Two new sesquiterpene lactone glycosides from Cnicus benedictus

Article abstract of DOI:10.1080/14786419.2017.1295239

Two new sesquiterpene lactone glycosides, namely melitensin 15-O-β-D-glucoside (1) and 11β,13-dihydrosalonitenolide 15-O-β-D-glucoside (2), along with eight known compounds (3–10) were isolated from the aerial part of Cnicus benedictus L. Their structures

Full text of DOI:10.1080/14786419.2017.1295239

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Two new sesquiterpene lactone glycosides from
Cnicus benedictus
Ying Peng, Yuqing Jian, Ali Zulfiqar, Bin Li, Kaiqiang Zhang, Fei Long, Caiyun
Peng, Xiong Cai, Ikhlas A. Khan & Wei Wang
To cite this article: Ying Peng, Yuqing Jian, Ali Zulfiqar, Bin Li, Kaiqiang Zhang, Fei Long, Caiyun
Peng, Xiong Cai, Ikhlas A. Khan & Wei Wang (2017): Two new sesquiterpene lactone glycosides
from Cnicus benedictus, Natural Product Research, DOI: 10.1080/14786419.2017.1295239
To link to this article: http://dx.doi.org/10.1080/14786419.2017.1295239
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Date: 14 March 2017, At: 00:35

Natural Product research, 2017
http://dx.doi.org/10.1080/14786419.2017.1295239
Two new sesquiterpene lactone glycosides from Cnicus
benedictus
Ying Penga,b‡, Yuqing Jiana‡, Ali Zulfiqarb, Bin Lia, Kaiqiang Zhanga,b, Fei Longa,b
Caiyun Penga, Xiong Caia, Ikhlas A. Khana,b and Wei Wanga
,
atcM and ethnomedicine Innovation & development laboratory, school of Pharmacy, hunan university
of chinese Medicine, changsha, hunan, china; bNational center for Natural Products research, division
of Pharmacognosy, department of BioMolecular sciences, school of Pharmacy, university of Mississippi,
university, Ms, usa
ABSTRACT
ARTICLE HISTORY
received 31 december 2016
accepted 5 February 2017
Two new sesquiterpene lactone glycosides, namely melitensin 15-O-β-
D-glucoside (1) and 11β,13-dihydrosalonitenolide 15-O-β-D-glucoside
(2), along with eight known compounds (3–10) were isolated from
the aerial part of Cnicus benedictus L. Their structures were elucidated
from analyses of extensive spectroscopic data. Compounds 1–6 all
possessed an α-methyl-γ-lactone moiety. Moreover, compound
5 exhibited moderate activity against the growth of Aspergillus
KEYWORDS
Cnicus benedictus;
asteraceae; sesquiterpene
lactone glycosides;
antimicrobial
fumigatus, with IC50 values of 17.67 μg mL−1
.
1. Introduction
Cnicus benedictus L. (Asteraceae), the sole species in the genus Cnicus, is an annual herb
native to the Mediterranean region, which is distributed all over Middle and South-East
Europe. It was well known as a traditional indigenous medicine in many countries for the
application of stomachic, tonic, antidepressant, anti-inflammatory, antibiotic and antiseptic.
In Algeria, the name ‘March grass’was given to this plant for its effective against burns and
wounds. It was also used in liquor and beer production (Tsankova et al. 1994; Paun et al.
2015).
CONTACT Ikhlas a. Khan
Ikhan@olemiss.edu; Wei Wang
wangwei402@hotmail.com
‡ these authors contributed equally to this work.
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2017.1295239.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
Y. PENG ET AL.
Previous phytochemical investigation into the plant had revealed the existence of lignans
(Vanhaelen & Vanhaelen-Fastré 1975; Sólyomváry et al. 2014), steroids and terpenoids
(Ulubelen & Berkan 1977; Kataria 1995). In addition, pharmacological studies of crude extracts
from the title plant have reported, such as neuroprotective and antioxidant activity (Paun
et al. 2015), anti-inflammatory (Schneider & Lachner 1987) and antimicrobial (Vanhaelen-
Fastre 1972). During our research for new natural products produced from medicinal plant,
two new sesquiterpene lactone glycosides, named melitensin 15-O-β-D-glucoside (1) and
11β,13-dihydrosalonitenolide 15-O-β-D-glucoside (2), as well as eight known compounds
were identified as melitensin (3) (Djeddi et al. 2008), 8α-hydroxy-11β,13-dihydro-onopord-
aldehyde (4) (Medjroubi et al. 1998), cnicin (5) (Marco et al. 2005), (2S,3S)-3-hydroxy-2-methyl-
4-butanolide (6) (Řezanka et al. 2004), cirsimaritin (7) (Liu et al. 1992), apigenin (8) (Miyazawa
& Hisama 2003), isokaempferide (9) (Collado et al. 1985) and kaempferol 3-methyl ether
7-O-β-D-glucoside (10) (Laggoune et al. 2008) were isolated from the methanol extract of
aerial part of C. benedictus. Among them, compounds 1–4, and 6–10 were isolated for the
first time from the plant. In this study, the isolation and structural elucidation of 1 and 2 were
described.
2. Results and discussion
Melitensin 15-O-β-D-glucoside (1) was isolated as a colourless plate. The molecular formula
of 1 was established as C21H32O9 by a quasi-molecular [M + Na]+ ion peak at m/z 451.1935
(calcd for C21H32O9Na: 451.1944) from its HRESIMS data. The IR spectrum of 1 afforded strong
absorption bands for hydroxy group (3352 cm−1) and carbonyl group (1760 cm−1). In the 1H
NMR spectrum, five olefinic protons [δH 5.98 (1H, dd, J = 17.1, 10.8 Hz, H-1), 5.54 (H-3a), 5.08
(H-3b) and 5.06 (2H, H-2)], two methyl [δH 1.39 (3H, d, J = 6.5 Hz, H3-13) and 1.18 (3H, s, H3-
14)] signals were observed. The 13C NMR spectrum revealed that 1 possessed 21 carbons
including a carbonyl at δC 181.5 (C-12), a monosubstituted vinyl group [δC 148.8 (C-1) and
112.3 (C-2)] and a terminal vinyl group [δC 142.7 (C-4) and 115.4 (C-3)]. Additionally, a set of
oxygenated carbon signals [δC 103.6 (C-1′), 77.8 (C-3′), 77.6 (C-5′), 75.0 (C-2′), 71.4 (C-4′) and
62.5 (C-6′)] suggested the characteristic of glucose moiety. The β-configuration was estab-
lished due to the coupling constants of the anomeric proton signals at δH 4.37 (1H, d,
3JH1′-H2′ = 6.8 Hz, H-1′). After acid hydrolysis and derivatisation of 1, HPLC analysis revealed
the presence of D-glucose unit (Tanaka et al. 2007). Comparison of the NMR data assigned
for the aglucon of 1 with those of melitensin (González et al. 1975; Cardona et al. 1989)
suggested that they shared a similar skeleton. The main difference between 1 and melitensin
was that 1 was glycoside of melitensin. Comprehensive interpretation of 1H-1H COSY, HSQC
and HMBC spectra led to the assignments of 1H and 13C NMR data as shown in Table S1.
1
The H-1H COSY spectrum of 1 revealed the presence of three spin systems (C-1 to C-2,
C-5/C-9 to C-13 and C-1′ to C-6′). In the HMBC spectrum, the correlations between H3-14 and
C-5/C-1/C-9, between H-2/H-8 and C-10, between H-5 and C-9/C-3, between H-15 and C-5/C-
3, as well as between H3-13 and C-12 clearly defined the structure of elemanolide type ses-
quiterpene skeleton. Moreover, the correlations between H-1′ of glucose moiety and C-15
demonstrated that β-D-glucose moiety attached to the C-15 position (Figure S1). The relative
configuration of 1 was deduced by NOESY experiment. The NOE correlations between H-6
and H3-14/H-8/H-11 indicated that these protons were located at the same side of the mol-
ecule, the NOE correlation between H3-14 and H-3b suggested that C3-14 and terminal vinyl

NATURAL PRODUCT RESEARCH
3
Figure 1 chemical structures of 1 and 2 isolated from Cnicus benedictus.
group has the same orientation. The NOE correlation between H-5 and H-7 indicated that the
two protons were cofacial (Figure S2). Therefore, the structure of 1 was elucidated as shown
in Figure 1, which is a new elemanolides glycoside named melitensin 15-O-β-D-glucoside.
11β,13-Dihydrosalonitenolide 15-O-β-D-glucoside (2) was isolated as a colourless plate,
its molecular formula was determined to be C21H32O9 by HRESIMS at m/z 473.2094
[M + HCOO]− (calcd. for C22H33O11: 473.2023). The IR spectrum showed absorption bands for
hydroxy group (3371 cm−1) and carbonyl group (1752 cm−1). The 1H NMR showed two olefinic
protons [δH 4.97 (1H, H-1) and 4.83 (1H, H-5)] and two methyl signal [δH 1.44 (3H, s, H3–14
)
and 1.39 (3H, d, J = 6.9 Hz, H3–13)]. The 13C NMR spectrum of 2 showed a carbonyl at δC 182.0
(C-12), two trisubstituted vinyl groups [δC 140.9 (C-4), 131.9 (C-5), 135.4 (C-10) and 129.3
(C-1)]. Additionally, a set of oxygenated carbon signals [δC 104.5 (C-1′), 78.2 (C-3′), 77.9 (C-5′),
75.1 (C-2′), 72.4 (C-4′) and 62.7 (C-6′)] suggested the characteristic of glucose moieties. The
β-configuration was established due to the coupling constants of the anomeric proton sig-
nals at δH 4.33 (1H, d, 3JH1′-H2′ = 7.8 Hz, H-1′). After acid hydrolysis and derivatisation of 2, HPLC
analysis revealed the presence of D-glucose unit (Tanaka et al. 2007). Comparison of the
NMR data assigned for aglycone of 2 with those of 11β,13-dihydrosalonitenolide (Marco
et al. 1992) was very similar, indicated that 2 possessed the same sesquiterpene skeleton
with the known one. The main difference between 2 and the known one was that 2 was
1
glycoside of 11β,13-dihydrosalonitenolide. Comprehensive interpretation of H-1H COSY,
HSQC and HMBC spectra allowed the assignments of all proton and carbon signals (Table
S1).
1
The H-1H COSY spectrum of 2 revealed the presence of three spin systems in bold as
shown in Figure S1. In the HMBC spectrum, the correlations between H3-14 and C-1/C-9,
between H-2/H-8 and C-10, between H-9 and C-1, between H-15 and C-3/C-5, between H-2
and C-4, between H-3 and C-5, as well as between H3-13 and C-12, also suggested the pres-
ence of the skeleton of germacranolide type sesquiterpene. Furthermore, the correlations
between H-1′ of glucose moiety and C-15 demonstrated that a β-D-glucose moiety con-
nected to the C-15 position of the sesquiterpene unit (Figure S1). The NOESY spectrum of 2
displayed NOE correlation between H-6 and H-8/H-11/H3-14, between H-8 and H-11 indi-
cating these protons H-8, H-6, H-11 and H3-14 should be cofacial, the NOE correlation
between H-7 and H-5 suggested that these protons are located in the same orientation.
Moreover, the geometries of the double bonds could be determined by the NOE correlation
between H-3b and H-5, as well as H-2a and H3-14 (Figure S2). Therefore, the structure of 2
was determined as 11β,13-dihydrosalonitenolide 15-O-β-D-glucoside (Figure 1).

4
Y. PENG ET AL.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a Rudolph Research Analytical Autopol IV automatic
polarimeter. The UV spectra were tested on a Varian Cary 50 Bio UV–Vis spectrophotometer.
IR spectra were acquired on an Agilent Technologies Cary 630 FTIR spectrometer. The NMR
spectra were measured by a Varian Mercury 400 MHz. ESI-MS data and HR-ESI-MS were
obtained using an Agilent Technologies 6520 Accurate Mass Q-TOF LC/MS. Thin layer chro-
matography (TLC) was carried out on silica gel sheets (Alugram® Sil G/UV254; Macherey-
Nagel, Germany) and reversed-phase plates (RP-18 F254S; Merck, Germany). Chromatography
was carried out using silica gel (32–63 μm for flash chromatography, Dynamic Adsorbents
Inc.), reversed-phase C18 silica (Polarbond, JT Baker), Sephadex LH-20 (Sigma).
3.2. Plant material
The aerial part of C. benedictus L was purchased from the online company of Frontier Co-op,
and was identified by Prof. Ikhlas A. Khan. A voucher specimen (No. 2114) was deposited at
National Center for Natural Products Research at the University of Mississippi.
3.3. Extraction and isolation
The dried aerial part of C. benedictus L (814 g) was extracted with methanol at room tem-
perature and the solution was evaporated in vacuum to yield a crude extract (118.0 g). The
extract was primary applied to ODS column chromatography (CC) using MeOH/H2O gradients
to give 15 fractions (Fr. 1–Fr. 15). Fr. 1 (64.0 g) was suspended in water and extracted with
ethyl acetate to yield ethyl acetate soluble part (2.1 g) which was subjected to silica gel CC
to afford 6 (11.0 mg). Fr. 3 (10.7 g) was subjected to Sephadex LH-20 and eluted with MeOH
to obtain 8 fractions (Fr. 3-1–Fr. 3-8). Fr. 3-3 (6.6 g) was applied to silica gel CC and eluted
with MeOH/CHCl3 to obtain 21 fractions (Fr. 3-3-1–Fr. 3-3-21). 3 (149.6 mg) was found in a
pure state in Fr. 3-3-6. Fr. 3-3-20 (0.9 g) was subjected to silica gel CC, Sephadex LH-20, ODS
CC to obtain 1 (13.7 mg) and 2 (8.6 mg). Fr. 4 (7.0 g) was applied to silica gel CC with MeOH/
CHCl3 gradient to give 13 fractions (Fr. 4-1–Fr. 4-13), 4 (8.1 mg) was crystallised out from Fr.
4-6. 10 (8.7 mg) was purified from Fr. 4-1 (1.9 g) by Sephadex LH-20 and eluted with MeOH.
Fr. 5 (1.6 g) was subjected to silica gel CC to obtain 5 (8.1 mg). Fr. 8 (1.2 g) was subjected to
Sephadex LH-20 and eluted with MeOH to obtain 9 (28.2 mg) and 8 (28.8 mg). Fr. 9 (0.3 g)
was also subjected to Sephadex LH-20 and eluted with MeOH to obtain 7 (4.7 mg).
3.3.1. Melitensin 15-O-β-D-glucoside (1)
Colourless plate;[ꢀ]2D5 = +1.7° (c 0.05, MeOH), IR (KBr) vmax: 3352, 2935, 2834, 1760, 1022 cm−1;
1
HRESIMS m/z: 451.1935 [M + Na]+ (calcd for C21H32O9Na: 451.1944). H NMR (MeOD-d4,
400 MHz): δH 5.98 (1H, dd, J = 17.1, 10.8 Hz, H-1), 5.06 (2H, H-2), 5.54 (1H, br s, H-3a), 5.08 (1H,
br s, H-3b), 4.39 (1H, d, J = 13.4 Hz, H-15a), 4.18 (1H, d, J = 13.4 Hz, H-15b), 4.47 (1H, dd,
J = 11.6, 11.2 Hz, H-6), 4.37 (1H, d, J = 6.8 Hz, H-1′), 3.98 (1H, m, H-8), 3.93 (1H, dd, J = 11.5,
1.0 Hz, H-6′a), 3.75 (1H, dd, J = 11.5, 4.5 Hz, H-6′b), 3.46 (1H, H-3′), 3.40 (1H, H-4′), 3.33 (1H,
H-5′), 3.31 (1H, H-2′), 2.79 (1H, H-11), 2.57 (1H, d, J = 11.6 Hz, H-5), 1.80 (1H, H-9a), 1.67 (1H,
t-like, J = 11.7 Hz, H-9b), 1.39 (3H, d, J = 6.5 Hz, H-13), 1.18 (3H, s, H-14); 13C NMR (MeOD-d4,

NATURAL PRODUCT RESEARCH
5
100 MHz): δC 181.5 (C-12), 148.8 (C-1), 142.7 (C-4), 115.4 (C-3), 112.3 (C-2), 103.6 (C-1′), 79.9
(C-6), 77.8 (C-3′), 77.6 (C-5′), 75.0 (C-2′), 73.4 (C-15), 71.4 (C-4′), 69.0 (C-8), 62.5 (C-6′), 59.2 (C-7),
51.5 (C-5), 50.1 (C-9), 42.6 (C-10), 42.3 (C-11), 19.1 (C-14), 14.6 (C-13).
3.3.2. 11β,13-dihydrosalonitenolide 15-O-β-D-glucoside (2)
Colourless plate; [ꢀ]2D5 = +26.2° (c 0.05, MeOH) IR (KBr) vmax: 3371, 2926, 2824, 1752, 1074,
1
1024 cm−1; HRESIMS m/z: 473.2023 [M + HCOO]− (Calcd for C22H33O11: 473.2094). H NMR
(MeOD-d4, 400 MHz): δH 4.97 (1H, H-1), 4.97 (1H, H-6), 4.83 (1H, H-5), 4.59 (1H, d, J = 12.2 Hz,
H-15a), 4.01 (1H, d, J = 12.2 Hz, H-15b), 4.33 (1H, d, J = 7.8 Hz, H-1′), 3.92 (1H, m, H-8), 3.89
(1H, dd, J = 11.8, 1.5 Hz, H-6′a), 3.69 (1H, dd, J = 11.8, 5.0 Hz, H-6′b), 3.39 (1H, H-3′), 3.34 (1H,
H-4′), 3.27 (1H, H-5′), 3.23 (1H, H-2′), 2.80 (1H, m, H-11), 2.66 (1H, m, H-3a), 1.96 (1H, m, H-3b),
2.56 (1H, dd, J = 12.5, 1.2 Hz, H-9a), 2.37 (1H, dd, J = 12.5, 10.6 Hz, H-9b), 2.21 (1H, m, H-2a),
2.17 (1H, m, H-2b), 1.44 (3H, s, H-14), 1.39 (3H, d, J = 6.9 Hz, H-13); 13C NMR (MeOD-d4,
100 MHz): δC 182.0 (C-12), 140.9 (C-4), 135.4 (C-10), 131.9 (C-5), 129.3 (C-1), 104.5 (C-1′), 78.2
(C-3′), 78.1 (C-6), 77.9 (C-5′), 75.1 (C-2′), 72.4 (C-4′), 71.5 (C-8), 68.6 (C-15), 62.7 (C-6′), 61.3 (C-7),
53.3 (C-9), 41.9 (C-11), 36.0 (C-3), 26.9 (C-2), 17.8 (C-13), 17.4 (C-14).
3.3.2.1. Acid hydrolysis and HPLC analysis. Compounds 1 and 2 (each 1 mg) were,
respectively, dissolved in 2 mol L−1 HCl (10 mL) and heated in a water bath (80°C) for 4 h.
The reaction mixtures were evaporated under reduced pressure. Each residue was dissolved
in H2O and extracted with EtOAc. Each aqueous layer was concentrated to dryness under
reduced pressure. Then, 1 mL of pyridine and 2 mg of L-cysteine methyl ester hydrochloride
(Sigma) were added to the residue, and the reaction mixture was heated at 60°C for 1 h.
Isothiocyanate (2 mg) was added to the mixture, then heated at 60°C for another 1 h. The
reaction mixtures were analysed by HPLC [column: (Agilent TC C18, 4.6 mm × 250 mm, 5 μm);
flow rate: 0.8 mL min−1; mobile phase: CH3CN-0.06% aq. formic acid (25:75, v/v); detection
wavelength: 250 nm]. The monosaccharides were identified to be D-glucose by comparison
of the retention times of monosaccharide derivatives with those of standard samples:
L-glucose (18.9 min) and D-glucose (20.3 min), respectively.
3.4. Microorganism
Micro-organisms were obtained from the American Type Culture Collection (Manassas,
VA, USA) and the bacteria [Cryptococcus neoformans, Staphylococcus aureus ATCC 33591
(standard methicillin-sensitive), Staphylococcus aureus ATCC 29213 (methicillin-sensitive),
Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and vancomycin-resistant
Enterococcus faecalis ATCC 29212, low-level vancomycin-resistant Enterococcus faecalis
ATCC 51299, MRSA (ATCC 3391) and fungal (Candida albicans and Aspergillus fumigatus)
strains]. All organisms were tested using modified versions of the CLSI (formerly National
Committee on Clinical Laboraory Standards, 2003) method. Drug controls [Amphotericin
B (92%) for fungi and Ciprofloxacin (99.3%) for bacteria] were obtained from ICN
Biomedicals, Aurora, OH. Solvents (Fisher brand), used for chromatographic procedures,
were of certified grades. All organisms were obtained from the American Type Culture
Collection (Manassas, VA).

6
Y. PENG ET AL.
4. Conclusion
Chemical investigation into the methanol extract of aerial part of C. benedictus led to the
isolation of two new sesquiterpene lactone glycosides, melitensin 15-O-β-D-glucoside (1)
and 11β,13-dihydrosalonitenolide 15-O-β-D-glucoside (2), together with eight known com-
pounds (3–10). Compounds 1–6 all possessed an α-methyl-γ-lactone moiety (Liu et al. 2015;
Bhat et al. 2016; Marzouk & Abd Elhalim 2016; Sosa et al. 2016; Yuan et al. 2017). All the
compounds exhibited weak antimicrobial activity against all these strains with the test con-
centration of 20 μg mL−1 and compound 5 exhibited moderate antifungal activity against
Aspergillus fumigatus with IC50 value of 17.67 μg mL−1.
Acknowledgements
This research was supported by Education Department of Hunan Province (No. 13k077), Hunan Science
and Technology Department Funds (No: 2014SK4037, 2014FJ1007), and TCM stem cell laboratory
of HUCM. We are thankful to Dr. Melissa R. Jacob and Dr. Bharathi Avula from NCNPR, University of
Mississippi for antimicrobial assays and HRESIMS experiments.
Disclosure statement
No potential conflict of interest was reported by the authors.
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