Six New Coumarin Glycosides from the Aerial Parts of Gendarussa vulgaris

Article abstract of DOI:10.3390/molecules24081456

Six new coumarin glycosides, genglycoside A–F (1–6), were isolated from the aerial parts of Gendarussa vulgaris, along with ten known analogues (7–16). Their structures were unambiguously established on the basis of extensive spectroscopic data and HPLC analysis. The cytotoxic activities of all isolated compounds were evaluated by MTT assay. Compound 12 showed the most potent cytotoxicity in Eca-109, MCF-7, and HepG2 cell lines. By the preliminary structure–activity relationships, it was firstly discovered that the glycosylation or esterification at 7,8-dihydroxy or 7-hydroxy drastically reduced the cytotoxic activity of the parent coumarin.

Full text of DOI:10.3390/molecules24081456

molecules
Article
Six New Coumarin Glycosides from the Aerial Parts
of Gendarussa vulgaris
Yanjun Sun 1 *, Meiling Gao 1,2, Haojie Chen 1,2, Ruijie Han 1,2, Hui Chen 1,2, Kun Du 1,2
,2,
,
Yanli Zhang 1 , Meng Li 1,2, Yingying Si 1,2 and Weisheng Feng
,2
1,2,
*
1
Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine
Development of Henan Province, Henan University of Chinese Medicine, Zhengzhou 450046, Henan, China;
gaoxiaomei6266@126.com (M.G.); CHj3928@126.com (H.C.); 18638221936@126.com (R.H.);
chenhuiyxy@hactcm.edu.cn (H.C.); qqninenine@hotmail.com (K.D.); zyl2013hnzy@163.com (Y.Z.);
limeng31716@163.com (M.L.); yingying8690@163.com (Y.S.)
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, Henan, China
Correspondence: sunyanjun2011@hactcm.edu.cn (Y.S.); fwsh@hactcm.edu.cn (W.F.);
Tel.: +86-371-6596-2746 (Y.S. & W.F.)
2
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 25 March 2019; Accepted: 10 April 2019; Published: 12 April 2019
Abstract: Six new coumarin glycosides, genglycoside A–F (1–6), were isolated from the aerial parts
of Gendarussa vulgaris, along with ten known analogues ( 16). Their structures were unambiguously
7–
established on the basis of extensive spectroscopic data and HPLC analysis. The cytotoxic activities of all
isolated compounds were evaluated by MTT assay. Compound 12 showed the most potent cytotoxicity
in Eca-109, MCF-7, and HepG2 cell lines. By the preliminary structure–activity relationships, it was
firstly discovered that the glycosylation or esterification at 7,8-dihydroxy or 7-hydroxy drastically
reduced the cytotoxic activity of the parent coumarin.
Keywords: Gendarussa vulgaris; coumarin glycoside; cytotoxic activity
1
. Introduction
Gendarussa vulgaris Nees, which belongs to the family of Acanthaceae, is an evergreen dwarf
shrub mainly distributed in China, India, Sri Lanka, and the Malay Peninsula [1]. As an important
medicinal plant, it has been described in Chinese Pharmacopoeia 2015, Supplement to the Compendium of
Materia Medica, Luchuan Materia Medica, Lingnan Medical Records, etc. Its aerial parts (called Xiaobogu
in Chinese) are frequently used for the treatment of fascia fracture, traumatic injury, rheumatism,
and ostalgia, blood stasis menstrual block, and postpartum abdominal pain [
investigations on G. vulgaris revealed the presence of bioactive alkaloids, flavonoids, phenylpropanoids,
steroids, and triterpenes [ ]. Naturally occurring coumarins have exhibited a broad spectrum of
pharmacological actions, including as an anticoagulant [ ], CNS stimulant [ ], antioxidant [ ], antiviral [ ],
hepatoprotective [ ], anti-inflammatory [ ], anticancer [ ], and cyclooxygenase, lipooxygenase,
cholinesterase (ChE), and monoamine oxidase (MAO) inhibitory activities [ 10], antimutagenic [10], etc.
2]. Previous chemical
1,2
3
4
5
6
7
8
9
9,
As potential therapeuticdrugsagainst cancer, coumarinshavenotonlyexhibitedobviousanti-proliferative
activity in malignant melanoma, prostate cancer, and renal cell carcinoma in some clinical trials [11],
but also very rare cardiotoxicity, nephrotoxicity, dermal toxicity, and other MDR (multi-drug resistance)
side effects [9]. In our search for cytotoxic natural products from the aerial parts of G. vulgaris,
six new coumarin glucosides (
1
–
6
) were obtained together with ten known analogues ( 16). Details of
7–
the isolation, structure elucidation, and cytotoxicity of all isolated compounds against Eca-109, MCF-7,
and HepG2 cell lines are described here (Figure 1).
Molecules 2019, 24, 1456; doi:10.3390/molecules24081456
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1456
2 of 10
5
4
H3CO
10
9
OH
3
O
7
6
''
6'''
5''
HO
O
O
2
O
HO
HO
O
8
''
O
1'''
O
O
1''
1'
OH
6
'
2
'''
OH
2''
H3CO
HO
HO
1
4
7
2
OH
6'
5
4
OH
''
H
3
CO
O
6
7
10
9
4
5
H
3
CO
HO
6
10
3
6
HO
O
3
4
O
5
HO
HO
7
H
3
CO
O
10
9
HO
1'
1
''
OH
6'
O
2
O
O
6'
OH
6''
6
3
3' 2'
8
O
2''
9
O
2
O
OH
3
''
OH
8
OH
6''
O
HO
HO
HO
O
O
2
O
O
7
O
HO 4'
HO
O
8
O
1
'
1'
1''
HO
HO
2'
2'
2''
3'
OH
3''
OH
OH
'' 2'' OH 1''
3
5
3
6
H
3
CO
5
4
H
3
CO
10
9
3
OH
R O
O
O
1
O
8'
O
2
O
6
'
OH
O
R2
HO
7'
1'
O
HO
HO
O
1
'
9'
OH
3
'
2
'
9
1
1
1
R
1
=β-D-glc, R
2
=OH
H
3
CO
6'
O
O
5
'
0 R
2 R
3 R
1
1
1
1
1
1
=β-D-glc, R =H
2
H
3
CO 3'
4'
OH
=H, R
2
2
=OH
=H
=H, R
8
14 R
=CH
=H, R
=CH , R
3
, R
=OCH
=H
2
=OH
11
1
1
5 R
6 R
2
3
3
2
Figure 1. The chemical structures of compounds 1–16 from Gendarussa vulgaris.
2
. Results and Discussion
The EtOH extract of the aerial parts of G. vulgaris was partitioned between PE, EtOAc, n-BuOH,
and water, respectively. The EtOAc and n-BuOH layers were fractionated and purified by repeated
column chromatography, allowing the isolation of sixteen coumarins ( 16), including six new coumarin
glycosides, genglycoside A–F ( ), along with ten known analogues. The known metabolites were
identified as indidene F ( ) [12], isofraxetin 6-O- -d-glucopyranoside ( ) [13], fraxin ( ) [14], scopoletin
-O- -d-glucopyranoside (10) [15], cleomiscosin A (11) [16], fraxetin (12) [17], scopoletin (13) [18],
1–
1–6
7
β
8
9
7
β
fraxidin (14) [19], isofraxidin (15) [17], scoparone (16) [20], by comparison of their spectroscopic data
with values reported in the literature.
Compound
1 was obtained as a white, amorphous powder. The positive HR-ESI-MS spectrum
+
revealed an [M + K] peak at m/z 721.1381 (calcd. 721.1382 for C H O K), suggesting a molecular
30
34 18
formula of C H O with fourteen degrees of unsaturation. Its IR spectrum showed the presence
3
0
34 18
−
1
−1
−1
of hydroxyl (3384 cm ), carbonyl (1712 cm ), and aromatic ring (1610, 1509 cm ). The UV
spectrum showed the maximum absorptions at 207, 291, and 335 nm. The H NMR spectrum
1
(Table 1, see Figure S1 in Supplementary Materials) showed two cis-olefinic protons δ 7.80 (1H, d,

Molecules 2019, 24, 1456
3 of 10
J = 9.5 Hz), 6.13 (1H, d, J = 9.5 Hz), and one aromatic proton
δ
6.93 (1H, s), suggesting the existence of
one 6,7,8-trisubstituted coumarin skeleton. One vanilloyl (4-hydroxy-3-methoxybenzoyl) group was
deduced by one ABX system of aromatic protons
δ
7.28 (1H, d, J = 1.9 Hz), 7.05 (1H, d, J = 8.5 Hz),
1
7
.18 (1H, dd, J = 8.5, 1.9 Hz), and one aromatic methoxy group
NMR spectrum also displayed one remaining aromatic methoxy group
anomeric protons 5.06 (1H, d, J = 5.3 Hz), 5.12 (1H, d, J = 5.3 Hz). d-glucose was identified by
acid hydrolysis and HPLC analysis. The -configuration of d-glucose was determined by the large
coupling constants (J = 5.3, 5.3 Hz) of the anomeric protons and the chemical shifts ( 103.2, 99.6) of
the anomeric carbons. The 13C-NMR spectrum (Table 2, see Figure S2 in Supplementary Materials)
also revealed one coumarin skeleton including one carbonyl group 160.1, two olefinic carbons 111.9,
44.6, one benzene ring 110.0, 104.8, 145.3, 148.4, 131.0, 142.8, besides one methoxyl group 56.1,
one vanilloyl group 122.8, 112.4, 148.4, 150.5, 114.0, 123.6, 165.0, 55.5, and two glucopyranosyl groups
103.2, 73.7, 76.2, 70.4, 74.2, 64.0, 99.6, 73.1, 76.8, 69.7, 77.3, 60.8. These spectroscopic data indicated
δ
3.74 (3H, s). Furthermore, the H
δ
3.77 (3H, s), two sugar
δ
β
δ
δ
δ
1
δ
δ
δ
δ
that compound 1 was a coumarin glucoside derivative. The aglycone was identified as fraxetin, by
comparison of its NMR data with those reported in the literature [17]. By the HMBC correlations
0
000
)
(
Figure 2) of the anomeric protons
δ
5.06 (1H, d, J = 5.3 Hz, H-1 ) and 5.12 (1H, d, J = 5.3 Hz, H-1
0
0
00
with C-8 (
δ
131.3) and C-4
(
δ
150.5), two glucopyranosyl groups were linked to C-7 and C-4
0
respectively. The HMBC correlation of the oxymethylene proton
with C-7 (
δ
4.19 (1H, dd, J = 11.8, 7.5 Hz, H-6 )
165.0), indicated that the 6-OH of inner glucopyranosyl group was esterified by vanillic
acid. The remaining methoxy group was located at C-6, based on the HMBC correlation between
the methoxy group protons 3.77 (3H, s) and C-6 ( 145.3). Thus, compound was established
as 8-[6-(4-O- -d-glucopyranosyloxy-3-methoxybenzoyl)]-O-
0
0
δ
δ
δ
1
β
β-d-glucopyranosyloxy-6-methoxy-7
-hydroxycoumarin, and named genglycoside A.
H CO
3
OH
OH
H
3
CO
HO
O
O
HO
O
O
O
HO
HO
HO
HO
O
O
O
O
O
OH
HO
O
O
O
OH
O
H CO
OH
3
HO
HO
HO
OH
1
5
OH
3
H CO
3
H CO
OH
HO
HO
OH
O
O
OH
O
O
HO
HO
O
HO
O
O
O
O
O
OH
HO
HO
O O
OH
OH
H
C
OH
3
6
based on HMBC
H
3
CO
H
3
CO
H
3
CO
HO
O
O
H C
3
HO
O
O
O
O
OH
O
O
O
HO
CO
HO
HO
O
O
O
O
O
H
3
CO
HO
HO
H
3
OH
HO
OH
HO
4
2
Figure 2. Key HMBC correlations of compounds 1–6.
Compound
2
was obtained as a white, amorphous powder. Its IR spectrum showed the presence
−
1
−1
−1
of hydroxyl (3359 cm ), carbonyl (1710 cm ), and aromatic ring (1602, 1505 cm ). The UV
1
13
spectrum showed the maximum absorptions at 205, 295, and 345 nm. Its H and C NMR (Tables 1
and 2, see Figures S5 and S6 in Supplementary Materials) were quite similar to those of , except
that one glucopyranosyl group disappeared in . This was further supported by its HR-ESI-MS,
1
2

Molecules 2019, 24, 1456
4 of 10
+
which gave an [M + Na] quasi-molecular ion peak m/z 543.1097 (calcd. for C H O Na, 543.1115),
24
24 13
being 162 mass-units less than that of
proton δ 4.94 (1H, d, J = 7.7 Hz, H-1 ) and C-8 (δ 131.5), indicated that the glucopyranosyl group was
1
. The HMBC correlation (Figure 2) between the sugar anomeric
0
also attached to C-8. In addition, the HMBC correlation between the oxymethylene proton
δ
4.14
165.3), suggested that the 6-OH of glucopyranosyl group was
esterified by vanillic acid. Thus, compound was identified as 8-[6-(3-hydroxy-4-methoxybenzoyl)]
0
00
δ
(
1H, dd, J = 11.8, 7.5 Hz, H-6 ) and C-7 (
2
-
β-d-glucopyranosyloxy-6-methoxy-7-hydroxycoumarin, and named genglycoside B.
1
Table 1. H NMR spectroscopic data (500 MHz) of 1–6.
4 b
5 a
6 a
No.
1 a
2 a
3 a
3
4
5
8
6.13 d (9.5)
7.80 d (9.5)
6.93 s
6.08 d (9.5)
7.79 d (9.5)
6.92 s
6.33 d (9.6) 5.97 d (9.3) 6.22 d (9.5) 6.39 d (9.5)
7.96 d (9.6) 7.56 d (9.3) 7.88 d (9.5) 7.94 d (9.5)
7.30 s
7.17 s
6.65 s
7.02 s
7.14 s
6
-OCH
3.77 s
5.06 d (5.3)
3.47 m
3.31 m
3.23 m
3.49 m
.43 dd (11.8, 2.0)
.19 dd (11.8, 7.5)
3.76 s
4.94 d (7.7)
3.44 m
3.31 m
3.23 m
3.81 s
3.80 s
3.81 s
3.81 s
3
0
0
0
0
0
1
2
3
4
5
5.21d (7.4)
3.50 m
3.50 m
3.23 m
3.47 m
3.61 m
3.48 m
4.35 d (7.8)
3.10 m
3.21 m
3.02 m
3.23 m
5.11 d (7.8) 4.97 d (7.8) 5.26 d (7.8)
3.59 m
3.60 m
4.30 m
3.51 m
4.60 m
4.41 m
3.37 m
2.97 m
3.24 m
2.89 m
3.87 m
3.60 m
3.38 m
3.20 m
3.10 m
3.09 m
3.58 m
3.41 m
3.47 m
4.46 dd (11.8, 1.9)
4.14 dd (11.8, 7.5)
4
4
0
6
0
0
0
0
0
0
1
2
3
4
5
4.06 d (7.8) 5.18 d (7.8)
0
0
0
0
7.28 d (1.9)
7.29 d (1.9)
7.03 s
2.84 m
3.24 m
2.99 m
3.32 m
3.38 m
3.57 m
3.38 m
3.20 m
3.10 m
3.12 m
3.58m
3.40 m
7.05 d (8.5)
6.79 d (8.2)
7.22 dd (8.2, 1.9)
3.75 s
3
3
.61 m
.43 m
0
0
6
7.18 dd (8.5, 1.9)
3.74 s
7.03 s
0
0
0
0
3
5
-OCH3
3.79 s
3.79 s
-OCH
3
0
0
0
0
0
00
00
00
00
00
1
2
3
4
5
5.12 d (5.3)
3.25 m
3.32 m
3.22 m
3.47 m
5.38 br.s
3.89 m
3.48 m
3.40 m
4.15 m
3
3
.71 m
.48 m
0
00
6
1.25 d (6.2)
a 1H NMR data (δ) were measured in DMSO-d6; b 1H NMR data (δ) were measured in CD3OD.
Compound
3 was obtained as a white, amorphous powder. The positive HR-ESI-MS spectrum
+
revealed an [M + Na] peak at m/z 539.1396 (calcd. 539.1377 for C H O Na), suggesting a molecular
22
28 14
formula of C H O with nine degrees of unsaturation. Its IR spectrum showed the presence of
2
2
28 14
−
1
−1
−1
hydroxyl (3333 cm ), carbonyl (1700 cm ), and aromatic ring (1612, 1512 cm ). The UV spectrum
showed the maximum absorptions at 204, 290, and 340 nm. The H NMR spectrum (Table 1,
1
see Figure S9 in Supplementary Materials) indicated the presence of one 6,7-disubstituted coumarin
skeleton
of two glucopyranosyl groups
group
3.81 (3H, s). The 13C NMR spectrum (Table 2, see Figure S10 in Supplementary Materials)
exhibited one coumarin skeleton including one carbonyl group 160.4, two olefinic carbons 113.4,
44.1, one benzene ring 112.4, 110.0, 146.0, 148.9, 103.1, 148.9, besides two sets of glucopyranosyl
groups 99.0, 71.8, 87.4, 67.9, 76.5, 61.1, 103.9, 73.8, 75.9, 70.1, 76.9, 60.4, one methoxy group 56.1.
d-glucose was also identified by the same analytical method as compound . The large coupling
constants (7.4, 7.8 Hz) of anomeric protons allowed the identification of two
moieties. The HMBC cross peaks (Figure 2) of the anomeric proton
δ
7.96 (1H, d, J = 9.6 Hz), 6.33 (1H, d, J = 9.6 Hz), 7.30 (1H, s), 7.17 (1H, s), anomeric protons
δ
5.21 (1H, d, J = 7.4 Hz), 4.35 (1H, d, J = 7.8 Hz), one methoxy
δ
δ
δ
1
δ
δ
δ
1
β
-glucopyranosyl
0
δ
5.21 (1H, d, J = 7.4 Hz, H-1 )

Molecules 2019, 24, 1456
5 of 10
0
0
0
δ
and 4.35 (1H, d, J = 7.8 Hz, H-1 ) with C-7 (
δ
148.9) and C-3 (
87.4), respectively, indicated that
0
one glucopyranosyl group was linked to C-7 of the aglycone and the other was substituted at C-3
of the inner glucopyranosyl group. The methoxy group was located at C-6, based on the HMBC
correlation between methoxy group protons
δ
3.81 (3H,s) and C-6 (δ 146.0). Thus, compound 3 was
established as 7-[(3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]-6-methoxycoumarin, and named
genglycoside C.
Compound
4 was obtained as a white, amorphous powder. Its IR spectrum showed the presence
−
1
−1
−1
of hydroxyl (3377 cm ), carbonyl (1709 cm ), and an aromatic ring (1603, 1501 cm ). The UV
1
13
spectrum showed the maximum absorptions at 208, 292, and 343 nm. Its H and C NMR (Tables 1
and 2, see Figures S13 and S14 in Supplementary Materials) were quite similar to those of , except for
the appearance of one syringoyl group and one rhamnopyranosyl moiety in , respectively, instead of
1
4
one vanilloyl group and one glucopyranosyl moiety found in 1. This was further supported by its
+
HR-ESI-MS, which gave an [M + Na] quasi-molecular ion peak m/z 719.1794 (calcd. for C H O Na,
31
36 18
7
19.1800), being 14 mass-units more than that of
group was deduced by two aromatic protons 7.03 (2H, s), two aromatic methoxy groups
combined with the HMBC correlation of the aromatic protons 7.03 (2H, s) with the carbonyl group
167.0. d-glucose and l-rhamnose were also identified by the same HPLC analysis as compound
1
. The syringoyl (4-hydroxy-3,5-dimethoxybenzoyl)
δ
δ
3.79 (6H, s),
δ
δ
1.
The large coupling constant (7.8 Hz) of anomeric proton allowed the identification of
moiety. The
its NMR data with the literature values [21]. The rhamnopyranosyl group was confirmed by
six aliphatic carbons 103.4, 72.3, 73.6, 72.2, 72.0, 18.0. By the HMBC correlations (Figure 2) of
anomeric protons 5.11 (1H, d, J = 7.8 Hz, H-1 ) and
β-glucopyranosyl
α
configuration for the l-rhamnopyranosyl unit was established by comparison of
δ
0
000
δ
δ
5.38 (1H, br.s, H-1 ) with C-8 (
δ
132.2)
0
0
00
and C-4 (
δ
139.7), the glucopyranosyl and rhamnopyranosyl groups were linked to C-8 and C-4 ,
respectively. The HMBC correlations of the methylene protons
0
δ
4.60 (1H, m, H-6 ), 4.41 (1H, m,
0
00
δ
H-6 ) with C-7 (
acid. The remaining methoxy group was located at C-6, based on the HMBC correlation between
methoxy group protons 3.80 (3H, s) and C-6 ( 147.2). Thus, compound was established
as 8-[6-(4-O- -l-rhamnopyranosyloxy-3,5-dimethoxybenzoyl)]-O- -d-glucopyranosyloxy-6-methoxy-7
hydroxycoumarin, and named genglycoside D.
Compound was obtained as a white, amorphous powder. The positive HR-ESI-MS spectrum
revealed an [M + Na] peak at m/z 555.1320 (calcd. 555.1326 for C H O Na), suggesting a molecular
167.0), indicated that the 6-OH of inner glucopyranosyl was esterified by syringic
δ
δ
4
α
β
-
5
+
22
28 15
formula of C H O with nine degrees of unsaturation. Its IR spectrum showed the presence of hydroxyl
2
2
28 15
−1
−1
−1
(
3378 cm ), carbonyl (1706 cm ), and aromatic ring (1607, 1509 cm ). The UV spectrum showed
the maximum absorptions at 206, 292, and 339 nm. The H NMR spectrum (Table 1, see Figure S17
1
in Supplementary Materials) indicated the presence of one 6,7,8-trisubstituted coumarin skeleton
1H, d, J = 9.5 Hz), 6.22 (1H, d, J = 9.5 Hz), 7.02 (1H, s), anomeric protons of two glucopyranosyl groups
4.97 (1H, d, J = 7.8 Hz), 4.06 (1H, d, J = 7.8 Hz), one methoxy group
3.81 (3H, s). The 13C NMR
spectrum (Table 2, see Figure S18 in Supplementary Materials) also exhibited one coumarin skeleton
including one carbonyl group 160.2, two olefinic carbons 111.2, 144.8, one benzene ring 110.1, 105.0,
45.3, 143.7, 131.3, 142.7, besides two sets of glucopyranosyl groups 103.6, 73.8, 76.4, 69.5, 76.5, 67.7,
03.0, 73.5, 76.2, 69.8, 76.6, 60.8, one methoxy group 56.1. d-glucose was also identified by the same
. The large coupling constants (7.8, 7.8 Hz) of anomeric protons allowed
-glucopyranosyl moieties. The HMBC cross peaks (Figure 2) of the anomeric
δ7.88
(
δ
δ
δ
δ
δ
1
1
δ
δ
analytical method as compound
the identification of two
protons 4.97 (1H, d, J = 7.8 Hz, H-1 ), 4.06 (1H, d, J = 7.8 Hz, H-1 ) with C-8 (δ131.3) and C-6 ( 67.7),
1
β
0
00
0
δ
δ
indicated that one glucopyranosyl group was linked to C-8 of the aglycone and the other was substituted
0
at C-6 of the inner glucopyranosyl group. The methoxy group was located at C-6, based on the HMBC
correlation between methoxy group
as 8-[6-( -d-glucopyranosyloxy)]-O-
genglycoside E.
δ
3.81 (3H,s) and C-6 (
δ145.3). Thus, compound 5 was established
β
β-d-glucopyranosyloxy-6-methoxy-7-hydroxycoumarin, and named

Molecules 2019, 24, 1456
6 of 10
Compound
6
was obtained as a white, amorphous powder. The positive HR-ESI-MS spectrum
+
revealed an [M + Na] peak at m/z 555.1320 (calcd. 555.1326 for C H O Na), suggesting a molecular
22
28 15
formula of C H O with nine degrees of unsaturation. Its IR spectrum showed the presence of
2
2
28 15
−
1
−1
−1
hydroxyl (3327 cm ), carbonyl (1682 cm ), and aromatic ring (1608, 1506 cm ). The UV spectrum
showed the maximum absorptions at 207, 292, and 338 nm. The H NMR spectrum (Table 1, see Figure
1
S21 in Supplementary Materials) indicated the presence of one 6,7,8-trisubstituted coumarin skeleton
δ
7.94 (1H, d, J = 9.5 Hz), 6.39 (1H, d, J = 9.5 Hz), 7.14 (1H, s), anomeric protons of two glucopyranosyl
groups 5.26 (1H, d, J = 7.8 Hz), 5.18 (1H, d, J = 7.8 Hz), one methoxy group
3.81 (3H, s). The 13
NMR spectrum (Table 2, see Figure S22 in Supplementary Materials) exhibited one coumarin skeleton
including one carbonyl group 159.9, two olefinic carbons 114.9, 144.2, one benzene ring 114.4,
102.6, 74.0, 76.4, 69.9, 77.6,
56.6. d-glucose was also identified by
. The large coupling constants (7.8, 7.8 Hz) of two anomeric
-glucopyranosyl moieties. The HMBC cross peaks (Figure 2)
δ
δ
C
δ
δ
δ
1
05.9, 149.6, 141.3, 136.1, 142.4, besides two sets of glucopyranosyl groups
δ
6
0.7, 102.5, 74.0, 76.4, 69.9, 77.5, 60.7, one methoxy group
δ
the same analytical method as compound
protons allowed the identification of
of the anomeric protons 5.26 (1H, d, J = 7.8 Hz, H-1 ) and 5.18 (1H, d, J = 7.8 Hz, H-1 ) with C-7
141.3) and C-8 ( 136.1), respectively, indicated that two glucopyranosyl groups were linked to C-7
and C-8 of the aglycone, respectively. The methoxy group was located at C-6, based on the HMBC
correlation between methoxy group 3.81 (3H,s) and C-6 ( 149.6). Thus, compound was established
1
β
0
00
δ
(δ
δ
δ
δ
6
as 7,8-bis(β-d-glucopyranosyloxy)-6-methoxycoumarin, and named genglycoside F.
Table 2. 13C NMR Spectroscopic Data (100 MHz) of 1–6.
No.
2
1a
2 a
3 a
4 b
5 a
6 a
No.
1 a
2 a
3 a
4 b
5 a
6 a
1
03.9
d
102.5
d
0
0
0
0
0
0
0
0
0
0
160.1 s 160.2 s 160.4 s 163.4 s 160.2 s 159.9 s
1
2
3
4
5
122.8 s 123.4 s
126.9 s 103.0 s
1
1
1
11.9
d
44.6
d
04.8
d
115.0
d
144.5
d
103.8
d
113.4
d
144.1
d
110.0
d
112.4
d
146.0
d
105.3
d
111.2
d
144.8
d
105.0
d
114.9
d
144.2
d
105.9
d
112.4
d
112.4
d
107.6
73.5 d
d
3
73.8 d
75.9 d
70.1 d
76.9 d
60.4 t
74.0 d
76.4 d
69.9 s
77.5 d
60.7 t
4
148.4 s 147.2 s
154.3 s
139.7 s
154.3 s
76.2 d
69.8 d
76.6 d
60.8 d
5
150.5 s 151.4 s
1
1
14.0
d
22.6
d
115.0
d
120.5
d
6
145.3 s 145.8 s 146.0 s 147.2 s 145.3 s 149.6 s
107.6
d
167.0 s
56.5 q
56.5 q
0
0
0
0
7
148.4 s 147.2 s 148.9 s 146.0 s 143.7 s 141.3 s
6
7
8
9
131.3 s 131.5 s 103.1d 132.2 s 131.3 s 136.1 s
142.8 s 143.2 s 148.9 s 144.6 s 142.7 s 142.4 s
110.0 s 112.4 s 112.4 s 110.2 s 110.1 s 114.4 s
165.0 s 165.3 s
55.5 q
0
0
0
0
3
5
-OCH
-OCH
3
3
55.5 q
10
1
03.4
d
0
00
6
-OCH
3
56.1 q
56.0 q
56.1 q
56.8 q
56.1 q
56.6 q
1
99.6 d
73.1 d
99.6 d
73.1 d
1
03.2
d
104.8
d
104.4
d
103.6
d
102.6
d
0
000
1
99.0 d
2
72.3 d
0
0
0
0
0
000
000
000
000
2
3
4
5
6
73.7 d
76.2 d
70.4 d
74.2 d
64.0 t
73.8 d
76.3 d
70.2 d
74.3 d
63.7 t
71.8 d
87.4 d
67.9 d
76.5 d
61.1 t
75.3 d
77.9 d
71.4 d
75.7 d
65.2 t
73.8 d
76.4 d
69.5 d
76.5 d
67.7 t
74.0 d
76.4 d
69.9 d
77.6 d
60.7 t
3
4
5
6
76.8 d
69.7 d
77.3 d
60.8 t
76.8 d
69.7 d
77.3 d
60.8 t
73.6 d
72.2 d
72.0 d
18.0 q
a 1H NMR data (δ) were measured in DMSO-d6; b 1H NMR data (δ) were measured in CD3OD.
According to the previous procedure [22], all isolated compounds were evaluated for cytotoxic
activities against Eca-109, MCF-7, and HepG2 cell lines, as well as a normal human umbilical vein
endothelial cell line (HUVEC) (Table 3). Etoposide was used as the positive control. All isolated
compounds exerted no cytotoxicity against the normal cell line. Compound 12 showed the highest
cytotoxicity against Eca-109, MCF-7, and HepG2 cell lines, with IC50 values of 20.38, 28.61, 30.27
respectively. Compounds 10 and 15 16 had no cytotoxicity with IC50 > 100 M. Furthermore,
,8-dihydroxy derivative 12 exhibited significantly higher activity compared to corresponding
glycosylation and etherification analogues 4–6 11 14, and 15, indicating that 7,8-dihydroxy
were structurally required for the cytotoxicity against Eca-109, MCF-7, and HepG cells lines. The same
µM,
1–
–
µ
7
1,
2
,
,
9,
,
2
effect was found between 7-hydroxy derivative 13 and corresponding analogues
3, 7, 10 and 16.
The glycosylation or esterification at 7,8-dihydroxy or 7-dihydroxy drastically reduced the cytotoxic

Molecules 2019, 24, 1456
7 of 10
activity of the parent coumarin. With the promising cytotoxicities against three cell lines, compound 12
may be the most valuable lead compound in all tested isolates.
Table 3. Cytotoxicities of compounds 11–14 against Eca-109, MCF-7, and HepG2 cell lines (IC50, µM).
Compound
Eca-109
MCF-7
HepG2
HUVEC
1
1
1
1
1
2
3
4
21.04 ± 1.85
20.38 ± 1.94
45.72 ± 3.55
41.09 ± 3.78
20.48 ± 1.82
35.29 ± 2.61
28.61 ± 1.37
61.59 ± 5.70
59.59 ± 5.24
5.82 ± 0.49
43.72 ± 3.97
30.27 ± 1.18
53.74 ± 4.09
>100
>100
>100
>100
>100
etoposide
1.15 ± 0.09
41. 65 ± 0.32
3
. Experimental Section
3
.1. General Experimental Procedures
The UV spectra were measured on a Shimadzu UV-1700 spectrometer (Shimadzu Corporation,
Kyoto, Japan). The IR spectra were measured on a Nicolet 10 Microscope Spectrometer
Thermo Scientific, San Jose, CA, USA). The 1D and 2D NMR spectra were recorded on
(
Bruker-AC (E)-500 spectrometer (Bruker AM 500, Fällanden, Switzerland) using TMS as an internal
standard. The HR-ESI-MS was determined on a Bruker microTOF-Q instrument (Bruker BioSpin,
Rheinstetten, Germany). Column chromatography was performed with silica gel (200–300 mesh;
Qingdao Marine Chemical Inc., Qingdao, China), sephadex LH-20 (GE Healthcare), ODS (50 µm; YMC Co.
LTD., Kyoto, Japan), AB-8 macroporous resin (Qinshi Science and Technology Ltd., Zhengzhou, China).
Thin layer chromatography (TLC) was carried out on silica gel GF254 precoated plates (Qingdao Marine
Chemical Inc., Qingdao, China), and spots were visualized under UV light. Preparative HPLC
separations were performed on a SEP system (Beijing Sepuruisi scientific Co., Ltd., China) equipped
with a variable-wavelength UV detector, using a YMC-Pack ODS-A column (250
×
20 mm, 5 µm).
Chemical reagents for isolation were of analytical grade and purchased from Tianjin Siyou Co., Ltd., China.
Biological reagents were from Sigma Company. Human heptocellular (HepG2), esophageal (Eca-109),
and breast (MCF-7) cancer cell lines were from Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, China.
3
.2. Plant Material
The plant materials were collected from Liping, Guizhou province, China, in September 2016,
and identified by Cheng-Ming Dong as the aerial parts of G. vulgaris, according to the Chinese
Pharmacopoeia 2015. A voucher specimen (GV 20160901) was deposited at the School of Pharmacy,
Henan University of Chinese Medicine.
3
.3. Extraction and Isolation
The dried aerial parts of G. vulgaris were ground into a power (20 kg) and refluxed with 95% EtOH
(
60 L
×
3). The filtrate was concentrated under reduced pressure to yield a dark-brown residue (1.1 kg).
The residue was suspended in water (4.4 L) and partitioned with petroleum ether (PE, 4.4 L
EtOAc (4.4 L × 3), and n-BuOH (4.4 L × 3), successively.
×
3),
The EtOAc extract (194 g) was fractionated using silica gel column chromatography (CC, 11
×
70 cm)
◦
with a gradient of PE (60–90 C)–acetone. The fractions were combined into ten main fractions E1–10
based on TLC results. Fraction E8 (3.05 g) was chromatographed over open ODS (2.5
×
45 cm) eluted
by methanol–H O (v/v 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30) to yield sub-fractions E–8–1~E–8–3.
2
Sub-fraction E–8–2 (0.95 g) was further submitted to silica gel CC (1.0
100:10) to give 15 (3.1 mg). Sub-fraction E–8–3 (1.44 g) was further applied to preparative HPLC,
eluted with methanol–H O (75: 25) at a flow rate of 7 mL/min to give 13 (7.8 mg, t 15 min), 14 (5.1 mg,
×
20 cm) eluted by CHCl –MeOH
3
(
2
R
tR 18 min), 16 (3.5 mg, t 26 min). Fraction E9 (2.75 g) was further chromatographed over open ODS
R

Molecules 2019, 24, 1456
8 of 10
(
2
×
40 cm) eluted with a gradient of methanol–H O (v/v 30:70, 60:40, 65:35, 70:30, 80:20, 90:10) to yield
2
sub-fractions E–9–1~E–9–2. Sub-fraction E–9–1 (0.85 g) was purified by preparative HPLC eluted with
methanol–H O (70:30) at a flow rate of 7 mL/min to give 12 (82.6 mg, t 22 min).
2
R
The n-BuOH extract (64 g) was fractionated by AB-8 CC (5
EtOH-H O; 0:100, 30:70, 95:5, each 5 L) to give fractions N1 and N2. Fraction N1 (15 g) was separated
2
×
90 cm, 900 g) with a gradient system
(
by silica gel CC (5
00:5, 100:7, 100:10, 100:20, 100:30, 100:50) to afford sub-fraction N-1-1~N-1-3. Sub-Fraction N-1-1 (1.5 g)
was subjected to sephadex LH-20 CC (2 50 cm) eluted by methanol to give compounds (4.2 mg)
and 10 (3.9 mg). Sub-Fraction N-1-3 (1.2 g) was purified by preparative HPLC eluted with MeOH–H O
×
50 cm, 150 g) with a gradient system of increasing polarity (CH Cl –MeOH; 100:3,
2 2
1
×
4
2
(
40:60) at 7 mL/min to yield
tR 21 min), (7.9 mg, t 25 min). Fraction N2 (6 g) was separated by sephadex LH-20 CC (2.0
eluted by methanol to yield sub-fraction N-2-1~ N-2-5. Sub-fraction N-2-1 (1.82 g) was purified by
silica gel CC (1.5 22 cm, 18 g) eluted with CH Cl –MeOH (100:3, 100:5, 100:7, 100:10, 100:30) to
compounds 2 (3.8 mg), 8 (4.3 mg), 9 (3.1 mg), 11 (3.5 mg).
1
(10.5 mg, t 12 min),
5
(7.6 mg, t 16 min),
6
(5.3 mg, t 18 min),
3
×
(4.5 mg,
90 cm),
R
R
R
7
R
×
2
2
3
.4. Spectroscopic and Physical Data
Genglycoside A ( ): white, amorphous powder; UV (MeOH)
35 (0.14) nm; IR νmax 3384, 2923, 2853, 1702, 1684, 1610, 1509, 1455, 1416, 1271, 1163, 1121 cm ;
1
λmax (log ε ) 207 (0.73), 291 (0.16),
−1
3
+
HR-ESI-MS (positive): m/z 721.1381 [M + K] (calcd. for C H O K, 721.1382); NMR data (DMSO-d ),
30
34 18
6
see Tables 1 and 2.
Genglycoside B (
2
): white, amorphous powder; UV (MeOH)
λ
max (log
ε
) 205 (1.08), 295 (0.22),
−
1
3
45 (0.19) nm; IR νmax 3359, 2920, 2851, 1710, 1602, 1505, 1418, 1286, 1217, 1124, 1073 cm ; HR-ESI-MS
(
positive): m/z 543.1097 [M + Na]+ (calcd. for C H O Na, 543.1115); NMR data (DMSO-d ),
24
24 13
6
see Tables 1 and 2.
Genglycoside C (
3
): white, amorphous powder; UV (MeOH)
λ
max (log
ε
) 204 (1.34), 290 (0.26),
−1
340 (0.27) nm; IR νmax 3333, 2922, 2853, 1700, 1612, 1564, 1512, 1422, 1394, 1281, 1248, 1199, 1076 cm ;
+
HR-ESI-MS (positive): m/z 539.1396 [M + Na] (calcd. for C H O Na, 539.1377); NMR data
22
28 14
(DMSO-d ), see Tables 1 and 2.
6
Genglycoside D (
4
): white, amorphous powder; UV (MeOH)
λ
max (log ε ) 208 (1.25), 292 (0.17), 343
−1
(
0.20) nm; IRνmax 3377, 2927, 2854, 1709, 1603, 1577, 1501, 1456, 1416, 1336, 1221, 1187, 1127, 1067 cm ;
HR-ESI-MS (positive): m/z 719.1794 [M + Na] (calcd. for C H O Na, 719.1800); NMR data (CD OD),
+
31
36 18
3
see Tables 1 and 2.
Genglycoside E (
5
): white, amorphous powder; UV (MeOH)
λ
max (log
ε
) 206 (1.09), 292 (0.30),
−1
3
39 (0.20) nm; IRνmax 3378, 2922, 2854, 1706, 1607, 1566, 1509, 1439, 1412, 1348, 1296, 1197, 1163 cm ;
+
HR-ESI-MS (positive): m/z 555.1320 [M + Na] (calcd. for C H O Na, 555.1326); NMR data
22
28 15
(
DMSO-d ), see Tables 1 and 2.
6
Genglycoside F (6): white, amorphous powder; UV (MeOH) λmax (log ε ) 207 (1.20), 292
(
1
0.31), 338 (0.21) nm; IR νmax 3327, 2924, 2853, 1682, 1608, 1569, 1506, 1487, 1439, 1414, 1294, 1207,
−
137, 1072 cm ; HR-ESI-MS (positive): m/z 555.1320 [M + Na] (calcd. for C H O Na, 555.1326);
22 28 15
1
+
NMR data (DMSO-d ), see Tables 1 and 2.
6
3
.5. Acid Hydrolysis and Sugar Analysis
According to the literature [23], the absolute configurations of the monosaccharide moieties
◦
were determined. Compounds
1
–
6
(2 mg) were hydrolyzed with 2 N HCl (5 mL) for 2 h at 90 C.
The HCl was removed by evaporation, and then extracted by EtOAc (7 mL
×
3). The aqueous layer
was evaporated to dryness under N to give a residue. The residue was dissolved in 0.5 mL anhydrous
2
◦
pyridine containing 2 mg l-cysteine methyl ester hydrochloride. The mixture was kept at 60 C for 1 h,
◦
and 20
µ
L of isothiocyanate was added, followed by heating at 60 C for another 1 h. Then, the reactant
was analyzed by an HPLC system (column: YMC-Triart C18 column (250
CH CN/0.1%H PO4; detection wavelength: 250 nm, injection volume: 10
×
4.6 mm, 5
L; flow rate: 1.2 mL/min).
µm); eluent:
µ
3
3

Molecules 2019, 24, 1456
9 of 10
The derivatives of d-glucose and l-rhamnose in compounds 1−6 were identified by comparison to
the retention times of authentic samples (t : d-glucose, 17.4 min; l-rhamnose, 26.9 min).
R
4
. Conclusions
With fewer adverse effects, natural products have played an important role in new drug
discovery. Extensive research has been focused on natural products with significant cytotoxic activities,
such as alkaloids, terpenoids [24], flavonoids [25], and lignans [26]. In contrast, the cytotoxic activities
of simple coumarins are rarely reported. Our research revealed that simple coumarins were the major
active constituents of G. vulgaris. However, until now, the chemical investigations of G. vulgaris were
inadequate, with only two studies [
three phenylpropanoids, two steroids, and one triterpene. The phytochemical investigation of G. vulgaris
resulted in the isolation of sixteen simple coumarins, including six new glucoside derivatives ( ).
Among the sixteen isolated coumarins, only compounds 11 14 showed certain cytotoxic activities against
Eca-109, MCF-7, and HepG2 cell lines. On the basis of the preliminary structure–activity relationship
SAR) studies, 7,8-dihydroxy and 7-hydroxy play a very important role in maintaining cytotoxicity
1,27] indicating that it contained seven alkaloids, eight flavonoids,
1–6
–
(
for simple coumarin. The glycosylation and etherification of the 7,8-dihydroxy and 7-hydroxy strongly
reduced the cytotoxic activity. This study not only enriches the chemical diversity of coumarin glycosides
in Gendarussa plants, but also broadens the application field of G. vulgaris.
Supplementary Materials: The following are available online. Figures S1–S24: NMR spectra of compounds 1–6.
Author Contributions: Y.J.S. and W.S.F. designed the research; M.L.G., H.J.C., R.J.H., H.C., K.D., Y.L.Z., M.L.,
and Y.Y.S. performed the research and analyzed the data; Y.J.S. wrote the paper. All authors read and approved
the final manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (No. 31300284), Basic Science
Foundation of Henan University of Chinese Medicine (No. 2014KYYWF-QN26), Science and Technology Innovation
Talent Support Scheme of Henan University of Chinese Medicine (No. 2016XCXRC01), Scientific and Technological Key
Project in Henan Province (No. 192102310438), Open Project of Henan Key Laboratory of Zhang Zhong-jing Formulae
and Herbs for Immunoregulation (No. kfkt201701).
Acknowledgments: The authors would like to thank Yanbin Guan for the technical assistance in MTT assay.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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