Molecular networking-driven isolation of 8′-Glycosylated biscoumarins from Cruciata articulata

Article abstract of DOI:10.1016/j.phytochem.2021.112856

A molecular networking-guided phytochemical investigation of Cruciata articulata led to the isolation of five unreported biscoumarins, four of which were characterized by a shared 6-methoxy-7,8′-dihydroxy-3,7′-biscoumarin aglycone. These were isolated alongside two known coumarin glycosides, daphnetin-8-O-β-D-glucoside and 6′-acetoxy-daphnetin-8-O-β-D-glucoside. Their structures were elucidated by extensive 1D and 2D NMR experiments, in combination with chemical transformation and MS/MS fragmentation analysis. Four of the biscoumarins were glycosylated at the 8′ position: these are the first examples of this substitution pattern to be described in nature. All compounds were tested for cytotoxic, antimicrobial, anti-inflammatory, and α-glucosidase inhibitory properties, but did not display significant activity.

Full text of DOI:10.1016/j.phytochem.2021.112856

Phytochemistry 190 (2021) 112856
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
′
Molecular networking-driven isolation of 8 -Glycosylated biscoumarins
from Cruciata articulata
a
a
b
c
d
Xueling Liu , Yuyu Dong , Valida Alizade , Manana Khutsishvili , Daniel Atha ,
a
a,*
Robert P. Borris , Benjamin R. Clark
a
School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin, 300072, China
Institute of Botany, Azerbaijan National Academy of Sciences, Baku, AZ1102, Azerbaijan
National Herbarium of Georgia, Ilia State University, Tbilisi, 100995, Georgia
New York Botanical Garden, Bronx, NY, USA
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
A molecular networking-guided phytochemical investigation of Cruciata articulata led to the isolation of five
Cruciata articulata
Rubiaceae
′
′
unreported biscoumarins, four of which were characterized by a shared 6-methoxy-7,8 -dihydroxy-3,7 -biscou-
marin aglycone. These were isolated alongside two known coumarin glycosides, daphnetin-8-O-β-D-glucoside
Structural elucidation
Molecular networking
Biscoumarins
′
and 6 -acetoxy-daphnetin-8-O-β-D-glucoside. Their structures were elucidated by extensive 1D and 2D NMR
experiments, in combination with chemical transformation and MS/MS fragmentation analysis. Four of the
′
biscoumarins were glycosylated at the 8 position: these are the first examples of this substitution pattern to be
MS/MS
described in nature. All compounds were tested for cytotoxic, antimicrobial, anti-inflammatory, and
dase inhibitory properties, but did not display significant activity.
α
-glucosi-
1
. Introduction
Additionally, both natural and synthetic coumarins can be used as
fluorescent probes or luminescent materials (Jiang et al., 2020; Maha-
patra et al., 2013).
Cruciata articulata (L.) Ehrend (Rubiaceae) has a range of distribution
including Egypt, the Mediterranean region, and western Asia (Abdel--
Khalik and Bakker., 2007). Even though volatile oils, coumarins, iri-
doids, flavonoids and terpenoids have been investigated in the genus
Cruciata (De Rosa et al., 2003, 2002; Ergun et al., 1984; Il’Ina et al.,
Over the past decade, analysis of MS/MS fragmentation patterns has
become increasingly useful in the identification of plant metabolites
from multiple structure classes (Aron et al., 2020; Gangopadhyay et al.,
2016; Wang et al., 2014; M. Watrous et al., 2012). Furthermore, MS/MS
molecular networking (MN) has emerged as a promising technique for
analyzing LC-based mass spectrometry and metabolomics data sets
based on its accessibility through the Global Natural Product Social
Network (GNPS; https://gnps.ucsd.edu) Web-based platform (Woo
et al., 2019; Wang et al., 2016b; Yang et al., 2013). MN analysis can
match MS/MS data against reference molecules, identifying known
compounds and clustering ions into families according to their spec-
trometric similarity (Kim et al., 2020; Naman et al., 2017).
2
013; Mitova et al., 1996; Tava et al., 2020), only a single phytochem-
ical investigation has focused on Cruciata articulata, describing the
isolation of anthraquinones from the species (Ushakov et al., 1988).
Coumarins are specialized metabolites widely distributed in plants,
especially in the Umbelliferae, Fabaceae, Asteraceae, and Thymelaea-
ceae families (Gaber et al., 2019; Hoult and Pay ´a ., 1996). Coumarins are
primarily biogenetically derived from shikimic acid pathways, though a
significant amount of them are formed from mixed biosynthetic routes
(
Knaggs, 2003). Previous reports have demonstrated that coumarins
In our study, biscoumarins were targeted for isolation from
C. articulata based on MS/MS analysis. Herein, we describe the isolation,
purification, structure elucidation, MS/MS fragmentation analysis and
biological testing of these compounds.
possess a wide range of biological properties (Liang et al., 2011; Pierson
et al., 2010), including antibacterial (da Cunha et al., 2020), anticancer
(
Song et al., 2020), anticoagulant (Greaves, 2005), HIV inhibitory (Liu
et al., 2020), antiviral (Mishra et al., 2020), antiglycation (Salar et al.,
019) and anti-inflammatory (Chougala et al., 2018) properties.
2
*
Corresponding author.
E-mail address: bclark@tju.edu.cn (B.R. Clark).
https://doi.org/10.1016/j.phytochem.2021.112856
Received 30 March 2021; Received in revised form 16 June 2021; Accepted 25 June 2021
Available online 6 July 2021
0
031-9422/© 2021 Elsevier Ltd. All rights reserved.

X. Liu et al.
Phytochemistry 190 (2021) 112856
2
. Results and discussion
located this group at C-6 of the trisubstituted coumarin, which was
further supported by a NOESY correlation between the methoxy group
and H-5 (δ 7.12). An HMBC correlation from H-8 (δ 6.82) to a downfield
The n-BuOH fraction (Figure S1 of Supporting Information) of a
methanol extract of C. articulata was subjected to untargeted LC-MS/MS
analysis. Analysis of MS/MS data with the GNPS database gave 21
tentative hits to known compounds. Nine of these corresponded to fla-
vonoids, while a single hit corresponded to the biscoumarin glycoside
rutarensin, suggesting flavonoids and coumarins could be part of the
chemical composition of C. articulata. The ions in one cluster, designated
G1 (Fig. 1), shared a common fragment ion: m/z 179, which was also
carbon at 150.4 ​ ppm suggested the presence of an OH group at C-7.
For the 7,8-substituted coumarin moiety, substitution was confirmed
in a similar manner: COSY, HMBC, and NOESY correlations confirming
′
the locations of the protons at C3 -C6’ (Fig. 3), confirming the presence
′
′
of substituents at the C-7 and C-8 positions. A characteristic anomeric
′′
proton signal at δ 5.17 (d, J
H-NMR spectrum, which correlated to a downfield carbon signal at δ
​
=
​ 7.6 ​ Hz, H-1 ) could be observed in the
1
2
observed in the MS spectrum for the hit of the biscoumarin rutatensin
104.1 in the HSQC spectrum, suggesting the presence of a sugar moiety
1
(
Figure S2 of Supporting Information). Based on their masses, this sug-
in compound 1. A cluster of signals between 3 and 4
NMR, and between 62 and 74
​
ppm in the H-
gested that these could be previously unreported coumarins or biscou-
marins, which were thus targeted for isolation.
​
ppm in the 13C-NMR (Table 1), supported
this hypothesis and were consistent with the presence of a glucose
moiety. Correlations from H-1’’ (δ 5.17) to C-8’ (δ 132.9) in the HMBC
spectrum demonstrated that the sugar was located at C-8’. Thus, the
linkage between the two coumarin moieties must necessarily be between
Based on the MN-guided isolation, five undescribed biscoumarins
(
1–5) along with two known coumarin glycosides (6, 7) (Fig. 2) were
isolated from the n-BuOH fraction of the methanolic extract of
′
′
′
C. articulata. The known compounds were identified as 6 -acetoxy-
the C-3 and C-7 carbons; the C3–O–C7 linkage was supported by the
′
daphnetin-8-O-β-D-glucoside (6) and daphnetin-8-O-β-D-glucoside (7)
by the combined analysis of 1D, 2D NMR, HR-ESI-MS, and by compar-
ison with previously reported NMR data (Shakeel-u-Rehman et al.,
chemical shift of C-3 (δ 137.2); characteristic of a C3–O–C7 linkage
(Sim o˜ es et al., 2009). By further comparison with the data in the liter-
ature (Wang et al., 2016a), the aglycone of compound 1 was confirmed
′
′
2
010; Zhu et al., 2008).
to be 6-methoxy-7,8 -hydroxy-3,7 -biscoumarin.
Compound 1 was isolated as a yellow amorphous powder. The mo-
Acid hydrolysis of compound 1, followed by purification of the
lecular formula was determined to be C27
H
24
O
14 due to the observation
resulting sugar, indicated the presence of D-glucose, which was
-
25
D
of an [M - H] quasi-molecular ion at m/z 571.1080 in the HR-ESI-MS.
confirmed by comparison of optical rotation ([
α
]
+61.7 (c 0.05, H
2
O))
Hz, H-
) confirmed the β-configuration of D-glucose (Hernandez et al., 2004).
The H-NMR spectrum showed the presence of seven aromatic 1H res-
1
and Rf values with a standard sample. The coupling constant (7.6
​
onances. The doublet proton signals at δ 6.42 (1H, d, J
​
=
​
9.6
​
Hz) and
′
′′
´
1
′
8
.03 (1H, d, J
​
=
​
9.6
​
Hz) were characteristic of H-3 and H-4 of a
Signals consistent with the presence of a single acetate group were
observed in both the 13C (δ 169.9, 20.2) and H (δ 1.73, 3Н) spectra.
Correlations from the oxygenated methylene proton H-6’’ (δ 4.03–4.10)
with the acetate carbonyl resonance (δ 169.9) in the HMBC spectrum
1
coumarin moiety, respectively (Wang et al., 2016a). Another pair of
aromatic proton signals at δ 7.44 (1H, d, J 8.9 Hz), and 7.10 (1H, m)
​
=
​
​
′
′
were assigned as H-5 and H-6 , respectively, of the same 7,8-disubsti-
tuted coumarin. Three additional aromatic resonances at δ 6.82, 7.12,
′′
confirmed that the acetyl group was located at C-6 of the glucose
and 7.49 ​ ppm were assigned as H-4, H-5 and H-8 of a second coumarin
moiety (Fig. 3). Assignment of all glucose signals (Table 1) was sup-
ported by the combined analysis of 1D NMR and 2D NMR spectra.
Therefore, the structure of compound 1 was determined to be
group, this one substituted at positions 3, 6 and 7. The presence of the
two coumarin groups was supported by observation of two characteristic
1
3
coumarin CO resonances at 156.8 and 159.3
​
ppm in the C NMR
′′
′
′
6
-O-acetyl-8 -O-β-D-glucopyranosyl-6-methoxy-7-hydroxy-3,7 -biscou-
spectrum. The positions of all substituents were supported by COSY
correlations, in addition to HMBC and NOESY data (Fig. 3). A key HMBC
correlation from the methoxy singlet at δ 3.76 (3H, s) to C-6 (δ 145.8),
marin (Fig. 2).
Compound 2 was isolated as a yellow amorphous powder. Based a
-
[
M - H] quasi-molecular ion at m/z 529.0981 in the HR-ESI-MS spec-
trum, the molecular formula was determined to be C25
H
22
O
13. Com-
1
13
parison of the H and C NMR spectroscopic data of compounds 2 and 1
(
Table 1), suggested the presence of the same 6-methoxy-7-hydroxy-
′
3
,7 -biscoumarin aglycone. The NMR data for 1 and 2 were mostly
identical, except for the loss of the acetyl group in compound 2. The
′′
anomeric proton signal at δ 5.22 (d, J
​
=
​ 7.2 ​ Hz, H-1 ) and its corre-
sponding carbon signal at δ 102.3 suggested the occurrence of one sugar
moiety, which was confirmed by acid hydrolysis experiments, again
indicating the presence of D-glucose. The β configuration of D-glucose
was deduced on the basis of a characteristic coupling constant (7.2 ​ Hz)
1
in the H-NMR spectrum. HMBC and NOESY correlations confirmed the
locations of glucose and methoxy groups were the same as for compound
′
1
. Thus, compound 2 was identified as 8 -O-β-D-glucopyranosyl-6-
′
methoxy-7-hydroxy-3,7 -biscoumarin (Fig. 2).
Compound 3 was obtained as a yellow amorphous powder. The
32
molecular formula was determined to be C31H O18 based on HR-ESI-MS
-
1
13
data (m/z 691.1502, [M - H] ). On the basis of the H and C NMR
spectroscopic data (Table 1), compound 3 possessed the same central
biscoumarin moiety as for compounds 1 and 2. The difference was that
compound 3 possessed two sugar moieties, as shown by observation of
′′
two anomeric proton signals at δ 5.24 (d, J
​
=
​
7.3
​ Hz, H-1 ) and 5.07 (d,
′
′′
1
J
​
=
​ 6.8 ​ Hz, H-1 ) in the H-NMR spectrum. Acid hydrolysis experi-
ments indicated the presence of only D-glucose, suggesting that com-
Fig. 1. Molecular network of Cluster G1. Red: Parent ions of isolated new
biscoumarins (1–4); Yellow: Parent ions for proposed structures. (For inter-
pretation of the references to color in this figure legend, the reader is referred to
the Web version of this article.)
pound 3 contained two β-D-glucose moieties. HMBC correlations from
′
′′
H-1’’ (δ 5.24) to C-8’ (δ 133.6) and from H-1 (δ 5.07) to C-7 (δ 148.4),
together with a correlation from δ 5.24 to H-8 in the NOESY spectrum,
2

X. Liu et al.
Phytochemistry 190 (2021) 112856
Fig. 2. Chemical structures of compounds 1–7.
Fig. 3. Selected HMBC (solid arrows) and NOESY (dashed arrows) correlations for compounds 1 and 3.
′
confirmed that the two β-D-glucose moieties were located at C-8 and C-7
the aglycone for compounds 1–3.
of the biscoumarin moiety (Fig. 3). All signals were assigned by 1D and
MS/MS analysis, in positive mode, revealed that compounds 1–7
shared similar fragment ions (Figures S3 of Supporting Information). A
fragmentation pathway for these biscoumarins was proposed based on
the MS/MS data (Fig. 4). For compound 1 in positive mode, the parent
2
D NMR spectroscopic analysis. Overall, the structure of compound 3
′
′
was determined to be 7, 8 -O-β-D-diglucopyranosyl-6-methoxy-3,7 -
biscoumarin (Fig. 2).
′
Compound 4 was isolated as a yellow amorphous powder. The mo-
ion at m/z 591.1504 can lose a 6 -acetoxy-β-D-glucopyranoside unit to
lecular formula was determined to be C24
H
20
O
13
12 based on HR-ESI-MS
yield a biscoumarin fragment ion at m/z 369.0602, which is consistent
with the structure of the aglycone 5. Subsequently, this biscoumarin
fragment can split into two different coumarin monomer ions at m/z
179.0339 and m/z 208.0367, which follow different subsequent frag-
mentation patterns. The coumarin monomer ion at m/z 179.0339 can
easily lose a CO group, which is consistent with previous reports (Bor-
kowski et al., 2015; Wang et al., 2014), affording an ion at m/z
151.0390. The other ion at m/z 208.0367, can lose a methyl group and a
CO unit to obtain an ion at m/z 164.0104. Compounds 2, 3 and 5 shared
very similar fragmentation patterns with compound 1. Compound 4,
lacking a 6-OMe group, showed a different fragmentation pattern. The
biscoumarin ion at m/z 339.0497 indicates the loss of one glucose
moiety from the parent ion at m/z 518.1290 of compound 4. Subse-
quently, the biscoumarin ion can fragment into either of two isomeric
coumarin monomer ions, both at m/z 179.0339 (Fig. 4). The 7,8-dihy-
droxylated coumarin fragment can then react in the same way as for
compound 1, losing a CO group and yielding an ion at m/z 151.0390, or
losing oxygen to give an ion at 163.0389 (Fig. 4). The 3-hydroxylated
ion can fragment to form isomeric ions, though it is not possible to
determine which of the two pathways predominates. Previous studies
have already elucidated possible MS/MS fragmentation pathways for
coumarins (Borkowski et al., 2015; Wang et al., 2014) and biscoumarins,
-
1
data (m/z 499.0879, [M - H] ). The H and C NMR data (Table 1)
showed that compound 4 possessed a different aglycone, lacking the 6-
1
OMe group of compounds 1–3. The H NMR spectrum showed one
′′
​ 7.4 ​ Hz, H-1 )
typical anomeric sugar proton signal at δ 5.20 (d, J
​
=
′
′
which correlated to a carbon signal at δ 102.4 (C-1 ) in the HSQC
spectrum. As for the previous compounds, acid hydrolysis confirmed the
presence of D-glucose. In the HMBC spectrum, correlations from H-1’’ (δ
.20) to C-8’ (δ 132.9) confirmed that the β-D-glucose moiety was
located at C-8 of the biscoumarin moiety (Fig. 2). All signals were
5
′
assigned by analysis of 1D and 2D NMR spectra, and the structure of 4
′
′
was determined to be 8 -O-β-D-glucopyranosyl-7-hydroxy-3,7 -biscou-
marin (Fig. 2).
Compound 5 was obtained as a white amorphous powder. Based on a
-
[
M - H] quasi-molecular ion at m/z 367.0449 in the HR-ESI-MS spec-
12 8
trum, the molecular formula was determined to be C19H O . The
spectroscopic features suggested that its skeleton was the same as for
compounds 1–3. However, no signals corresponding to a sugar were
observed. Instead, an additional hydroxy group at δ 10.19 (br, s) was
located at C-8’. Correlations from methoxy group protons at δ 3.75 to C-
(δ 145.6) in the HMBC spectrum and correlations from 3.75 (s) to H-5
δ 7.16) in the NOESY spectrum indicated that methoxy group was
located at C-6 of the biscoumarin moiety. Thus, the structure of 5 was
6
(
2
which revealed similar fragments (Wang et al., 2016a). MS analysis
′
′
determined to be 6-methoxy-7,8 -dihydroxy-3,7 -biscoumarin (Fig. 2),
results indicated that both biscoumarins and coumarin monomers
3

X. Liu et al.
Phytochemistry 190 (2021) 112856
Table 1
Table 1 (continued)
1
13
H (600
​
MHz) and C NMR (150
​
MHz) data for compounds 1–5 in DMSO d (δ
6
Position
3
4
5
(
J, Hz)).
H
C
H
C
H
C
Position
1
2
3
.17–3.22
a
H
C
H
C
(
m )
′
′
′
′
′
′
a
4
5
6
3.10 (m )
69.6
76.5
60.7
3.08–3.09
69.8
77.4
62.7
2
3
4
5
6
7
8
9
156.8
137.2
126.9
109.1
145.8
150.4
102.7
146.7
109.8
55.9
157.0
137.5
126.9
109.1
145.8
150.3
102.7
146.7
109.9
56.0
a
(
m )
3.15–3.19
3.08–3.09
(m )
7.49 (1H, s)
7.56 (1H, s)
a
a
a
a
(m )
7.12 (m )
7.12 (m )
a
3.70 (1H, d,
3.56 (m );
1
3
0.5);
3.36–3.38
(m )
a
.39–3.47
6.82 (1H, s)
3.76 (3H, s)
6.81 (1H, s)
3.76 (3H, s)
a
(
m )
7
-Glu-
’’’
5.07 (1H, d,
6.8)
99.8
73.1
77.1
69.7
76.8
60.6
1
6
2
3
4
5
6
7
8
9
1
8
0
1
-OMe
′
2’’’
3’’’
4’’’
5’’’
6’’’
3.28–3.33
159.3
114.4
144.2
123.7
114.8
150.6
132.9
147.8
115.9
102.3
159.4
114.4
144.3
123.5
115.2
150.7
133.3
147.6
116.0
102.3
a
′
′
′
′
′
′
′
(m )
6.42 (1H, d, 9.6)
8.03 (1H, d, 9.6)
7.44 (1H, d, 8.9)
6.42 (1H, d, 9.6)
8.03 (1H, d, 9.6)
7.43 (1H, d, 8.9)
3.39–3.47
a
(m )
a
a
3.15–3.19
7.10 (m )
7.09 (m )
a
(m )
3.28–3.33
a
(m )
′
3.56 (1H, d,
14.4);
0
′
-Glu-
5.17 (1H, d, 7.6)
5.22 (1H, d, 7.2)
′′
3.39–3.47
1
a
′
′
′
′
′
′
′
′
′
′
a
a
(m )
2
3
4
5
6
3.20 (m )
73.9
76.3
69.9
74.1
62.8
3.18 (m )
74.0
76.5
69.7
77.5
60.6
a
a
3.20 (m )
3.18 (m )
a
Multiplicity undetermined due to overlapping signals.
Observed only in HMBC spectrum; br-broad.
a
3.09 (1H, m)
3.30 (1H, m)
4.03–4.10 (2H,
m)
3.08 (m )
b
a
3.08 (m )
3.56 (1H, d, 11.0); 3.37 (1H, dd,
4.0, 11.0)
shared a common fragment ion at m/z 179. This, combined with the
molecular networking (MN) analysis, providing an opportunity to
further explore the molecular structures of the remaining coumarins in
the molecular network. Based on parent ions and fragmentation pat-
terns, we have proposed some tentative molecular structures for other
parent ions observed in cluster G1 (Figures S4 and S5 of Supporting
Information), though it should be noted that definitive assignment of
positional isomers is not usually possible with MS/MS data alone (Leber
et al., 2020). No ions corresponding to daphnoretin itself was not
detected in the molecular network, nor in the LC-MS/MS data.
′
′
′
′
7
8
169.9
20.2
-Me
1.73 (3H, s)
Position
3
4
5
H
C
H
C
H
C
2
3
4
156.8
139.2
124.9
157.2
135.7
128.6
156.7
138.3
124.0
7.50 (1H, s)
7.21 (1H, s)
7.65 (1H, s)
7.37 (1H, d,
7.34
(
1H, s)
5
6
109.3
146.3
128.9
114.7
7.16
109.1
145.6
8
.5)
6.71 (1H, d,
.5)
(1H, s)
Biscoumarins are a relatively uncommon structure class, in which
the two coumarin moieties can be linked in a variety of ways, including
simple O- or C-linkages (Tanemossu et al., 2014), or more complex
linkages involving spiro centers or isoprenoid linkers (Hussain et al.,
8
b
7
8
148.4
103.0
163.1
102.3
149.4
102.6
7.22 (1H, s)
3.77 (3H, s)
6.66 (1H, d,
.5)
6.82
1
(1H, s)
′
9
145.7
112.3
56.0
153.4
109.3
146.1
110.2
55.9
2012). While a small number of 3,7 -O-linked biscoumarins have been
1
6
0
previously reported in the literature, this is the first report of biscou-
-OMe
3.75
′
marins hydroxylated at the 8 -position. One of the best-known 3,
(
3H, s)
10.19
br, s)
′
7
-O-linked biscoumarins is daphnoretin (Cordell, 1984), the structure
7
-OH
8.46 (br, s)
of which was first proposed in 1963 (Tschesche et al., 1963). Previous
studies have showed that daphnoretin possesses multiple biological
properties such as anti-tumor, antiviral, and antioxidant activities, and
the ability to activate PKC (Deiana et al., 2003; Hussain et al., 2012;
Yang et al., 2014). There are very few reports of more-closely related
natural products to compounds 1–5 in the literature: the only similar
(
′
′
2
3
159.3
114.6
159.6
114.2
159.7
114.5
6.46 (1H, d,
6.40 (1H, d,
9.5)
6.42
9
.5)
(1H, d,
9
.5)
′
′
′
4
5
6
8.06 (1H, d,
.5)
144.3
123.6
115.9
8.01 (1H, d,
9.5)
144.4
123.5
114.7
8.02
144.6
118.5
115.5
9
(1H, d,
′
9
.5)
compounds with 8 -sustituents are a group of bi- and tri-coumarins
7.47 (1H, d,
.6)
7.40 (1H, d,
8.6)
7.18
′
incorporating 8 -methoxy groups, reported from Chimonanthus salicifo-
8
(1H, d,
lius (Wang et al., 2016a).
8
.6)
Compounds 1–7 were tested for a range of biological activities:
cytotoxicity against HeLa, MDA-MB-231, HepG2 and MCF-7 ​ cell lines;
7.17 (1H, d,
.6)
7.07 (1H, d,
8.6)
7.05
8
(1H, d,
8
.6)
anti-inflammatory activity via inhibition of NO production in
RAW264.7 cells; antimicrobial activity against a small suite of test
microbes; and -glucosidase inhibition. For the antibacterial assay, at
40 g per well (20 L, 2 mg/mL), none of the compounds showed any
growth inhibition against multiple test microbes. For the cytotoxicity
assay, anti-inflammatory activity and -glucosidase inhibitory assay,
none of the compounds 1–7 showed any inhibition activity at a final
concentration of 200 M. None of the compounds showed activity in
any of these assays. Given the reports of cytotoxic properties in other
′
′
′
7
8
9
1
8
150.0
133.6
147.5
116.4
102.3
151.5
132.9
147.6
115.6
102.4
145.6
135.7
143.9
116.3
​
α
′
​
μ
​
μ
​
0
′
-Glu-
5.24 (1H, d,
7.3)
5.20 (1H, d,
7.4)
′
′
1
α
′
′
2
3
3.15–3.19
74.0
77.4
3.17–3.22
74.0
76.5
a
a
(
m )
(m )
′
′
a
​
μ
3.10 (m )
4

X. Liu et al.
Phytochemistry 190 (2021) 112856
Fig. 4. Proposed fragmentation pathway of compounds 1 and 4; positive mode.
biscoumarins, the absence of cytotoxic activity for compounds 1–7 was
UV–vis spectra were measured using a HITACHI U3900 spectropho-
tometer (HITACHI, Kyoto, Japan). High-resolution electrospray ioniza-
tion mass spectra (HR-ESI-MS) were measured using a Q Exactive HF
Orbitrap LC-MS (Thermo Fisher Scientific, Waltham MA, USA). NMR
unexpected; the presence of the additional OH or glycoside group at
′
position 8 may reduce the activity of these compounds.
Based on the guidance of MN analysis, we have successfully isolated
seven coumarin derivatives from Cruciata articulata, five of which were
undescribed biscoumarins. The presence of five new biscoumarins,
spectra were recorded on an Avance III 600 ​ MHz spectrometer (Bruker
BioSpin, Billerica MA, USA) using TMS or the residual solvents as in-
ternal standard. For open column chromatography, Diaion HP-20
(Mitsubishi Chemical Industries, Tokyo, Japan) was used. Analytical
and semipreparative HPLC separations were performed on an Agilent
1260 series HPLC system (G1311B quaternary pump, G1329B auto-
sampler, G1316A thermostatted column compartment and G1315D
photodiode array detector; Agilent Technologies, Santa Clara CA, USA),
′
along with 6 -acetoxy-daphnetin-β-D-glucoside (6) and daphnetin-8-O-
glucoside (7), constitutes the first description of coumarins and bis-
coumarins from this species. Though several coumarins have already
been reported from the Rubiaceae family (Benevides et al., 2004; Bua-
thong et al., 2019; Dao et al., 2015; De Rosa et al., 2002; Farid et al.,
2
002; Mfonku et al., 2020; Ralambonirina Rasoarivelo et al., 2018;
Singh and Sharma, 2020; Wolff et al., 2019), most of them are coumarin
monomers. Biscoumarins are not common in this family, and this is the
using a Pursuit XRs C18 column (5
​
μ
m, 4.6
​
×
​ 150 ​ mm, Agilent
Technologies, USA). Semipreparative HPLC separation was carried out
on a Shimadzu LC-20AR series HPLC instrument equipped with a UV
′
first time that 3,7 -biscoumarins have been found in the family Rubia-
ceae; these are also the first biscoumarins to be reported which are
glycosylated at the 8 position. On the basis of MS2 fragmentation
pathway analysis, we have proposed potential structures for some of the
minor biscoumarin glycosides appearing in the cluster G1 (Figure S5).
According to analysis of the G1 cluster (Fig. 1), there are still several
remaining coumarins that have not been isolated and identified, sug-
gesting that C. articulata could be further studied in order to expand the
knowledge of the phytochemistry of this under-studied species, and to
expand the known chemical diversity of biscoumarin derivatives.
detector and a reversed-phase C18 column (Pursuit XRs-C18, 10
​ m,
μ
′
21.2 250 mm). All solvents used were of HPLC grade (Concord
​
×
​
​
Technologies, Tianjin, China). Thin-layer chromatography (TLC) was
carried out on silica gel GF254 plates (Haiyang Chemicals Corp., Qing-
dao, China). TLC spots were visualized by heating silica gel plates
sprayed with 5% H
RAW264.7 cells were purchased from Type Culture Collection of the
Chinese Academy of Sciences (Shanghai, China). -Glucosidase from
Saccharomyces cerevisiae, acarbose and p-nitrophenyl- -glucopyranoside
PNPG) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2 4
SO in EtOH (v/v). The living HeLa, MCF-7 and
​
α
α
(
3
. Experimental
3
.2. Plant material
3
.1. General
Samples comprising entire plants of Cruciata articulata (L.) Ehrend,
(Rubiaceae) were collected at Kosmalyan Village near Zarin Gala in the
Lerik District of Azerbaijan (38.680310 N, 48.370432 E, 1412
elevation) in August 2006. Herbarium specimens documenting the
Melting points were measured on a Micro-melting point apparatus
with corrections. Optical rotations were recorded on an AUTOPOL II
Polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA).
​
​
​ m
5

X. Liu et al.
Phytochemistry 190 (2021) 112856
collection (Kerimov 58) have been deposited in the herbaria of the
Institute of Botany, Azerbaijan National Academy of Sciences (BAK) and
the New York Botanical Garden (NY). The whole plant, including roots,
was freed of extraneous matter and air dried in the shade prior to being
milled to a coarse powder.
and affording compounds 3 (11.5
59.8 min).
The 100% MeOH–H
RP-18 column (10 m, 21.2
v) at a flow rate of 10 mL/min and afforded compound 5 (4.8
R R
​ mg; t 68.2 ​ min) and 7 (8.3 ​ mg; t
​
2
O fraction (0.4
​
g) was separated on preparative
O–MeCN (73:27, v/
mg; t
​
μ
​
×
​
250 mm) using H
​
2
​
​
R
9
0.2 ​ min).
3
.3. LC-MS/MS-based molecular networking analysis
3
.4.1. Compound 1
Yellow, amorphous powder; mp 209–211 C; [
◦
25
D
A Q Exactive HF orbitrap mass spectrometer coupled with an Ulti-
α
]
-49.9 (c 0.01,
) 217 (5.15), 314 (3.70); 1H-NMR
mate 3000 RSLC nano (Thermo Fisher Scientific, USA) was used for LC-
MS/MS analysis in both positive and negative ion modes. An ODS col-
MeOH); UV (MeOH) λmax (log
Methanol-d 600 MHz), see Table 1;
MHz), see Table 1; HRESIMS m/z 571.1080 [M - H] (calcd. for
14, 571.1082).
ε
1
3
(
4
,
​
4
C-NMR (Methanol-d ,
-
umn (Pursuit XRs C18, 5
ogies, USA) was used for LC separation. The separation was performed
as follows: isocratic elution of 23% MeCN for 25 min and then ramping
up to 100% MeCN from 25 min to 40 min. Mass resolutions of 120,000
​
μ
m, 4.6
​
mm
​
×
​ 150 ​ mm, Agilent Technol-
1
50
​
C
27
H
23
O
​
​
​
3
.4.2. Compound 2
and 30,000 were applied for precursors and fragments. Data dependent
MS/MS acquisition with fragmentation of the top five most intense
precursors was performed in the analysis. MS/MS spectra were acquired
with stepped normalized collision energies (NCE) of 20, 40 and 60. Ion
◦
25
D
Yellow, amorphous powder; mp 210–212 C; [
α
]
-62.4 (c 0.01,
) 217 (5.12), 339 (3.60); 1H-NMR
MeOH); UV (MeOH) λmax (log
(
ε
13
DMSO d , 600 ​ MHz), see Table 1; C-NMR (DMSO d , 150 ​ MHz), see
6
6
-
21 13
Table 1; HRESIMS m/z 529.0981 [M - H] (calcd. for C25H O ,
source parameters are as follows: spray voltage, 3.8
​
kV for positive;
5
29.0977).
◦
capillary temperature, 250 C; sheath gas flow rate, 45; aux gas flow
◦
rate, 12; probe heater temperature, 300 C; mass range (m/z),
3
.4.3. Compound 3
1
00–1500. The data processing of LC-MS/MS raw data was performed
25
D
◦
Yellow, amorphous powder; mp 208–210 C; [
α
]
-54.3 (c 0.02,
using MSConvert software. A molecular network was created using the
GNPS Web platform (https://gnps.ucsd.edu). All the results and pa-
rameters could be accessed with the GNPS job id for MN analysis
MeOH); UV (MeOH) λmax (log
DMSO d , 600 MHz), see Table 1; C-NMR (DMSO d
Table 1; HRESIMS m/z 691.1502 [M - H] (calcd. for C31
ε
) 212 (5.11), 344 (3.41); 1H-NMR
1
3
(
6
​
6
, 150 ​ MHz), see
-
31 18
H O ,
(
http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=e5add1d3da2e45fa
6
91.1505).
8
f46d93775a03119).
3
.4.4. Compound 4
3
.4. Extraction and isolation
◦
25
D
Yellow, amorphous powder; mp 205–206 C; [
α
]
-45.2 (c 0.05,
MeOH); UV (MeOH) λmax (log
DMSO d , 600 MHz), see Table 1; C-NMR (DMSO d
Table 1; HRESIMS m/z 499.0879 [M - H] (calcd. for C24
ε
) 212 (4.97), 341 (3.27); 1H-NMR
A 1
extracted with 3
24 h each time), and plant material was macerated without stirring.
The pooled methanol extracts were evaporated to dryness in vacuo to
afford a tarry residue. Then, the methanolic extract (56.8 g) was
dispersed in MeOH:H O (9:1, 50 mL) and defatted with n-hexane
100 mL), which yielded the n-hexane fraction. The hydro-
​
kg sample of the dried and powdered plant material was
1
3
(
6
​
6
, 150 ​ MHz), see
​
×
​ 4 ​
L of methanol (MeOH) at ambient temperature
-
H
19
O
12
,
(
​
4
3
2
99.0871).
​
.4.5. Compound 5
2
​
◦
White, amorphous powder; mp 202–204 C; UV (MeOH) λmax (log
ε
)
(
3
​
×
​
​
1
13
12 (4.80), 343 (3.11); H-NMR (DMSO d
, 150 MHz), see Table 1; HRESIMS m/z 367.0449 [M -
H] (calcd. for C19 , 367.0448).
6
, 600 ​ MHz), see Table 1; C-
alcoholic phase was freed of solvent, dispersed in water and successively
NMR (DMSO d
6
​
extracted with dichloromethane (DCM) and n-BuOH (each
-
11 8
H O
3
​
×
​
100
​
mL) to afford n-hexane (1.5
g) fractions.
g) was subject to open column chroma-
tography on Diaion HP-20 (120 g), eluting with a H
O–MeOH step
gradient (each 3 200 mL) to give sub-fractions H O (2.1 g), 20%
MeOH–H O (1.4 g), 50% MeOH–H O (0.8 g), 80% MeOH–H O (6.7 g),
00% MeOH–H O (0.4 g), successively. Losses during purification are
attributed to imperfect dryness of the initial sample.
The 80% MeOH–H O fraction (6.7 g) was separated on a semi-
preparative HPLC (Pursuit XRs-C18, 10 m, 21.2 250 mm) at a
flow rate of 10
mL/min, by using isocratic elution of 20% MeCN–H
for the first 40 min, with subsequent washing with 100% MeCN, which
afforded fractions 1–9.
Fraction 5 (0.8 g; t
ative RP-18 column (10
O–MeCN (83:17, v/v), which afforded compound 2 (10.5
0.0 min), compound 4 (5.6 mg; t 84.7 min). Similarly, fraction 3
0.7 g; t min) was fractionated using the same RP-18
15.00–20.00
column by using 15% MeCN–H O with a flow rate at 10 mL/min, and
afforded compound 6 (6.2 mg; t 48.4 min). Fraction 9 (0.5 g; t
2.50–55.00 min) was separated on the RP-18 column using 25%
MeCN–H O of a flow rate at 8 mL/min, yielding compound 1 (6.8 mg;
29.3 min).
The 20% MeOH–H
MeOH–H O fraction (0.8
column (10 m, 21.2
​ g), DCM (2.2 ​ g), n-BuOH
(
14.5
​
g) and water (26.5 ​
3
.4.6. Acid hydrolysis of 1–4 and sugar analysis
A solution of each of compounds 1–4 (2 mg) in 3% HCl (2
heated for 4 h. Then, the solution was evaporated several times to
The n-BuOH fraction (14.5
​
​
​ mL) was
​
2
​
​
×
​
​
2
​
dryness with methanol until neutral. The neutralized solutions were
centrifuged, filtrated and further purified on a LC-2030 C Prominence-I
system (Shimadzu, Kyoto, Japan) equipped with a RID-20 A refractive
2
​
2
​
2
​
1
2
​
index detector, using
10 250 mm, 5 m). The flow rate was 3
phase was MeCN–H O (75:25) (Zhao et al., 2019; Liu et al., 2021). The
identity of D-glucose was confirmed by comparison of optical rotations
a
Waters XBridge BEH Amide column
2
​
(
​
×
​
​
​
μ
​
mL/min, and the mobile
​
μ
​
×
​
​
2
​
2
O
​
2
D
5
with authentic samples (D-glucose, sample: [
α
]
+61.7 (c 0.05, H
2
O);
2
D
5
​
R
20.00–30.00
m, 21.2
​
min) was separated on a prepar-
250 mm, 10 mL/min) using
mg; t
authentic D-glucose: [
α
]
+61.9 (c 0.05, H
2
O) and by comparing their
​
μ
​
×
​
​
​
TLC behavior with standard samples [Si gel, developed with
H
2
​
R
CHCl –MeOH–H O (8:5:1)] (Zha et al., 2015), both the test sugar and
authentic D-glucose shared the same Rf value at 0.35.
3
2
9
​
​
R
​
(
​
R
​
2
​
3.4.7. Antimicrobial testing
​
R
​
​
R
Antimicrobial assays were carried out according to literature pro-
tocols (Li and Clark, 2020) against the following test microbes: Staphy-
lococcus aureus (ATCC 12600), Pseudomonas fluorescens (ATCC 13525),
Enterococcus hirae (ATCC 8043), Streptococcus mutans (ATCC 25175),
Moraxella catarrhalis (ATCC 25238), Staphylococcus epidermidis (ATCC
14990), Pseudomonas aeruginosa (ATCC 15692), Bacillus subtilis (ATCC
4
​
2
​
​
t
R
​
2
O fraction (1.4
g) were combined, then separated on a RP-18
250 mm, 10 O,
mL/min) using 12% MeCN–H
​ g), together with the 50%
2
​
​
μ
​
×
​
​
​
2
6

X. Liu et al.
Phytochemistry 190 (2021) 112856
6
633), Saccharomyces kudriavzevii (ATCC 2601) and Candida albicans
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​
μ
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◦
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​
×
​ 10 ​ cells
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μ
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.4.9. Anti-inflammatory activity and NO production
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◦
cells were cultured in 96-well plates at 37 C for 24
​
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​
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​
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.4.10. Assay for
The -glucosidase inhibitory activity was conducted according to
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.1 M phosphate buffer), 5 L of the indicated concentration of acar-
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-glucosidase inhibitory activity
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.0
◦
​
α
​
​
​
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​
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