Phytochemistry 170 (2020) 112196

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Sesquiterpene lactones from Sonchus palustris L. (Asteraceae, Cichorieae) T

a a b c c
Oleksandr Shulha , Serhat Sezai Çiçek , Simona Piccolella , Lucie Rárová , Miroslav Strnad ,
Frank Sönnichsend, Severina Pacificob, Christian Zidorna,∗
a
Pharmazeutisches Institut, Abteilung Pharmazeutische Biologie, Christian-Albrechts-Universität zu Kiel, Gutenbergstraße 76, 24118, Kiel, Germany
b
Department of Environmental Biological and Pharmaceutical Sciences and Technologies, University of Campania Luigi Vanvitelli, Via Vivaldi 43, 81100, Caserta, Italy
c
Laboratory of Growth Regulators, Faculty of Science, Palacký University, and Institute of Experimental Botany of the Czech Academy of Sciences, Šlechtitelů 27, CZ-
78371, Olomouc, Czech Republic
d
Otto Diels Institute for Organic Chemistry, University of Kiel, Otto-Hahn-Platz 4, 24118, Kiel, Germany

A R T I C LE I N FO A B S T R A C T

Keywords: Seven previously undescribed sesquiterpene lactones, three known sesquiterpene lactones (ixerin D, 15-p-hy-
Sonchus palustris droxyphenylacetyllactucin, and 15-p-hydroxyphenylacetyllactucin-8-sulfate), and two known quinic acid deri-
Sesquiterpene lactones vatives (3-O-feruloylquinic acid and 3,5-di-O-caffeoylquinic acid) were isolated from Sonchus palustris L. roots.
Quinic acid derivatives Four formerly undescribed compounds were elucidated to be 3β,14-dihydroxycostunolide-3-O-β-D-glucopyr-
Chemophenetics
anosyl-(2-O-p-hydroxyphenylacetyl)-14-O-p-hydroxyphenylacetate, 15-p-methoxyphenylacetyllactucin, 15-p-
Schleswig-Holstein
Biological activities
methoxyphenylacetyllactucin-8-sulfate, and 8-p-hydroxyphenylacetyllactucin-15-sulfate. Additionally, three
undescribed conjugates of lactucin and a eudesmanolide type sesquiterpenic acid, sonchpalustrin, 4″-O-me-
thylsonchpalustrin, and isosonchpalustrin, were characterized. The structures of the newly discovered natural
products were elucidated using 1D and 2D NMR spectroscopy and UHPLC-HRMS. 15-p-
Hydroxyphenylacetyllactucin and 15-p-methoxyphenylacetyllactucin showed significant in vitro cytotoxicity
against CEM and BJ cells with IC50 values ranging from 3.9 to 9.8 μM. Compounds 3 and 4 showed also strong
anti-inflammatory activity in vitro.

1. Introduction recent review on the chemistry of the Cichorieae of Schleswig-Holstein,

Sonchus palustris L., English vernacular name marsh sow thistle, is a
tall (80–200 cm) perennial herb which is native to temperate parts of
Europe, Russia, and Central Asia (Kirpicznikov, 2001). S. palustris pre-
fers wet and nitrogen rich habitats; the species tolerates mildly brackish
water (Jäger, 2017).
Existing data on the natural products and bioactivity of Sonchus
species were summarized by Li and Yang (2018). Some Sonchus species
have been used in folk medicine to treat fever, bronchitis, and in-
flammation. Pharmacological studies of different plant extracts showed
anticancer, antioxidant, anti-inflammatory, antimicrobial, and hepato-
protective activities. Phytochemical investigations identified sesqui-
terpene lactones, quinic acid esters, flavonoids, triterpenes, and cou-
marins in different Sonchus species. Previous phytochemical studies
regarding sesquiterpene lactones within the genus Sonchus have been
compiled by Zidorn (2008) and Shulha and Zidorn (2019). Within the
genus Sonchus, eudesmanolides are the main type of sesquiterpene
lactones, but some taxa also contain guaianolide type sesquiterpene
lactones. S. palustris has not been phytochemically investigated yet. A
where plants for the study presented here were collected, also contains
no data for S. palustris, but summarizes natural products occurring in
related taxa from the same region (Zidorn, 2019a).
The purpose of this investigation was to chemophenetically (Zidorn,
2019b) characterize the roots of S. palustris. Moreover, we wanted to
assess the main constituents for their potential bioactivity. Thus, this
paper describes isolation and structure elucidation of seven undescribed
sesquiterpene lactones (1, 4, and 6–10) together with five known
compounds (2, 3, 5, 11, and 12) (Fig. 1). UHPLC traces recorded for
acetone and methanol extracts from roots of Sonchus palustris, with as-
signed compounds 1-12, are depicted in Fig. 2. Cytotoxic activity of
3β,14-dihydroxycostunolide-3-O-β-D-glucopyranosyl-(2-O-p-hydro-
xyphenylacetyl)-14-O-p-hydroxyphenylacetate (1), ixerin D (2), 15-p-
hydroxyphenylacetyllactucin (3), 15-p-methoxyphenylacetyllactucin
(4), and 15-p-hydroxyphenylacetyllactucin-8-sulfate (5) was also as-
sessed.

∗
Corresponding author.
E-mail address: czidorn@pharmazie.uni-kiel.de (C. Zidorn).

https://doi.org/10.1016/j.phytochem.2019.112196
Received 18 June 2019; Received in revised form 30 October 2019; Accepted 1 November 2019
0031-9422/ © 2019 Elsevier Ltd. All rights reserved.
O. Shulha, et al.
Fig. 1. Structures of sesquiterpene lactones 1–10 isolated from roots of Sonchus palustris.
2. Results and discussion
2.1. Structure elucidation
Compound 1 was isolated as a yellow powder. Its molecular formula
was determined as C37H42O13 by HR-ESI-MS (m/z 693.2563 [M–H]–;
calculated for C37H41O13, 693.2553). The 1H NMR spectrum (all shift
values in ppm) showed twelve olefinic protons at δH 7.11 (2H, dt,
J = 8.5, 2.0 Hz), 7.06 (2H, dt, J = 8.5, 2.0 Hz), 6.75 (2H, dt, J = 8.5,
2.0 Hz), 6.69 (2H, dt, J = 8.5, 2.0 Hz), 6.20 (1H, d, J = 3.5 Hz), 5.58
(1H, d, J = 3.5 Hz), 5.09 (1H, dd, J = 4.0, 12.0 Hz), and 5.01 (1H, dd,
J = 10.5, 1.0 Hz); six oxygenated methines at δH 4.78 (1H, dd, J = 8.0,
9.5 Hz), 4.54 (1H, m), 4.48 (1H, m), 3.52 (1H, m), 3.36 (1H, m), and
3.26 (1H, m); one anomeric proton at δH 4.37 (1H, d, J = 8.0 Hz); four
oxygenated methylene protons at δH 4.56 (1H, m), 4.42 (1H, m), 3.88
(1H, dd, J = 2.5, 12.0 Hz), and 3.66 (1H, m); ten methylene protons at
δH 3.61 (2H, d, J = 5.5 Hz), 3.56 (2H, s), 2.46 (1H, m), 2.41 (1H, m),
2.23 (1H, m), 1.98 (1H, m), 1.92 (1H, m), 1.45 (1H, m); one methine at
δH 2.58 (1H, m), and one methyl group at δH 1.30 (3H, d, J = 1.0 Hz).
13
C NMR (all shift values in ppm) spectra, which were interpreted
taking also into account data from HSQC and HMBC experiments, in-
dicated three carboxylic carbons at δC 173.8, 173.2, and 172.7; signals
assignable to two largely overlapping p-substituted aromatic systems at
δC 157.9 (representing one quaternary carbon), 157.8 (representing one
quaternary carbon), 131.7 (representing four tertiary carbons), 126.3
(representing four tertiary carbons), and 116.6 (representing two qua-
ternary carbons); six olefinic carbons at δC 142.0, 141.6, 136.7, 132.3,
128.3, and 121.0; one anomeric carbon at δC 100.4; six oxygenated
methines at δC 84.0, 83.0, 78.3, 76.3, 75.7, 72.0; two oxygenated me-
thylenes at δC 63.0, and 62.8; one methine carbon at δC 50.8; five
methylene carbons at δC 41.7, 41.4, 38.0, 33.8, and 30.2, and one
methyl carbon at δC 11.9. These data were compatible with a 12,6-
germacranolide substituted with a glucose moiety and two p-hydro-
xyphenylacetyl moieties. Closer inspection of the signals assignable to
the sesquiterpene lactone moiety revealed that they were very similar
to data reported for 3β,14-dihydrocostunolide-3-O-β-glucopyranoside
and quasi identical (apart from slight differences caused by the different
solvent used for NMR measurements) to 3β,14-dihydrocostunolide-3-O-
2
Phytochemistry 170 (2020) 112196
β-glucopyranosyl-14-O-p-hydroxyphenylacetate reported by Bitam
et al. (2008) for compounds isolated from Launaea arborescens (Batt.)
Murb. (Asteraceae, tribe Cichorieae, subtribe Hyoseridinae) (Kilian
et al., 2009). The coupling constant of H-1‴ (8.0 Hz) proved β-config-
uration of the anomeric proton. GC analysis of the octylated derivative
of the sugar moiety, after hydrolysis, confirmed the sugar to be D-glu-
cose (section 4.4). The HMBC correlation from H-1‴ to C-3 confirmed
that the glucose was connected to the aglycone at C-3. Four doublet of
triplets (at δH 7.11, 7.06, 6.75, and 6.69) confirmed the presence of two
para-substituted aromatic rings. Signals at δC 157.9 and 157.8 indicated
the presence of two phenol moieties. COSY correlations from H-2'/H-6′
to H-7′ and from H-2''/H-6″ to H-7″, as well as HMBC signals from H-7′
to C-8′ and from H-7″ to C-8″, suggested two p-hydroxyphenylacetyl
moieties. HMBC correlations from H-14α/H-14β to C-8′ and from H-2‴
to C-8″ established the connections of the two p-hydroxyphenylacetyl
moieties to O-14 of the aglycone and to O-2‴ of the glucose moiety,
respectively. Thus, the structure of 1 was elucidated to be 3β,14-di-
hydroxycostunolide-3-O-β-D-glucopyranosyl-(2-O-p-hydro-
xyphenylacetyl)-14-O-p-hydroxyphenylacetate. Double bond geometry
in the ten-membered ring was confirmed to be identical with the one
assumed by Bitam et al. (2008), C-1 (10) and C-4 (5) Z and E, respec-
tively, by NOESY correlations between H-14 and H-2a and between H-6
and H-15. Stereochemistry at C-6 as assumed by Bitam et al. (2008) was
in line with the observed coupling constants. Moreover, both the ste-
reochemistry at C-3 and C-6 was confirmed by NOEs between protons at
C-3 and C-5 on the one hand and between protons at C-6 and C-15 on
the other hand. The stereochemistry of the previously undescribed
sesquiterpene lactone derivative is thus not only identical with its im-
mediate mono- and di-deacylated analogues 3β,14-dihydrocostunolide-
3-O-β-glucopyranosyl-14-O-p-hydroxyphenylacetate and 3β,14-dihy-
drocostunolide-3-O-β-glucopyranoside but also identical to all other so
far described more than 20 costunolide type germacranolides found in
the Cichorieae tribe of the Asteraceae family so far (Shulha and Zidorn,
2019).
Compounds 2 and 3 were identified as ixerin D (Asada et al., 1984)
and 15-p-hydroxyphenylacetyllactucin (Abdel-Mogib et al., 1993), re-
spectively, using NMR and HR-MS spectral data. These compounds
have been reported from the Cichorieae genera Hypochaeris, Ixeridium,
O. Shulha, et al. Phytochemistry 170 (2020) 112196

Fig. 2. UHPLC traces recorded for acetone and methanol extracts from roots of Sonchus palustris (experimental parameters are indicated in section 4.5.), respectively.
Compound numbers correspond to compounds 1–10 in Fig. 1 and 11 and 12 in the text, respectively.

Ixeris, Nabalus, Picris, and Taraxacum; and the genus Reichardia, re-
spectively (Shulha and Zidorn, 2019).
Compound 4 was isolated as a white powder. The molecular for-
mula C24H24O7 was determined by HR-ESI-MS (m/z 423.1451 [M–H]–;
calculated for C24H23O7, 423.1449). The 1H NMR spectrum indicated
seven olefinic protons at δH 7.22 (2H, dt, J = 8.5, 2.0 Hz), 6.89 (2H, dt,
J = 8.5, 2.0 Hz), 6.17 (1H, d, J = 1.5 Hz), 6.14 (1H, dd, J = 1.5,
3.0 Hz), and 6.03 (1H, dd, J = 1.5, 3.0 Hz); two oxygenated methylene
protons at δH 5.25 (1H, dd, J = 1.5, 17.5 Hz) and 4.90 (1H, d,
J = 17.5 Hz); two methines at δH 3.86 (1H, d, J = 10.0 Hz) and 3.04
(1H, tt, J = 3.0, 10.0 Hz); two oxygenated methines at δH 3.76 (1H, t,
J = 10.0 Hz) and 3.72 (1H, m); one methoxy group at δH 3.73 (3H, s);
four methylene protons at δH 3.72 (2H, m), 2.74 (1H, dd, J = 10.5,
13.5 Hz) and 2.30 (1H, dd, J = 2.0, 13.5 Hz), and a methyl group at δH
2.33 (3H, s). The 13C NMR spectral data revealed three carboxylic
carbon at δC 193.9, 170.9, and 168.6; twelve olefinic carbons at δC
167.1, 158.3, 147.8, 137.8, 132.5, 131.6, 130.5, 126.1, 121.7, and
113.8; three oxygenated carbons at δC 80.4, 66.4, and 63.1, and six
aliphatic carbons 56.2, 55.0, 48.4, 48.0, 39.2, and 21.2. These signals
suggested the presence of an acylated lactucin derivative similar to
compound 3 (Abdel-Mogib et al., 1993). Two doublets of triplets (at δH
7.22 and 6.89) suggested a 1,4-substituted benzene ring. COSY corre-
lations between H-2'/H-6′ and H-7′, H-2'/H-6′ and H-3'/H-5′, as well as
HMBC correlations from H-9′ to C-4′ and from H-7′ to C-8′ confirmed
the presence of a p-methoxyphenylacetyl moiety. Another HMBC cor-
relations from H-15 to C-8′ proved that lactucin is substituted at C-15.
Using all above mentioned data, the structure of 4 was elucidated as 15-
p-methoxyphenylacetyllactucin. This compound is a formerly un-
described natural product and is the 15-O-methyl derivative of 15-p-
hydroxyphenylacetyllactucin (3), which was so far only reported from

3
O. Shulha, et al.
whole plants of Reichardia tingitana (L.) Roth (Abdel-Mogib et al.,
1993).
The structure of compound 5 was elucidated to be 15-p-hydro-
xyphenylacetyllactucin-8-sulfate after comparing its NMR and HR-MS
spectra with literature data (Sessa et al., 2000).
Compound 6 was isolated as a yellow powder. Its molecular formula
was confirmed to be C24H24O10S using HR-ESI-MS (m/z 503.1017
[M–H]–; calculated for C24H23O10S, 503.1017). The NMR spectra were
very similar to those for 4, with a more de-shielded signal assignable to
proton H-8 (δH 4.42 in 6 instead of δH 3.72 in 4). ESI Q-TOF HRMS
spectra confirmed the presence of sulfate group in the molecule, while
the higher value of H-8 signal confirmed the position of this additional
moiety. Therefore, the structure of 6 was elucidated as 15-p-methox-
yphenylacetyllactucin-8-sulfate, a formerly undescribed natural pro-
duct.
Compound 7 was isolated as a yellow powder. The molecular for-
mula C38H44O14S was established using HR-ESI-MS (m/z 755.2398
[M–H]–; calculated for C38H43O14S, 755.2379). The NMR spectra
showed signals similar to compound 5, indicating the presence of lac-
tucin and p-hydroxyphenylacetyl moieties. In addition, MS and NMR
data indicated the presence of a second sesquiterpene moiety. This was
identified as a eudesmanic acid; 1H NMR data assignable to this moiety
displayed two olefinic protons at δH 6.29 (1H, m) and 5.74 (1H, s); two
hydroxylated methines at δH 5.19 (1H, td, J = 5.0, 11.0 Hz) and 3.23
(1H, d, J = 10.0 Hz); two methines at δH 2.52 (1H, tt, J = 3.5, 12.0 Hz)
and 1.25 (1H, dd, J = 2.5, 12.5 Hz); eight methylene protons at δH 2.01
(1H, dd, J = 5.0, 13.0 Hz), 1.96 (1H, dt, J = 3.5, 13.0 Hz), 1.79 (1H, br
d, J = 13.0 Hz), 1.68 (1H, dd, J = 2.5, 13.0 Hz), 1.52 (1H, m), 1.47
(2H, m), and 1.19 (1H, m); and two methyl groups at δH 1.15 (3H, s)
and 1.10 (3H, s). The 13C NMR spectrum indicated a carboxylic carbon
at δC 169.5; two olefinic carbons at δC 147.0 and 124.5; three oxyge-
nated carbons at δC 82.1, 73.6, and 73.3; seven aliphatic carbons at δC,
52.1, 45.7, 41.4, 41.1, 40.6, 28.1, and 27.6; and two methyl carbons at
δC 30.0 and 14.2. Positions of two methyl groups were confirmed by
HMBC correlations from H3-14′ to C-1′, C-5′, and C-9′, and from H3-15′
to C-3′, C-4′, and C-5'. NOESY correlations between H-5′ and H-7′
suggested α-orientation of H-5'. Additional NOESY correlations between
H-1′ and H-5′ suggested α-orientation of H-1'. NOESY correlations be-
tween H3-14'/H3-15′ and H-2′, as well as the absence of correlations
between H3-14'/H3-15′ and H-5′ suggested beta-orientation for the
proton H-2′ and the methyl groups H3-14′ and H3-15'. The multiplet
type and the coupling constant of the signal assignable to H-1' (d,
10.0 Hz) together with the absence of a NOESY correlation between H-
1′ and H3-14′ confirmed the α-orientation of H-1'. In contrast, H-2′ had
to be β-oriented in order to explain the coupling constant of the signal
assignable to H-1'. Thus, the structure of the eudesmanic acid moiety
was established as 1β,2α-dihydroxyilicic acid. The most similar natural
products described so far are 1β-hydroxyilicic acid and 2α-hydro-
xyilicic acid (Abu Zarga et al., 1998, 2003). HMBC correlations from
the two protons at C-15 to C-12′ and from H-2′ to C-8″ suggested that
the additional acyl moiety was connected via O-15 of the lactucin
moiety and to the C-8′ of p-hydroxyphenylacetyl moiety. Based on these
data, compound 7 was identified as ((3aS,4S,9aS,9bR)-6-methyl-3-me-
thylene-2,7-dioxo-4-(sulfooxy)-2,3,3a,4,5,7,9a, 9b-octahydroazuleno
[4,5-b]furan-9-yl)methyl 2-((2R,4aR,5S,6S,8R,8aR)-5,8-dihydroxy-6-
(2-(4-hydroxyphenyl)acetoxy)-4a, 8-dimethyldeca-hydronaphthalen-2-
yl)acrylate. A related conjugate between lactucin and a eudesmanic
acid moiety, lactucin-8-O-hypoglabrate, was previously found in Hy-
pochaeris glabra L. (Asteraceae, Cichorieae) (Bohlmann et al., 1981).
Because of the prohibitively long systematic name and because even a
semi-trivial name based on lactucin, 1β-hydroxyilicic acid, p-hydro-
xyphenylacetic acid, and sulfate would be very long, we here propose
the new trivial name sonchpalustrin for this compound.
Compound 8 was obtained as a yellow powder. The molecular for-
mula C39H46O14S was established by HR-ESI-MS (m/z 769.2538 [M -
H]-; calculated for C39H45O14S, 769.2536). The 1H NMR spectra of 8
4
Phytochemistry 170 (2020) 112196
was similar to 7, but displaying an additional signal assignable to a
methoxy group at δH 3.77 (3H, s). The HMBC correlation from H3-9″ to
C-4″ established that the methoxy group was attached to O-4''. Thus, the
structure of 8 was elucidated as ((3aS,4S,9aS,9bR)-6-methyl-3-methy-
lene-2,7-dioxo-4-(sulfooxy)-2,3,3a,4,5,7,9a, 9b-octahydroazuleno [4,5-
b]furan-9-yl)methyl 2-((2R,4aR,5S,6S,8R,8aR)-5,8-dihydroxy-6-(2-(4-
methoxyphenyl)acetoxy)-4a, 8-dimethyldeca-hydronaphthalen-2-yl)ac-
rylate. As this compound is the 4″-O-methyl derivative of sonchpalus-
trin 7, we name compound 8 4″-O-methylsonchpalustrin.
Compound 9 was obtained as a yellow powder; its molecular for-
mula was established as C23H22O10S using HR-ESI-MS (m/z 489.0870
[M–H]–; calculated for C23H21O10S, 489.0861). The NMR and HR-MS
data were nearly identical to those of compound 5 (Sessa et al., 2000).
However, an HMBC correlation from H-8 to the carboxy group (C-8) of
the p-hydroxyphenylacetyl moiety proved that p-hydroxyphenylacetyl
and sulfate moieties were connected via O-8 (p-hydroxyphenylacetyl)
and O-15 (sulfate moiety), respectively, and thus had inverse positions
in comparison to compound 5. Conclusively, the structure of 9 was
elucidated as 8-p-hydroxyphenylacetyllactucin-15-sulfate, which re-
presents a formerly undescribed natural product.
Compound 10 was isolated as a yellow powder. Its molecular for-
mula C38H44O14S was established using HR-ESI-MS (m/z 755.2369
[M–H]–; calculated for C38H43O14S, 755.2379). The molecular weight
of 10 was identical to compound 7, but it showed higher shift at H-8 (δH
5.06, 1H, td, J = 2.0, 10.5 Hz) and lower shift at H-15 (δH 5.32, 1H, d,
J = 17.0 Hz; δH 4.90, 1H, m) in comparison with the respective signals
of 7. Absolute shift values of protons in positions 8 and 15 in com-
parison with compound 7 indicated that the substituents O-8 and O-15
were exchanged in compound 10 (in comparison to compound 7).
Unfortunately, in the HMBC spectra measured at our 400 MHz NMR
spectrometer, no direct HMBC correlation from H-8 to C-12′ was de-
tectable. In order to proof the assumed linkage, an additional experi-
ment using a 600 MHz NMR spectrometer (Department of Organic
Chemistry, Kiel University) was performed. In this HMBC experiment, a
correlation between H-8 and C-12′ was detectable and proved that the
eudesmanic acid moiety was connected via O-8 of the lactucin moiety.
Conclusively, 10 was identified as (3aR,4S,9aS,9bR)-6-methyl-3-me-
thylene-2,7-dioxo-9-((sulfooxy)methyl)-2,3,3a,4,5,7,9a, 9b-octahy-
droazuleno [4,5-b]furan-4-yl 2-((2R,4aR,5S,6S,8R,8aR)-5,8-dihydroxy-
6-(2-(4-hydroxyphenyl)acetoxy)-4a, 8-dimethyldecahydronaphthalen-
2-yl)acrylate, another formerly undescribed natural product. As com-
pound 10 is an isomer of compound 7 with exchanged substituents at
the lactucin core, we propose the name isosonchpalustrin for compound
10.
Two additional compounds were isolated and based on comparisons
of their MS and NMR data with literature data identified as 3-O-fer-
uloylquinic acid (11) (Dokli et al., 2013) and 3,5-di-O-caffeoylquinic
acid (12) (D'Amelio et al., 2015), respectively. All twelve compounds
reported here, were found in the S. palustris for the first time.
2.2. Cytotoxicity testing
Cytotoxicity assays of 3β,14-dihydroxycostunolide-3-O-β-D-gluco-
pyranosyl-(2-O-p-hydroxyphenylacetyl)-14-O-p-hydroxyphenylacetate
(1), ixerin D (2), 15-p-hydroxyphenylacetyllactucin (3), 15-p-methox-
yphenylacetyllactucin (4), and 15-p-hydroxyphenylacetyllactucin-8-
sulfate (5) against cancer and normal human cells in vitro revealed that
compounds 3 and 4 have a moderate activity against all three tested
cancer cell lines as well as against normal human fibroblasts.
Cytotoxicity assay results are summarized in Table 4 (see Fig. 2)
Potential decrease in the expression of ELAM was observed after
treatment with compounds 1, 2, 3, 4, and 5. Only compounds 3 and 4
inhibited effectively the level of cell surface adhesion protein ELAM by
41% (3) and 61% (4), respectively. The cell viability of endothelial cells
after 30 min of treatment and another 4 h of induction of inflammation
by TNFα was also weakly affected (Fig. 3).
O. Shulha, et al. Phytochemistry 170 (2020) 112196

Fig. 3. Expression of E-selectin using ELISA on the cell surface of endothelial cells pretreated with the compounds 3 and 4, and curcumin (10 μM) for 30 min and then
stimulated with 10 ng/mL TNFα for another 4 h was determined and compared with cell viability. Experiments were repeated three times in triplicates.

3. Conclusions investigated cancer cells, as well as for normal human skin fibroblasts
(BJ). These compounds also decreased the level of E-selectin on the cell
In total, twelve natural products were isolated from the roots of S. surface of HUVECs. Compound 4 was more active in comparison with
palustris in the first phytochemical investigation of this species. Seven compound 3 in the cytotoxicity test and also in the anti-inflammatory
compounds represent previously undescribed sesquiterpene lactones, assay. This might be explained by a lower polarity of compound 4 in
including three lactucin derivatives esterified with a eudesmanic acid comparison with 3 due to the additional methoxy group in its structure.
moiety (7, 8, and 10). So far dimeric sesquiterpenoids have been re- Compounds 1, 2, and 5 were significantly less active, again, probably
ported only from a limited number of Cichorieae species. Dimers in- because of their higher polarity, due to glucose and sulfate moieties,
clude compounds, which are linked via ester, ether, and C–C bonds respectively.
(Shulha and Zidorn, 2019).
Sulfated sesquiterpene lactones are quite rare in Cichorieae species,
4. Experimental
and only nine sesquiterpene lactones with a sulfate moiety were pre-
viously found in this tribe (Shulha and Zidorn, 2019). In this paper, six
4.1. General experimental procedures
sulfated sesquiterpene lactones were isolated, and five among them
were previously undescribed. It seems that modern analytical separa-
Acetonitrile, acetone, ethyl acetate, dichloromethane, and methanol
tion and detection techniques favor the discovery of sulfated com-
were obtained from VWR (Darmstadt, Germany). Sephadex LH-20 (GE
pounds and thus, it can be expected that many more such compounds
Healthcare) was used for a column chromatography. Semi-preparative
will be detected in the future.
liquid chromatography was done using Waters e2695 separation
Chemophenetically, the genus Sonchus is in general characterized by
module equipped with DAD 2998 detector, Waters Fraction Collector III
a prevalence of eudesmanolides in its sesquiterpene lactone profiles
and VP 250/10 Nucleodur C18 5 μm column (Macherey-Nagel). AB
(Zidorn, 2008, 2019b; Shulha and Zidorn, 2019). In our phytochemical
SCIEX TripleTOF 4600 system coupled with Shimadzu Nexera X in-
investigation, only three of the ten isolated sesquiterpene lactones
strument and equipped with Phenomenex Luna Omega C18, 1.6 μm,
contained a eudesmanolide structure, while the majority of the ses-
50 × 2.1 mm column was used for UHPLC-HRMS/MS (ESI; +,–)
quiterpenoids, eight out of ten compounds, comprised (in three in-
analyses. A Bruker Ascend 400 spectrometer was used for NMR mea-
stances together with eudesmanolic acid moieties) a lactucin moiety as
surements. A Bruker Avance 600 spectrometer was used for the addi-
the basic structure. Our data presented here, confirm that even in re-
tional measurements of NMR spectra for compound 10. Plants were
gions with poor biodiversity and even in Europe, whose flora has been
ground using an IKA MF 10 basic mill. Ultrasonic treatment was per-
historically well studied, chemophenetic studies have the potential to
formed using Bandelin Sonorex RK 1028 CH bath. Welch LVS201T
detect new bioactive natural products.
vacuum pumps, Büchi B-100 bathes and Büchi R-100 evaporators were
Five of the isolated sesquiterpenoids (1–5) were tested with regards
used for evaporation. Merck TLC plates (Silica gel 60 F254) were used
to their cytotoxic activity against human acute lymphoblastic leukemia
for analysis. Detections were performed using a UV lamp at 254 nm and
(CEM), human breast adenocarcinoma (MCF7), and human cervical
366 nm followed by spraying with sulfuric acid-vanillin reagent (and
carcinoma (HeLa) cells and for anti-inflammatory activity in vitro.
then interpretation in day light). Small scale silica gel separations were
Results showed that compounds 3 and 4 are cytotoxic for all
performed at Büchi PrepChrom C-700 instrument equipped with a

5
O. Shulha, et al. Phytochemistry 170 (2020) 112196

Table 1 the roots were macerated with 5 l (5 × 1 l) of methanol during 24 h

NMR data of compound 1a. followed by 15 min of ultrasonic bath. Extracts were filtrated and
Position δC, type δH (J in Hz) evaporated under vacuum to obtain 4.60 g (acetone) and 28.8 g (me-
thanol) of residue.
sesquiterpene lactone moiety The acetone extract was suspended in methanol to receive 0.57 g of
1 132.3, CH 5.09, dd (4.0, 12.0)
methanol-soluble fraction. This fraction was applied on a Sephadex
2 33.8, CH2 2.23, m
2.41, mb
column (methanol, 1 m × 1 cm) to obtain 8 subfractions (S1–S8).
3 84.0, CH 4.48, dd (10.5, 6.0) Fraction S5 (133 mg) was separated using the same Sephadex column to
4 142.0, C receive 5 subfractions (S5A-S5E). Fraction S5C (40.0 mg) was subjected
5 128.3, CH 5.01, br d (10.5) to Phenomenex Aqua C18 250 × 10 mm column and eluted with
6 83.0, CH 4.54, dd (10.5, 8.5)
MeCN:H2O 55:45 v/v to obtain 13.0 mg of 1.
7 50.8, CH 2.58, m
8 30.2, CH2 1.92, mb A part of acetone extract, which was not soluble in methanol
1.45, mb (4.03 g) was subjected to Sephadex column (methanol, 1 m × 2 cm) to
9 eq. ax 38.0, CH2 2.46, mb collect 13 subfractions (A1-A13). Fraction A11 (32.0 mg) was fractio-
1.98, mb
nated on C18 Nucleodur 250 × 10 mm column using MeOH:H2O 65:35
10 136.7, C
11 141.6, C
v/v as a mobile phase to obtain 6.3 mg of 4. Fraction A13 (50.0 mg) was
12 172.7, C separated on C18 Nucleodur 250 × 10 mm column eluted with
13 121.0, CH2 6.20, d (3.5) MeOH:0.025% formic acid 55:45 v/v to gain 7.0 mg of 3. Fraction A14
5.58, d (3.5) (70.0 mg) was subjected to Sephadex column (MeOH:Acetone:H2O
14 63.0, CH2 4.56, d (12.5)
3:1:1, 1 m × 1 cm) to receive 12.0 mg of 5.
4.42, d (12.5)
15 11.9, CH3 1.30, d (1.0) Methanolic extract (28.84 g) was dispensed in water and partitioned
para-hydroxyphenylacetyl moiety 1 between H2O and EtOAc. The organic layer was evaporated under va-
1′ 126.3, Cb cuum yielding 3.5 g of residue. The EtOAc extract was subjected to si-
2′ 131.7, CHb 7.06, dt (8.5, 2.0) lica gel 35 g cartridge (dry load) and eluted with a gradient of
3′ 116.6, CHb 6.69, dt (8.5, 2.0)
4′ 157.9, Cb
CH2Cl2:MeOH (flow rate: 20 ml/min; 0 min–100% CH2Cl2, 5 min–95%
5′ 116.6, CHb 6.69, dt (8.5, 2.0) CH2Cl2, 30 min–95% CH2Cl2, 60 min–90% CH2Cl2, 80 min–80%
6′ 131.7, CHb 7.06, dt (8.5, 2.0) CH2Cl2, 110 min–50% CH2Cl2, 120 min–100% MeOH, 140 min–100%
7′ 41.7, CH2b 3.56, s MeOH) to obtain 16 subfractions (B1A-B1P). Fraction B1J (103 mg) was
8′ 173.8, C
separated on C18 Nucleodur 250 × 10 mm column eluted with
para-hydroxyphenylacetyl moiety 2
1″ 126.3, Cb MeOH:0.025% formic acid gradient (flow rate: 2 ml/min; 0 min–35%
2″ 131.7, CHb 7.11, dt (8.5, 2.0) MeOH, 15 min–35% MeOH, 20 min–45% MeOH, 30 min–45% MeOH)
3″ 116.6, CHb 6.75, dt (8.5, 2.0) to receive 17.8 mg of 2 and 14.9 mg of 11. Fractions B1N (102 mg) and
4″ 157.8, Cb B1O (155 mg) were combined, subjected to C18 Nucleodur
5″ 116.6, CHb 6.75, dt (8.5, 2.0)
6″ 131.7, CHb 7.11, dt (8.5, 2.0)
250 × 10 mm column and separated using gradient elution with
7″ 41.4, CH2b 3.61, d (5.5) MeOH:0.025% formic acid (flow rate: 2 ml/min; 0 min–35% MeOH,
8″ 173.2, C 15 min–35% MeOH, 20 min–45% MeOH, 30 min–45% MeOH) to re-
glucose moiety ceive 13.5 mg of 12. Fraction B1K (149 mg) was applied on Sephadex
1''' 100.4, CH 4.37, d (8.0)
column (acetone, 1 m × 2 cm) to obtain 11 subfractions (B1K1– B1K11).
2''' 75.7, CH 4.78, dd (8.0, 9.5)
3''' 76.3, CH 3.52, mb Fractions B1K6–B1K8 were combined and subjected to Sephadex column
4''' 72.0, CH 3.36, mb (methanol, 1 m × 1 cm) to receive 7.4 mg of 8 and 9.4 mg of 6. Fraction
5''' 78.3, CH 3.26, mb B1M (169 mg) was subjected to Sephadex column (methanol,
6''' 62.8, CH2 3.88, dd (2.5, 12.0) 1.7 m × 2 cm) to obtain 9.4 mg of 10, 9.0 mg of 7 and 8.3 mg of 9.
3.66, mb

a
Spectra were referenced to solvent residual and solvent signals of CD3OD at
3.31 ppm (1H NMR) and 49.15 ppm (13C NMR), respectively.
b
Overlapping signals.
Büchi FlashPure EcoFlex silica gel 35 g cartridge.
4.2. Plant material
Whole plants of Sonchus palustris L. were collected on the 13th of
July, 2016 at the Windebyer Noor, Eckernförde, Rendsburg-
Eckernförde, Schleswig-Holstein, Germany at 0 m above mean sea level
(coordinates: N 54°28′12.9''; E 09°47′36.0″). A voucher specimen was
deposited in the private herbarium of CZ (CZ-20160713A-1) and in the
Kiel University Herbarium (KIEL0005010). Whole plants for natural
product isolation were separated into aerial parts and roots and dried at
room temperature in dark.
4.3. Extraction and isolation
Roots were powdered with a mill. 176 g of powdered roots were
exhaustively extracted with 3 l of acetone (3 × 1 l) by 1 h maceration
and 15 min ultrasonic treatment each cycle. After acetone extraction
4.4. Sugar identification
The sugar part of 1 was identified using GC analysis of octylated
derivative according to Leontein et al. (1978) with some modifications.
5.0 mg of 1 and 1 ml 2M CF3COOH were mixed in a tightly closed glass
vial. The vial was heated at 120 °C for 1 h. Afterwards the mixture was
evaporated under reduced pressure three times, adding 5 ml water each
time. 1 ml (R)-(−)-2-octanol and one drop of conc. CF3COOH were
added for octylation. Then the mixture was again heated at 120 °C for
12 h. Subsequently, the mixture was transferred to a separation funnel
and separated three times between 20 mL MeOH and 20 ml n-hexane.
The methanolic part was evaporated under vacuum; for acetylation step
0.5 ml acetic anhydride and 0.5 ml pyridine were added and the mix-
ture was heated in a vial at 100 °C for 20 min. The mixture was then
cooled to the room temperature; 10 ml H2O, 1 ml CH2Cl2, and 1 ml
0.1 M H2SO4 were added to the reaction mixture and the glass was
actively shaken. The CH2Cl2 layer was transferred to a 1.5 ml vial for
GC analysis. For comparison, 5.0 mg of D-glucose were treated using
the same procedure. GC analysis was executed using a TG-5 GC column
(Thermo Scientific, 15 m × 0.25 mm × 0.25 μm) with the following
settings: injection volume 1 μl; flow rate 1.2 ml/min; mobile phase
helium; split ratio 1:10; ion source temperature 250 °C; injector tem-
perature 280 °C; MS transfer line temperature 250 °C; scanning range

6
O. Shulha, et al. Phytochemistry 170 (2020) 112196

Table 2
NMR data of compound 4a, 6b, and 9b.
Position 4 6 9

δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz)

lactucin moiety
1 131.6, CH 134.0, C 134.3, C
2 193.9, C 196.4, C 196.7, C
3 132.5, CH 6.17, br d (1.5) 134.6, CH 6.16, mc 134.8, CH 6.47, br d (1.5)
4 167.1, C 168.9, C 170.2, C
c
5 48.0, CH 3.86, d (10.0) 49.5, CH 3.68, m 49.9, CH 3.93, d (10.0)
6 80.4, CH 3.76, t (10.0) 82.5, CH 3.69, mc 82.4, CH 3.79, t (10.0)
7 56.2, CH 3.04, tt (3.0, 10.0) 56.5, CH 3.00, tt (2.0, 10.0) 55.4, CH 3.45, tt (3.0, 10.0)
8 66.4, CH 3.72, mc 74.8, CH 4.42, td (2.0, 11.0) 71.1, CH 4.88, mc
9 48.4, CH2 2.74, dd (10.5, 13.5) 45.8, CH2 3.10, dd (2.0, 13.5) 45.1, CH2 2.85, dd (10.5, 13.5)
2.30, dd (2.0, 13.5) 2.74, dd (11.0, 13.5) 2.45, mc
10 147.8, C 149.9, C 148.4, C
11 137.8, C 137.6, C 138.1, C
12 168.6, C 170.6, C 170.0, C
13 121.7, CH2 6.14, dd (1.5, 3.0) 123.9, CH2 6.40, dd (0.5, 3.0) 122.2, CH2 5.91, d (3.0)
6.03, dd (1.5, 3.0) 6.16, mc 5.26, mc
14 21.2, CH3 2.33, s 21.6, CH3 2.41, s 21.6, CH3 2.41, s
15 63.1, CH2 5.25, dd (1.5, 17.5) 64.8, CH2 5.25, dd (1.5, 17.0) 68.1, CH2 5.28, ddc (2.0, 17.0)
4.90, dd (1.0, 17.5) 5.06, dd (1.5, 17.0) 4.86, mc
para-hydroxy-/para-methoxy-phenylacetyl moiety
1′ 126.1, C 127.5, C 125.8, C
2′ 130.5, CHc 7.22, dt (8.5, 2.0) 131.7, CHc 7.22, dt (8.5, 2.0) 131.7, CHc 7.14, dt (8.5, 2.0)
3′ 113.8, CHc 6.89, dt (8.5, 2.0) 115.2, CHc 6.87, dt (8.5, 2.0) 116.6, CHc 6.75, dt (8.5, 2.0)
4′ 158.3, CH 160.6, C 158.1, C
5′ 113.8, CHc 6.89, dt (8.5, 2.0) 115.2, CHc 6.87, dt (8.5, 2.0) 116.6, CHc 6.75, dt (8.5, 2.0)
6′ 130.5, CHc 7.22, dt (8.5, 2.0) 131.7, CHc 7.22, dt (8.5, 2.0) 131.7, CHc 7.14, dt (8.5, 2.0)
7′ 39.2, CH2 3.72, mc 41.3, CH2 3.68, mc 41.7, CH2 3.63, s
8′ 170.9, C 172.8, C 172.7, C
9′ 55.0, CH3 3.73, s 55.9, CH3 3.79, s

a
Spectra were referenced to solvent residual and solvent signals of DMSO‑d6 at 2.50 ppm (1H NMR) and 39.51 ppm (13C NMR), respectively.
b
Spectra were referenced to solvent residual and solvent signals of CD3OD at 3.31 ppm (1H NMR) and 49.15 ppm (13C NMR), respectively.
c
Overlapping signals.

for full scan: (m/z) 45–700; ionization mode EI; temperature gradient:
0 min 60 °C, 2 min 60 °C, 4.6 min 180 °C, 5.1 min 180 °C, 38.5 min
280 °C, and 39.5 min 280 °C. Peaks with the characteristic m/z value
(m/z = 331) on chromatograms of D-glucose sample and compound 1
sample represented the same retention times (t = 20.6 min) and MS
spectra.
4.5. UHPLC analyses
UHPLC experiments were performed using a VWR Hitachi
Chromaster UltraRS liquid chromatograph equipped with ELSD 100
and DAD 6430 detectors. A Phenomenex Luna Omega C18, 1.6 μm,
100 × 2,1 mm column was used for the analyses with the following
settings: mobile phase A: 0.1% formic acid in water; mobile phase B:
100% acetonitrile; linear gradient: 0 min 5% B, 25 min 40% B, 35 min
60% B, 40 min 95% B, 50 min 95% B, 50.1 min stop, post time 10 min;
flow rate: 0.200 ml/min; injection volume: 2 μl; oven temperature:
30 °C.
4.6. Cytotoxicity testing
4.6.1. Cell cultures
The screening cell lines: T-lymphoblastic leukemia CEM; breast
carcinoma MCF7; cervical carcinoma cell line HeLa were purchased
from European Collection of Authenticated Cell Cultures (ECACC,
London, UK). Human foreskin fibroblasts BJ were obtained from the
American Type Culture Collection (Manassas, VA, USA). Cells were
cultured in DMEM (Dulbecco's Modified Eagle Medium) or RPMI 1640
medium (Sigma Aldrich, MO, USA). HUVEC cells were a kind gift from
Professor Jitka Ulrichová, Faculty of Medicine and Dentistry, Palacky
University, Olomouc. HUVECs were cultivated in ECPM medium
(Provitro, Berlin, Germany). Media used were supplemented with 10%
or 20% (CEM) fetal bovine serum, 2 mM L-glutamine, and 1% peni-
cillin-streptomycin. The cell lines were maintained under standard cell
culture conditions at 37 °C and 5% CO2 in humid environment. Cells
were sub-cultured twice or three times a week using the standard
trypsinization procedure.
4.6.2. Cytotoxicity assay
Cytotoxicity of tested compounds in above mentioned cell lines and
normal cells was determined by resazurin assay using manufacturer's
protocol (Sigma Aldrich, St. Louis, MO, USA). The procedure was de-
scribed earlier (Rárová et al., 2016), but it was performed using another
dye (resazurin). The data shown are means ± standard deviation (SD)
obtained from at least three independent experiments performed in
triplicates.
4.6.3. Cell-surface ELISA CD62E (E-Selectin, ELAM)
Enzyme-linked activity assay (ELISA) was used to detect the levels
of cell adhesion molecule ELAM on HUVECs after 30 min of incubation
with tested compounds and 4 h of stimulation with TNFα as described
earlier (Morrogh-Bernard et al., 2017). Experiments were repeated
three times in triplicate.
Cytotoxicity Testing. Calcein AM (Molecular Probes, Invitrogen,
Karlsruhe, Germany) cytotoxicity assays after 4 h of treatment in the
HUVECs were used to measure the cytotoxicity of extracts, fractions or
compounds for ELAM expression assay as described previously
(Morrogh-Bernard et al., 2017). Triplicates of at least three independent
experiments were used.
4.7. Compound 1 - 3β,14-dihydroxycostunolide-3-O-β-D-glucopyranosyl-
(2-O-p-hydroxyphenylacetyl)-14-O-p-hydroxyphenylacetate
1
Yellow powder; [α]20
D = + 28 (c 0.11, MeOH) for H NMR (CD3OD,

7
O. Shulha, et al. Phytochemistry 170 (2020) 112196

Table 3
NMR data of compound 7a, 8a, and 10a.
Position 7 8 10

δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz)

lactucin moiety
1 133.9, C 133.9, C 134.3, C
2 196.4, C 196.4, C 196.7, C
3 134.2, CH 6.29, mb 134.2, CH 6.29, mb 134.8, CH 6.49, mb
4 169.5, C 169.5, C 170.2, C
5 50.2, CH 3.96, d (10.0) 50.2, CH 3.95, d (10.0) 50.0, CH 3.97, d (10.0)
6 82.6, CH 3.84, mb 82.6, CH 3.80, mb 82.4, CH 3.87, t (10.0)
7 56.6, CH 3.33, mb 56.6, CH 3.33, mb 55.5, CH 3.63, mb
8 74.9, CH 4.48, td (2.0, 10.5) 74.9, CH 4.48, td (2.0, 10.5) 71.0, CH 5.06, td (2.0, 10.5)
9 45.8, CH2 3.15, dd (2.0, 13.5) 2.88, mb 45.9, CH2 3.15, dd (2.0, 13.0) 2.87, dd (10.0, 45.2, CH2 2.94, dd (11.0, 13.5) 2.48, dd (2.0, 13.5)
13.0)
10 150.2, C 150.2, C 148.3, C
11 137.6, C 137.6, C 138.7, C
12 170.6, C 170.6, C 169.9, C
13 124.0, CH2 6.43, mb 6.19, d (3.0) 124.0, CH2 6.43, d (2.5) 6.18, d (3.0) 121.7, CH2 6.11, d (3.0) 5.61, d (3.0)
14 21.7, CH3 2.45, s 21.7, CH3 2.45, s 21.6, CH3 2.45, s
15 65.0, CH2 5.43, dd (1.5, 17.5) 5.10, dd (1.0, 65.0, CH2 5.43, dd (2.0, 18.0) 5.10, dd (1.0, 68.1, CH2 5.32, d (17.0) 4.90, mb 1β,2α-dihydroxyilicic acid
18.0) 18.0) moiety
1′ 82.1, CH 3.23, d (10.0) 82.1, CH 3.23, d (10.0) 82.1, CH 3.23, d (10.0)
2′ 73.6, CH 5.19, td (5.0, 11.0) 73.6, CH 5.19, td (5.0, 11.0) 73.5, CH 5.19, td (5.0, 11.0)
3′ 45.7, CH2 2.01, dd (5.0, 13.0) 1.47, mb 45.7, CH2 2.00, mb 1.43, mb 45.7, CH2 2.01, mb 1.47, mb
4′ 73.3, C 73.3, C 73.2, C
5′ 52.1, CH 1.25, dd (2.5, 12.5) 52.1, CH 1.25, dd (3.0, 12.0) 52.1, CH 1.27, mb
6′ 27.6, CH2 1.79, br d (13.0) 1.52, mb 27.6, CH2 1.79, br d (13.0) 1.55, mb 27.8, CH2 1.78, mb1.52, mb
7′ 41.4, CH 2.52, tt (3.5, 12.0) 41.4, CH 2.52, tt (3.5, 12.0) 41.4, CH 2.53, mb
8′ 28.1, CH2 1.68, dd (2.5, 13.0) 1.47, mb 28.1, CH2 1.67, dd (3.0, 13.5) 1.49, mb 28.1, CH2 1.67, mb 1.47, mb
9′ 40.6, CH2 1.96, dtb (3.5, 13.0) 1.19, mb 40.7, CH2 1.96, dtb (3.5, 13.5) 1.21, mb 40.6, CH2 1.98, mb 1.19, mb
10′ 41.1, C 41.1, C 41.1, C
11′ 147.0, C 147.0, C 147.1, C
12′ 167.9, C 167.9, C 167.3, C
13′ 124.5, CH2 6.29, mb 5.74, s 124.5, CH2 6.29, mb 5.74, s 124.8, CH2 6.25, s 5.78, s
14′ 14.2, CH3 1.10, s 14.2, CH3 1.10, s 14.2, CH3 1.11, s
15′ 30.0, CH3 1.15, s 30.0, CH3 1.14, s 30.0, CH3 1.15, s
para-hydroxy-/para-methoxy-phenylacetyl moiety
1″ 126.7, C 128.0, C 126.7, C
2″ 131.5, CHb 7.10, dt (8.5, 2.0) 131.5, CHb 7.20, dt (8.0, 2.0) 131.5, CHb 7.10, dt (8.5, 2.0)
3″ 116.3, CHb 6.72, dt (8.5, 2.0) 115.0, CHb 6.86, dt (8.0, 2.0) 116.3, CHb 6.72, dt (8.5, 2.0)
4″ 157.6, C 160.3, C 157.6, C
5″ 116.3, CHb 6.72, dt (8.5, 2.0) 115.0, CHb 6.86, dt (8.0, 2.0) 116.3, CHb 6.72, dt (8.5, 2.0)
6″ 131.5, CHb 7.10, dt (8.5, 2.0) 131.5, CHb 7.20, dt (8.0, 2.0) 131.5, CHb 7.10, dt (8.5, 2.0)
7″ 41.6, CH2 3.55, s 41.6, CH2 3.59, s 41.6, CH2 3.55, s
8″ 174.2, C 174.0, C 174.2, C
OCH3″ 55.8, CH3 3.77, s

a
Spectra were referenced to solvent residual and solvent signals of CD3OD at 3.31 ppm (1H NMR) and 49.15 ppm (13C NMR), respectively.
b
Overlapping signals.

Table 4
Cytotoxicity (IC50, μM) after 72 h of treatment by compounds 1–5 in human acute lymphoblastic leukemia (CEM), human breast adenocarcinoma (MCF7), human
cervical carcinoma (HeLa) cells, and normal human skin fibroblasts (BJ). Staurosporine was used as a positive control.
Compound CEM MCF7 HeLa BJ

1 > 50 > 50 > 50 > 50

2 > 50 > 50 > 50 > 50
3 5.1 ± 0.7 17.1 ± 0.3 34.6 ± 2.8 9.8 ± 2.1
4 3.9 ± 0.2 15.2 ± 0.3 30.2 ± 1.3 8.4 ± 2.0
5 > 50 > 50 > 50 > 50
Staurosporine 0.023 ± 0.002 0.064 ± 0.002 0.175 ± 0.007 0.002 ± 0.000

300 MHz) and 13C NMR (CD3OD, 75 MHz) data, see Table 1; HRESIMS 425.1595 [M+H]+ (calcd for C24H25O7, 425.1595).
m/z 693.2563 [M–H]– (calcd for C37H41O13, 693.2553); m/z 717.2528
[M+Na]+ (calcd for C37H42O13Na, 717.2523).
4.9. Compound 6 - 15-p-methoxyphenylacetyllactucin-8-sulfate

4.8. Compound 4 - 15-p-methoxyphenylacetyllactucin D = – 16 (c 0.06, MeOH); for H NMR (CD3OD,

Yellow powder; [α]20 1

400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Table 2; HRESIMS
1
White powder; [α]20
D = + 25 (c 0.05, MeOH); for H NMR (DMSO- m/z 503.1017 [M–H]– (calcd for C24H23O10S, 503.1017); m/z 505.1171
D6, 400 MHz) and 13C NMR (DMSO-D6, 100 MHz) data, see Table 2; [M+H]+ (calcd for C24H25O10S, 505.1163).
HRESIMS m/z 423.1451 [M–H]– (calcd for C24H23O7, 423.1449); m/z

8
O. Shulha, et al.
4.10. Compound 7 – ((3aS,4S,9aS,9bR)-6-methyl-3-methylene-2,7-dioxo-
4-(sulfooxy)-2,3,3a,4,5,7,9a,9b-octahydroazuleno[4,5-b]furan-9-yl)
methyl 2-((2R,4aR,5S,6S,8R,8aR)-5,8-dihydroxy-6-(2-(4-hydroxyphenyl)
acetoxy)-4a,8-dimethyldecahydronaphthalen-2-yl)acrylate
D = – 12 (c 0.04, MeOH); for H NMR (CD3OD,
1
Yellow powder; [α]20
400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Table 3; HRESIMS
m/z 755.2398 [M–H]– (calcd for C38H43O14S, 755.2379); m/z 795.2093
[M+K]+ (calcd for C38H44O14SK, 795.2083).
4.11. Compound 8 – ((3as,4s,9as,9br)-6-methyl-3-methylene-2,7-dioxo-4-
(sulfooxy)-2,3,3a,4,5,7,9a,9b-octahydroazuleno[4,5-b]furan-9-yl)methyl
2-((2R,4aR,5S,6S,8R,8aR)-5,8-dihydroxy-6-(2-(4-methoxyphenyl)
acetoxy)-4a,8-dimethyldecahydronaphthalen-2-yl)acrylate
D = – 10 (c 0.05, MeOH); for H NMR (CD3OD,
1
Yellow powder; [α]20
400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Table 3; HRESIMS
m/z 769.2538 [M–H]– (calcd for C39H45O14S, 769.2536); m/z 809.2242
[M+K]+ (calcd for C39H46O14SK, 809.2240).
4.12. Compound 9 - 8-p-hydroxyphenylacetyllactucin-15-sulfate
1
Yellow powder; [α]20
D = + 21 (c 0.04, MeOH); for H NMR (CD3OD,
400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Table 2; HRESIMS
m/z 489.0870 [M–H]– (calcd for C23H21O10S, 489.0861); m/z 529.0564
[M+K]+ (calcd for C23H22O10SK, 529.0565).
4.13. Compound 10 – (3aR,4S,9aS,9bR)-6-methyl-3-methylene-2,7-dioxo-
9-((sulfooxy)methyl)-2,3,3a,4,5,7,9a,9b-octahydroazuleno[4,5-b]furan-4-
yl 2-((2R,4aR,5S,6S,8R,8aR)-5,8-dihydroxy-6-(2-(4-hydroxyphenyl)
acetoxy)-4a,8-dimethyldecahydronaphthalen-2-yl)acrylate
1 Rárová, L., Steigerová, J., Kvasnica, M., Bartůněk, P., Křížová, K., Chodounská, H., Kolář,
Yellow powder; [α]20
D = + 41 (c 0.05, MeOH); for H NMR (CD3OD,
400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Table 3; HRESIMS
m/z 755.2369 [M–H]– (calcd for C38H43O14S, 755.2379); m/z 795.2087
[M+K]+ (calcd for C38H44O14SK, 795.2083).
Acknowledgements
The authors wish to thank Thomas Stegemann (Botanical Institute,
Kiel University) for GC-MS support and Dr. Ulrich Girreser (Kiel) and
his team for NMR measurements. This work was also supported by the
European Regional Development Fund - Project ENOCH (No.
CZ.02.1.01/0.0/0.0/16_019/0000868).
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
Phytochemistry 170 (2020) 112196
doi.org/10.1016/j.phytochem.2019.112196.
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