization and antimicrobial activities of polyacetylene glycosides from Coreopsis tinctoria Nutt.

Article abstract of DOI:10.1016/j.phytochem.2016.12.023

Polyacetylene glycosides, (6Z, 12E)-tetradecadiene-8,10-diyne-1-ol-3(R)-O-β-D-glucopyranoside (trivially named coreoside E) and (6Z, 12E)-tetradecadiene-8,10-diyne-1-ol-3(R)-O-β-L-arabinopyranosyl-(1?→?2)-β-D-glucopyranoside (trivially named coreoside F),

Full text of DOI:10.1016/j.phytochem.2016.12.023

Phytochemistry xxx (2017) 1e5
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Isolation, characterization and antimicrobial activities of
polyacetylene glycosides from Coreopsis tinctoria Nutt.
a
a
a
a
,
b
a
a
a
Jia Guo , Ao Wang , Ke Yang , Hao Ding , Yimin Hu , Yumeng Yang , Siqi Huang ,
a
a
b
a, *
Jingguo Xu , Tianxing Liu , Haiyan Yang , Zhihong Xin
Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People's
Republic of China
a
b
College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 August 2016
Received in revised form
Polyacetylene glycosides, (6Z, 12E)-tetradecadiene-8,10-diyne-1-ol-3(R)-O-
b
-D-glucopyranoside (trivi-
-L-arabinopyranosyl-
b-D-glucopyranoside (trivially named coreoside F), were isolated from buds of Coreopsis tinctoria
ally named coreoside E) and (6Z, 12E)-tetradecadiene-8,10-diyne-1-ol-3(R)-O-
1 / 2)-
b
(
16 December 2016
Nutt., together with one known compound, coreoside B. Their chemical structures were elucidated by
extensive spectroscopic analysis and on the basis of their chemical reactivities. Coreoside E exhibited
high levels of antimicrobial activity against Staphylococcus aureus and Bacillus anthracis with minimum
Accepted 29 December 2016
Available online xxx
inhibitory concentrations of 27 ± 0.27 and 18 ± 0.40
B showed weak antimicrobial activity against S. aureus and B. anthracis.
mM, respectively, whereas coreoside F and coreoside
Keywords:
Coreopsis tinctoria Nutt.
Asteraceae
©
2016 Elsevier Ltd. All rights reserved.
Polyacetylene glycosides
Coreoside E
Coreoside F
Coreoside B
Antimicrobial activity
1
. Introduction
family and is a small, glabrous, aromatic annual plant that is
distributed globally (Dias et al., 2010). It is known as “snow chry-
santhemum” or “snow tea” in China, and grows on the Karakorum
Mountains in southern Xinjiang at an altitude above 3000 m
(Zhang et al., 2013). C. tinctoria contains a variety of bioactive
phytochemicals, including phenolics, flavonoids, phenyl-
propanoids, sterols and polyacetylene glycosides (Ma et al., 2016;
Wang et al., 2015; Z aꢀ laru et al., 2014). C. tinctoria is traditionally
Polyacetylene glycosides are derivatives of polyacetylenes, a
class of compounds that contain two or more carbonecarbon triple
bonds in their carbon skeleton (Negri, 2015). Polyacetylene glyco-
sides are widely distributed in a number of vegetables and me-
dicinal plants. They are especially found in the families Asteraceae,
Apiaceae, Araliaceae, and Campanulaceae, with representative ex-
amples such as Daucus carota L., Apium graveolens L., Petroselinum
crispum Mill., Panax ginseng, and Carthamus tinctorius, Atractylodes
lancea (Baranska et al., 2005; He et al., 2011; Ji et al., 2010; Lee et al.,
used in folk medicine to treat several diseases, such as hyperten-
sion, hyperlipidemia, diarrhea and internal pain (Dias et al., 2010;
Z aꢀ laru et al., 2014). Although a few dozen papers have focused on
2
009; Silva et al., 2015; Zidorn et al., 2005). Previous studies re-
the bioactivities of C. tinctoria, the precise chemical identification of
the bioactives remain unclear.
ported that polyacetylene glycosides have a diverse range of bio-
logical effects, such as cytotoxicity (Park et al., 2002), anti-
inflammatory (Zhang et al., 2013), antimicrobial (Pellati et al.,
2
2
The studies herein showed that the methanol extract of
C. tinctoria buds exhibited significant antimicrobial activity against
S. aureus, and this extract was further investigated. Two new (and
one known) polyacetylene glycosides were isolated from the
bioactive extract and evaluated for antimicrobial activity. Described
are the separation process of the bioactives, and elucidation of their
structures based on their spectroscopic properties and chemical
reactivity.
006), anti-HIV (Zhang et al., 2002) and antiallergic (Wang et al.,
001) activities.
Coreopsis tinctoria Nutt. belongs to the Asteraceae/Compositae
*
Corresponding author.
E-mail address: xzhfood@njau.edu.cn (Z. Xin).
http://dx.doi.org/10.1016/j.phytochem.2016.12.023
031-9422/© 2016 Elsevier Ltd. All rights reserved.
0
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tinctoria Nutt., Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2016.12.023

2
J. Guo et al. / Phytochemistry xxx (2017) 1e5
2
. Results and discussion
Correlation) spectra of 1 allowed the proton and carbon resonances
to be fully assigned (Fig.1). Key correlations from H-1 to C-3 and H-
0
2
.1. Structural elucidation of the polyacetylene glycosides
3 to C-1prime, were observed in the HMBC experiments, indicating
that the sugar was connected directly to the C-3 position of the
aglycone. The double bond between C-6 and C-7 was determined to
be in a cis configuration, because of the strong NOESY correlation
between H-6 and H-7, while the double bond between C-12 and C-
13 was assigned a trans configuration on the basis of the NOESY
Compound 1 was isolated as a brown amorphous powder. Its
28 7
molecular formula was determined to be C20H O based on high-
resolution electrospray ionization mass spectrometry (HRESIMS)
analysis of the pseudomolecular ion peak at m/z 403.1742 [M þ
þ
Na] (calcd. 403.1757), indicating seven degrees of unsaturation. Its
correlation between H-12 and H-14. The sugar unit was identified
3
UV spectrum showed strong absorption bands at 230, 262, 276, 294
and 313 nm, which are characteristic of an ene-diyne chromophore
as a
b
-glucopyranosyl group on the basis of its JH-1
0
,
H-2
0
coupling
constant of 7.84 Hz. Acid hydrolysis of 1 with 1 M HCl liberated
aglycone 1a and D-glucose (Fig. 1), which were identified using gas
chromatography-mass spectrometry (GC-MS) analysis of the cor-
responding trimethylsilyl L-cysteine derivatives that were directly
compared with authentic samples of these materials prepared in
the same manner. Both the sample prepared from 1 and D-glucose
provided a compound with the same retention time of 17.82 min,
indicating that compound 1 contained a D-glucose moiety. The
(Zhou et al., 2006). Its IR spectrum displayed strong absorption
ꢀ1
bands at 3326, 2311 and 1603 cm , corresponding to hydroxyl
groups, triple bonds and double bonds, respectively (Zhou et al.,
2
006). These data suggested that 1 was an ene-diyne compound.
1
The H NMR spectroscopic data showed four olefinic protons at
6.34 (m, 1H), 5.65 (m, 1H), 5.61 (m, 1H) and 6.32 (m, 1H), an
anomeric proton at
4.38 (d, J ¼ 7.84 Hz, 1H), and several sugar or
oxygenated protons between 3.1 and 4.0 (Fig. S1 and Table 1).
d
d
d
d
absolute configuration of compound 1 at C-3 was assigned as R by
13
ꢁ
The C NMR spectrum of 1 (Fig. S2) showed the presence of 20
carbon signals. Six of these were assigned to a sugar moiety,
comparison of its [
a]
D
value (ꢀ10.2 , c ¼ 1.08, MeOH) with that of
reference data (Umeyama et al., 2010). Based on these results,
including the anomeric carbon at
remaining 14 assigned to the aglycone moiety, the latter corre-
sponded to four acetylenic carbons at 78.9 (C-8), 71.8 (C-9), 71.7
C-10), and 79.1 (C-11), four olefinic carbons at 148.2 (C-6), 108.4
C-7), 109.4 (C-12), and 142.9 (C-13), two oxygenated carbons at
58.1 (C-1) and 76.5 (C-3), one methyl carbon at 17.4 (C-14), and
three methylene carbons at 36.8 (C-2), 34.0 (C-4), and 28.6 (C-5),
respectively. Taken together, these data indicated that 1 was a
d
102.5 (C-1prime), with the
compound 1 was elucidated to be (6Z, 12E)-tetradecadiene-8,10-
diyne-1-ol-3(R)-O-b-D-glucopyranoside, and trivially named cor-
eoside E (Fig. 1).
d
(
(
d
d
Compound 2 was isolated as a brown amorphous powder and
its molecular formula was determined to be C25 11 based on
36
H O
d
HRESIMS analysis of the pseudomolecular ion peak at m/z
þ
d
551.0295 [M þ K] (calcd. 551.0250). Its UV spectrum showed
typical ene-diyne absorption bands at 230, 262, 276, 294 and
313 nm. Its IR spectrum had absorbtion corresponding to hy-
droxyl, carboxyl, olefinic and glycosidic functionalities at 3425,
1
1
typical polyacetylene glycoside. Analysis of the He HeCOSY
Correlation Spectroscopy), HSQC (Heteronuclear Singular Quan-
(
ꢀ1
tum Correlation) and HMBC (Heteronuclear Multiple Bond
1719, 1653 and 1023 cm , respectively. Direct comparison of the
1
13
H and C NMR data for 2 (Fig. S3 and Fig. S4) with that of 1
indicated that they shared the same skeleton, with the sole dif-
ference being the monosaccharide sugar unit of 1 was replaced by
a disaccharide unit in 2. This assignment was supported by the
Table 1
1
NMR spectroscopic data for compounds 1 and 2 in DMSO-d
6
at 400 ( H) and
1
00 MHz (13C).
presence of two anomeric proton signals at
d
4.59 (d, J ¼ 6.72 Hz,
1
H), 4.49 (d, J ¼ 7.68 Hz, 1H), and two corresponding anomeric
1
2
0
00
carbon signals at 100.9 (C-1 ), 104.2 (C-1 ) (Table 1). The full
H
C
H
C
1
1
structure of compound 2 was further confirmed by He H COSY,
1
1
2
3
4
5
6
7
8
9
1
1
1
1
1
a
b
3.68 (m, 1H)
3.79 (m, 1H)
1.79 (m, 1H)
3.89 (m, 1H)
1.71 (m, 1H)
2.33 (m, 1H)
6.34 (m, 1H)
5.65 (m, 1H)
58.1
3.61 (m, 1H)
3.70 (m, 1H)
1.77 (m, 1H)
3.95 (m, 1H)
1.73 (m, 1H)
2.30 (m, 1H)
6.32 (m, 1H)
5.64 (m, 1H)
58.0
0
00
HSQC, and HMBC experiments. Key correlations from H-2 to C-1
00
0
and H-1 to C-2 were observed in the HMBC experiment, indi-
36.8
76.5
34.0
28.6
148.2
108.4
78.9
71.8
71.7
79.1
109.4
142.9
17.4
36.6
76.2
33.7
28.6
148.3
109.4
79.1
71.7
71.7
78.9
108.3
142.9
0
cating that the second sugar unit was connected to the C-2 po-
3
3
sition of the first sugar unit. The JH-1
constants of 6.72 and 7.68 Hz of the anomeric protons indicated
that the two sugar units were -glucopyranosyl and -arabino-
H-2
, ,
0 0 and JH-100 H-200 coupling
b
b
pyranosyl groups, respectively. The absolute configurations of the
two sugar units were identified as D-glucopyranosyl and L-ara-
binopyranosyl groups by the same procedures as used for com-
0
1
2
3
4
pound 1. The C-3 configuration was also assigned as R by
5.61 (m, 1H)
6.32 (m, 1H)
1.84 (d, J ¼ 6.76 Hz, 3H)
5.64 (m, 1H)
6.32 (m, 1H)
ꢁ
comparison of the [
a
]
D
value (ꢀ7.2 , c ¼ 1.15, MeOH) with that of
reference data (Umeyama et al., 2010). Consequently, compound 2
1.83 (d, J ¼ 6.84 Hz, 3H)
17.4
Glc
was determined as (6Z, 12E)-tetradecadiene-8,10-diyne-1-ol-
0
0
0
0
0
0
0
1
2
3
4
5
6
6
4.38 (d, J ¼ 7.84 Hz, 1H)
3.18 (t, J ¼ 8.20 Hz, 1H)
3.39 (m, 1H)
3.30 (m, 1H)
3.29 (m, 1H)
102.5
73.9
76.7
70.2
76.3
61.3
4.49 (d, J ¼ 7.68 Hz, 1H)
3.26 (m, 1H)
100.9
81.3
76.3
70.0
76.8
61.2
3(R)-O-
trivially named coreoside F (Fig. 1).
Compound 3 was also isolated as a brown amorphous powder.
Its molecular formula was established as C25 12 based on the
b-L-arabinopyranosyl-(1 / 2)-b-D-glucop-yranoside, and
3.40 (m, 1H)
3.36 (m, 1H)
3.28 (m, 1H)
3.79 (m, 1H)
36
H O
a
b
3.71 (m, 1H)
3.87 (m, 1H)
HRESIMS analysis of the pseudomolecular ion peak at m/z 551.1372
þ
3.92 (m, 1H)
[
M þ Na] (calcd. 551.1360) and NMR data analysis. Its UV spectrum
Ara
showed strong absorption bands at 239, 247, 278, 294 and 313 nm,
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
5
4.59 (d, J ¼ 6.72 Hz, 1H)
3.60 (m, 1H)
104.2,
72.7
68.0
71.8
65.5
indicating that 3 was also a polyacetylene glycoside. A careful
1
13
3.53 (m, 1H)
3.86 (m, 1H)
3.71 (m, 1H)
3.55 (m, 1H)
comparison of the H and C NMR spectra of 2 and 3 indicated very
similar signals, with the exception of a doublet corresponding to
a
b
the 14-CH
3
proton of 2 at 1.83 (d, J ¼ 6.84 Hz, 3H) being replaced by
a hydroxymethyl signal at 4.05 (s, 1H) in the spectra of 3.
Please cite this article in press as: Guo, J., et al., Isolation, characterization and antimicrobial activities of polyacetylene glycosides from Coreopsis
tinctoria Nutt., Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2016.12.023

J. Guo et al. / Phytochemistry xxx (2017) 1e5
3
Fig. 1. Compounds isolated from the methanol extract of C. tinctoria and key COSY and HMBC correlations for compounds 1 and 3.
Furthermore, the carbon signal at 17.4 (C-14) in the spectrum of 2
2.2. Antimicrobial bioassays
had been replaced by a carbon signal at 61.2 in the spectrum of 3,
indicating that the methyl group at the C-14 position of 2 had been
All three compounds were tested for antimicrobial activity
against nine pathogenic strains and the MICs values of the com-
pounds are summarized in Table 2. Compound 1 showed high
levels of antimicrobial activity d as compared to positive controls,
ampicillin and gentamicin d against Staphylococcus aureus and
replaced with a hydroxymethyl moiety in 3. Four olefinic protons in
1
the H NMR at
d
6.39 (m, 2H), 5.87 (d, J ¼ 15.76 Hz, 1H), and 5.70 (d,
J ¼ 15.68 Hz, 1H) were assigned to trans double bonds because of
their large coupling constants, suggesting that 3 possesses a (6E,
1
2E)-tetradecadiene-8,10-diyne-1,3-diol aglycone. The anomeric
Bacillus anthracis, with MICs values of 27 ± 0.27
18 ± 0.40 M, respectively. Compounds 2 and 3 both showed weak
antimicrobial activity against S. aureus and B. anthracis.
Separation of the methanol extract of C. tinctoria buds allowed
the isolation of two new and one known polyacetylene glycoside.
The structures of these compounds were elucidated using a com-
bination of spectroscopic methods and chemical analysis. To the
best of our knowledge, this is the first report concerning isolation of
compounds 1 and 2 from C. tinctoria.
mM and
carbon configuration of the two sugar units were determined as
glucopyranosyl and
and JH-100
The
b
0 0
,
H-2
-
m
3
b
-arabinopyranosyl groups from their JH-1
H-200 coupling constants of 7.76 and 6.56 Hz, respectively.
-glucopyranosyl and -arabinopyranosyl groups of compound
were confirmed as D-glucose and L-arabinose using the same
3
,
b
b
3
procedures as those used for compounds 1 and 2. The absolute
configuration of C-3 in compound 3 was assigned as R by com-
parison of the [
D
a] value with that of reference data. Based on these
data and a comparison with data from the literature (Zhang et al.,
2
013), compound 3 was identified as coreoside B (Fig. 1).
As the main source of polyacetylenes, a large number of poly-
Table 2
Antimicrobial activity of the compounds isolated from C. tinctoria.
acetylene glycosides have been isolated from the plants of the
Asteraceae family over the last 15 years (Negri, 2015). Most of these
compounds have 10,13 or 15 carbon atoms in their carbon skeleton,
namely C10, C13, and C15 polyacetylene glycosides (Negri, 2015). It is
relatively uncommon to isolate C14 polyacetylene glycosides,
especially with cis double bonds, from plants of the Asteraceae
family. Previous studies have shown that plant species of this
family can synthesize the same kind of polyacetylenes (Zhang et al.,
Microorganisms Minimum inhibitory concentration (MIC, mM)
1
2
3
Ampicillin Gentamicin
M. smegmatis
S. aureus
e
e
e
43 ± 0.65 14 ± 0.35
27 ± 0.27 93 ± 0.34 85 ± 0.55 43 ± 0.27 28 ± 0.37
B. subtilis
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
86 ± 0.28 28 ± 0.78
22 ± 0.44 14 ± 0.31
86 ± 0.46 14 ± 0.61
43 ± 0.35 7 ± 0.28
86 ± 0.48 14 ± 0.19
22 ± 0.40 14 ± 0.35
C. perfringens
M. tetragenus
C. albicans
M. phlei
2
013). The results of this study broaden the information of poly-
acetylene glycosides from plants of the Asteraceae family and may
help to identify new polyacetylene glycosides from this family.
E.coli
B. anthracis.
18 ± 0.40 73 ± 0.52 68 ± 0.36 43 ± 0.62 14 ± 0.26
Note: The minus (ꢀ) means no inhibition was observed.
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tinctoria Nutt., Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2016.12.023

4
J. Guo et al. / Phytochemistry xxx (2017) 1e5
B. anthracis is the causative agent of anthrax, which is a highly
was extracted with equal volumes of MeOH (X 3) at room tem-
lethal zoonotic disease found around the world (Wenner and
Kenner, 2004). Recently, B. anthracis has been used as a bio-
terrorist agent in the USA and Europe (Hicks et al., 2012; Irenge and
Gala, 2012). Although front-line antibiotics have been found that
can treat anthrax, B. anthracis strains are increasingly becoming
resistant (Bouzianas, 2010) and it is crucial to find new antibiotics.
All compounds in this study showed antimicrobial activity against
B. anthracis and among them compound 1 showed potent activity,
with the potential to be used as a new antibiotic against B. anthracis.
The hydrolysates of these polyacetylene glycosides have structural
features similar to the parent compounds and may also show
antimicrobial activity (Zhang et al., 2013). However, the mechanism
of antibacterial activity and the toxicological effects of these com-
pounds remain unknown and require further study.
perature. The combined MeOH extraction solutions were filtered
and concentrated in vacuo at 45 C to give an extract (32 g).
ꢁ
The latter was separated into three fractions (Fr.1e3) using silica
gel CC (100 g silica gel, 300e400 mesh) by a step-wise gradient
elution of petroleum ether:acetone (100:0 to 0:100, v/v). Fractions
with antimicrobial activity were subsequently separated by a
bioassay-guided method.
Fraction 1 (7.6 g) was separated into two subfractions (Fr. 1-1
and Fr. 1-2) by silica gel CC using CHCl
3
:MeOH (10:1, v/v) as an
eluent. Fr. 1-1 (with antimicrobial activity) was then passed
through a silica gel column with a CHCl :MeOH eluent (5:1, v/v) to
3
yield compound 1 (4.5 mg).
Fraction 2 (9.4 g) was subjected to silica gel CC with a
CHCl :MeOH eluent (1:1, v/v) to obtain two subfractions, Fr. 2-1 and
Fr. 2-2 (both with weak antimicrobial activity). Fr. 2-1 was sepa-
rated by Sephadex LH-20 CC with a CHCl :MeOH eluent (1:1, v/v) to
give compound 2 (3.6 mg). Fr. 2-2 was purified by silica gel CC using
a CHCl :MeOH eluent (5:1, v/v) to obtain compound 3 (9.2 mg).
3
3
. Conclusions
3
Three polyacetylene glycosides with antimicrobial activity
3
against B. anthracis have been isolated from the buds of C. tinctoria
and chemically identified and characterized. Further studies are
needed to elucidate the mechanism of antibacterial activity of these
compounds.
4.4. Acid hydrolysis of compounds and absolute configuration
determination
The method used to determine the absolute configuration was
carried out as described (Deyrup et al., 2007) with minor modifi-
cations. Briefly, pure compound (2 mg) was individually dissolved
in MeOH prior to addition of 1 M HCl (2 mL). Each mixture was
4
. Experimental
4.1. General experimental procedures
ꢁ
heated at 85 C for 15 h, with the resulting solution extracted with
1H, 13C NMR spectra and two-dimensional NMR spectra,
equal volume of EtoAc. Each EtoAc solution was then individually
evaporated to remove approximately 90% of the solvent. Then each
remaining mixture was individually subjected to silica gel CC using
CHCl as eluent to give polyacetylene (1a obtained from compound
3
1 and 3a obtained from compound 3). Each aqueous layer of the
extraction was evaporated in vacuo to provide a residue, which was
including COSY, HSQC and HMBC, were obtained on Bruker Avance
00, 400, or 500 spectrometers (Bruker BioSpin GmbH, Beijing,
China), using tetramethylsilane (TMS) as an internal standard. The
chemical shifts in the NMR spectra are recorded as values. UV
spectra were measured on a Beckman DU640 spectrophotometer
Beckman Coulter, Beijing, China). IR spectra were acquired using
3
d
(
2
repeatedly distilled to dryness from H O in vacuo until a neutral
KBr discs on a Nicolet Nexus 470 spectrophotometer (Thermo Sci-
entific, Beijing, China). Optical rotations were recorded on a Perkin-
Elmer 243B digital polarimeter. Electrospray ionization mass
spectrometry (ESI-MS) analyses were obtained using on a Q-Tof
Ultima Global GAA076 LC mass spectrometer (Waters Asia, Ltd.,
Singapore). Thin-layer chromatography (TLC) and column chro-
matography (CC) were performed on plates precoated with silica
residue was obtained. L-Cysteine methyl ester hydrochloride (2 mg
in 1 mL of anhydrous pyridine) was next added to each mixture and
stirred at 60 C for 1 h. A mixture of HMDS-TMCS (hexamethyldi-
ꢁ
silazane: trimethylchlorosilane ¼ 3:1, 300
the resulting mixtures stirred individually for another 30 min, with
hexane (3 mL) and H O (1 mL) then added. The hexane layer was
dried (anhyd. Na SO ) before being analyzed by GC-MS. Analysis
conditions were as follows: capillary column, HP-5MSi
(30 m ꢂ 0.25 mm, with a 0.25 m film, Agilent, USA); detection,
FID; injection temperature, 250 C; initial temperature 160 C,
mL) was also added, with
2
2
4
gel GF254 (10e40
Qingdao Marine Chemical Factory, Qingdao, China), reversed phase
C18 (Octadecylsilyl, ODS) silica gel (Silicycle, 50 m, Parc-
mm), and using silica gel (300e400 mesh,
m
ꢁ
ꢁ
m
ꢁ
ꢁ
ꢁ
Technologique Blvd, Canada) and Sephadex LH-20 (Sigma-
Aldrich, St. Louis, MO, USA), respectively.
ramped to 250 C at 15 C/min, and held at 250 C for 10 min.
Dimethyl sulfoxide-d
6
(DMSO-d
6
) and MeOH were purchased
4.5. Coreoside E (1)
from Merck (Darmstadt, Germany). All of the other chemicals and
solvents used in this study were of analytical grade.
Brown amorphous powder. [
a
]
D
: ꢀ10.2 (MeOH; c ¼ 1.08). UV
(
MeOH) max (ε): 230, 262, 276, 294, and 313 nm. IR (KBr) 3326,
l
ꢀ
1
1
13
4.2. Plant materials
2311 and 1603 cm . For H NMR and C NMR spectroscopic data,
þ
see Table 1. HRESI-MS m/z 403.1742 [M þ Na] (calcd. 403.1757 for
C. tinctoria was obtained from the Beiyuanchun farmers market
20 28 7
C H O ).
in Urumchi city of the Xinjiang Uygur Autonomous Region, China in
September 2012. Voucher specimens of C. tinctoria were identified
by Thomas Nuttall and are deposited in the Key Laboratory of Food
Processing and Quality Control, Nanjing Agricultural University,
with an index number of XJ8126.
1a. Hydrolysate of compound 1 (Fig. 1). Brown amorphous
1
powder. H NMR (300 MHz, MeOH, TMS):
d 6.44 (m, 2H), 5.92 (d,
J ¼ 15.92 Hz, 1H), 5.71 (d, J ¼ 16.00 Hz, 1H), 4.20 (m, 2H), 3.74 (m
13
5H), 2.30 (m, 2H), 2.06 (m, 1H), 1.64 (m, 2H), 1.53 (m, 2H). C NMR:
d
17.4, 36.4, 39.6, 59.7, 61.4, 68.7, 72.2, 73.5, 79.3, 80.1, 106.7, 108.2,
þ
1
47.7, 149.1; ESI-MS m/z 241.1 [M þ Na] .
4.3. Extraction and isolation
4.6. Coreoside F (2)
Fresh buds of C. tinctoria (2.5 kg) were dried, homogenized with
a Polytron homogenizer and extracted (X 3) with equal volumes of
cyclohexane at room temperature to remove oil. Then the residue
Brown amorphous powder. [
(MeOH) lmax (ε): 230, 262, 276, 294, and 313 nm. IR (KBr) 3425,
a
]
D
:ꢀ7.2 (MeOH; c ¼ 1.15). UV
Please cite this article in press as: Guo, J., et al., Isolation, characterization and antimicrobial activities of polyacetylene glycosides from Coreopsis
tinctoria Nutt., Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2016.12.023

J. Guo et al. / Phytochemistry xxx (2017) 1e5
5
ꢀ
1
1
13
1
719, 1653 and 1023 cm . For H NMR and C NMR spectroscopic
Appendix A. Supplementary data
þ
data, see Table 1. HRESI-MS m/z 551.0295 [M þ K] (calcd. 551.0250
for C25
36
H O
11).
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2016.12.023.
4.7. Coreoside B (3)
References
Brown amorphous powder. UV (MeOH)
l
max (ε): 239, 247, 278,
94, and 313 nm. H NMR (300 MHz, MeOH, TMS): 6.39 (m, 2H),
.87 (d, J ¼ 15.76 Hz, 1H), 5.70 (d, J ¼ 15.68 Hz, 1H), 4.59 (d,
Baranska, M., Schulz, H., Baranski, R., Nothnagel, T., Christensen, L.P., 2005. In situ
simultaneous analysis of polyacetylenes, carotenoids and polysaccharides in
carrot roots. J. Agric. Food. Chem. 53 (17), 6565e6571.
Bouzianas, D.G., 2010. Current and future medical approaches to combat the
anthrax threat. J. Med. Chem. 53 (11), 4305e4331.
1
2
5
d
J ¼ 6.56 Hz, 1H), 4.50 (d, J ¼ 7.76 Hz, 1H), 4.15 (m, 2H), 4.05 (s, 1H),
3
2
3
.89 (m, 6H), 3.70 (m, 4H), 3.40 (dd, J ¼ 6.30, 12.63 Hz, 2H), 3.27 (m,
Deyrup, S.T., Gloer, J.B., O'Donnell, K., Wicklow, D.T., 2007. Kolokosides A-D: tri-
terpenoid glycosides from a Hawaiian isolate of Xylaria sp. J. Nat. Prod. 70,
13
H), 2.33 (m, 2H), 1.81 (m, 3H), 1.68 (m, 3H). C NMR:
d 28.6, 33.7,
378e382.
6.6, 58.0, 61.2, 65.5, 68.0, 70.0, 71.7, 71.7, 72.7, 73.5, 74.1, 76.2, 76.3,
Dias, T., Bronze, M.R., Houghton, P.J., Mota-Filipe, H., Paulo, A., 2010. The flavonoid-
rich fraction of Coreopsis tinctoria promotes glucose tolerance regain through
pancreatic function recovery in streptozotocin-induced glucose-intolerant rats.
J. Ethnopharmacol. 132 (2), 483e490.
Gomez, S., Cosson, C., Deschamps, A.M., 1997. Evidence for a bacteriocin-like sub-
stance produced by a new strain of Streptococcus sp., inhibitory to gram-positive
foof-borne pathogens. Res. Microbiol. 1997 (148), 757e766.
He, J., Shen, Y., Jiang, J.S., Yang, Y.N., Feng, Z.M., Zhang, P.C., Yuan, S.P., Hou, Q., 2011.
New polyacetylene glucosides from the florets of Carthamus tinctorius and their
weak anti-inflammatory activities. Carbohydr. Res. 346 (13), 1903e1908.
Hicks, C.W., Sweeney, D.A., Cui, X., Li, Y., Eichacker, P.Q., 2012. An overview of
anthrax infection including the recently identified form of disease in injection
drug users. Intensive. Care. Med. 38 (7), 1092e1104.
7
6.8, 78.3, 79.8, 81.3, 100.9, 104.2, 107.6, 108.2, 145.9, 148.6; HRESI-
þ
MS m/z 551.1372 [M þ Na] (calcd. 551.1360 for C25
36
H O12).
3
a. Hydrolysate of compound 3 (Fig. 1). Brown amorphous
1
powder. H NMR (300 MHz, MeOH, TMS):
d 6.35 (m, 2H), 5.68 (d,
J ¼ 15.54 Hz, 1H), 3.65 (m 1H), 2.27 (m, 1H), 1.89 (m, 1H), 1.82 (dd,
13
J ¼ 1.83, 6.84 Hz, 1H), 1.62 (m, 2H), 1.54 (m, 2H). C NMR:
d
36.4,
3
1
9.6, 59.7, 61.4, 68.7, 72.2, 73.5, 74.1, 79.3, 80.1, 106.7, 108.2, 147.7,
ꢀ
49.1; ESI-MS m/z 233.1 [M e H] .
4
.8. Antimicrobial bioassays
Irenge, L.M., Gala, J.-L., 2012. Rapid detection methods for Bacillus anthracis in
environmental samples:
411e1422.
a review. Appl. Microbiol. Biotechnol. 93 (4),
1
Antimicrobial bioassays was carried out individually with each
pure compound using the disc diffusion method described by
Gomez et al. (1997). Briefly, each agar cup (6 mm diameter) was
Ji, Y., Feng, X., Xiao, C.C., Dong, Y.F., Wang, Q.Z., Wang, M., Zhao, Y.Y., 2010. A new
polyacetylene glycoside from the rhizomes of Atractylodes lancea. Chin. Chem.
Lett. 21 (7), 850e852.
Lee, S.M., Bae, K.H., Sohn, H.J., 2009. Panaxfuraynes A and B, two new tetrahy-
drofuranic polyacetylene glycosides from Panax ginseng C. A. Meyer. Tetrahe-
dron Lett. 50 (4), 416e418.
filled with a 100
mL solution of one of the test compounds dissolved
in 5% DMSO at concentrations of 2, 4, 8, 16, 32, 64, 128 and 256
m
M.
Cups containing DMSO and commercial antimicrobial agents,
ampicillin and gentamicin (National Institute for the Control of
Pharmaceutical and Biological products, China), were used as
negative and positive controls, respectively. The nine test micro-
organisms used were Mycobacterium smegmatis (CMCC 93321),
S. aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), Clostridium
perfringens (ATCC 13124), Micrococcus tetragenus (ATCC 35098),
Candida albicans (CMCC 98001), Mycobacterium phlei (AS 4.1180),
Escherichia coli (ATCC 25922) and B. anthracis (NX 1.1230). The
tested strains were cultivated in LuriaeBertani (LB) agar plates for
Ma, Z., Zheng, S., Han, H., Meng, J., Yang, X., Zeng, S., Zhou, H., Jiang, H., 2016. The
bioactive components of Coreopsis tinctoria (Asteraceae) capitula: antioxidant
activity in vitro and profile in rat plasma. J. Funct. Foods 20, 575e586.
Negri, R., 2015. Polyacetylenes from terrestrial plants and fungi: recent phyto-
chemical and biological advances. Fitoterapia 106, 92e109.
Park, J., Min, B., Jung, H., Kim, Y., Lee, H., Bae, K., 2002. Polyacetylene glycosides from
Gymnaster koraiensis. Chem. Pharm. Bull. 50, 685e687.
Pellati, F., Cal oꢁ , S., Benvenuti, S., Adinolfi, B., Nieri, P., Melegari, M., 2006. Isolation
and structure elucidation of cytotoxic polyacetylenes and polyenes from Echi-
nacea pallida. Phytochemistry 67 (13), 1359e1364.
Silva, D.B., Rodrigues, E.D., da Silva, G.V., Lopes, N.P., de Oliveira, D.C., 2015. Post-
column sodiation to enhance the detection of polyacetylene glycosides in LC-
DAD-MS analyses: an example from Bidens gardneri (Asteraceae). Talanta 135,
87e93.
Umeyama, A., Matsuoka, N., Mine, R., Nakata, A., Arimoto, E., Matsui, M., Shoji, N.,
Arihara, S., Takei, M., Hashimoto, T., 2010. Polyacetylene diols with anti-
proliferative and driving Th1 polarization effects from the marine sponge
Callyspongia sp. J. Nat. Med. 64 (1), 93e97.
Wang, N., Yao, X., Ishii, R., Kitanaka, S., 2001. Antiallergic agents from natural
sources.3. Structures and inhibitory effects on nitric oxide production and
histamine release of five novel polyacetylene glucosides from Bidens parviflora
willd. Chem. Pharm. Bull. 49 (8), 938e942.
ꢁ
the bacteria and in YPD agar plates for C. albicans at 37 C. After
incubating for 24 h, the zones of inhibition (mm in diameter) were
recorded and the minimal inhibitory concentrations (MICs) values
determined.
4.9. Statistical analysis
Wang, W., Chen, W., Yang, Y., Liu, T., Yang, H., Xin, Z., 2015. New phenolic com-
pounds from Coreopsis tinctoria Nutt. and their antioxidant and angiotensin i-
converting enzyme inhibitory activities. J. Agric. Food. Chem. 63 (1), 200e207.
Wenner, K.A., Kenner, J.R., 2004. Anthrax. Dermatol. Clin. 22 (3), 247e256.
Z aꢀ laru, C., Cri s¸ an, C.C., C ꢀa linescu, I., Moldovan, Z., T¸ a^ rcomnicu, I., Litescu, S.C.,
The resulting data are presented as means ± standard deviation
in the Supporting information.
Tatia, R., Moldovan, L., Boda, D., Iovu, M., 2014. Polyphenols in Coreopsis tinctoria
Nutt. fruits and the plant extracts antioxidant capacity evaluation. Central Eur. J.
Chem. 12 (8), 858e867.
Notes
The authors declare no competing financial interest.
Zhang, H., Lu, Z., Tan, G.T., Qiu, S., Farnsworth, N.R., Pezzuto, J.M., Fong, H.H.S., 2002.
Polyacetyleneginsenoside-Ro, a novel triterpene saponin from Panax ginseng.
Tetrahedron Lett. 43 (6), 973e977.
Acknowledgments
Zhang, Y., Shi, S., Zhao, M., Chai, X., Tu, P., 2013. Coreosides A-D, C14-polyacetylene
glycosides from the capitula of Coreopsis tinctoria and its anti-inflammatory
activity against COX-2. Fitoterapia 87, 93e97.
Zhou, Y.-Z., Ma, H.-Y.C.H., Qiao, L., Yao, Y., Cao, J.-Q., Pei, Y.-H., 2006. New acetylenic
glucosides from Carthamus Tinctorius. Chem. Pharm. Bull. 54 (10), 1455e1456.
Zidorn, C., J o€ hrer, K., Ganzera, M., Schubert, B., Sigmund, E.M., Mader, J., Greil, R.,
This work was financially sponsored by the Chinese National
Natural Science Fund (Grant number 31260377), and by the Fund
for Qing Lan Project of Jiangsu Province, and by special funds of
agro-product quality safety risk assessment of Ministry of Agri-
culture of the People's Republic of China (GJFP201601106).
Ellmerer, E.P., Stuppner, H., 2005. Polyacetylenes from the Apiaceae vegetables
carrot, celery, fennel, parsley, and parsnip and their cytotoxic activities. J. Agric.
Food Chem. 53 (7), 2518e2523.
Please cite this article in press as: Guo, J., et al., Isolation, characterization and antimicrobial activities of polyacetylene glycosides from Coreopsis
tinctoria Nutt., Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2016.12.023