Bioactive gentixanthone and gentichromone from the whole plants of Gentianella acuta (Michx.) Hulten

Article abstract of DOI:10.1016/j.fitote.2016.08.001

Five new compounds, gentixanthones A₁ (1), A₂ (2), and gentichromones A₁–A₃ (3–5), together with thirteen known xanthones (6–18) were obtained from the whole plants of Gentianella acuta (Michx.) Hulten. Their structures were elucidated by chemical and spectroscopic methods. Among them, compounds 6, 8, 13, 14, and 17 were obtained from Gentianella genus firstly, and 7, 12, 15, 16, and 18 were isolated from this plant for the first time. Meanwhile, inhibitory effects of 1–18 on motility of mouse isolated intestine tissue were determined. As results, xanthones 1, 2, 6, 7, 9, 10 and 14 were found to have significant reduce effect on intestine contraction tension, and structure-activity relationship was discussed.

Full text of DOI:10.1016/j.fitote.2016.08.001

Fitoterapia 113 (2016) 164–169
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Bioactive gentixanthone and gentichromone from the whole plants of
Gentianella acuta (Michx.) Hulten
Yanxia Liu , Yajuan Ni , Jingya Ruan , Lu Qu , Haiyang Yu a,b, Lifeng Han a,b, Yi Zhang a,b,⁎, Tao Wang
a
b
a
a
a,b,
⁎
a
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193, China
Tianjin Key Laboratory of TCM Chemistry and Analysis, Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, 312 Anshan Road, Nankai District, Tianjin
00193, China
b
3
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 June 2016
Received in revised form 3 August 2016
Accepted 5 August 2016
Available online 08 August 2016
Five new compounds, gentixanthones A1 (1), A2 (2), and gentichromones A –A3 (3–5), together with thirteen
known xanthones (6–18) were obtained from the whole plants of Gentianella acuta (Michx.) Hulten. Their struc-
1
tures were elucidated by chemical and spectroscopic methods. Among them, compounds 6, 8, 13, 14, and 17 were
obtained from Gentianella genus firstly, and 7, 12, 15, 16, and 18 were isolated from this plant for the first time.
Meanwhile, inhibitory effects of 1–18 on motility of mouse isolated intestine tissue were determined. As results,
xanthones 1, 2, 6, 7, 9, 10 and 14 were found to have significant reduce effect on intestine contraction tension, and
structure-activity relationship was discussed.
Keywords:
Gentianella acuta (Michx.) Hulten
Xanthone
© 2016 Elsevier B.V. All rights reserved.
Chromone
Intestine motility
Structure-activity relationship
1
. Introduction
1,3,5-trihydroxyxanthone (6) [8], 1,3,5,8-tetrahydroxyxanthone
(
7) [9], 1-O-β-D-glucopyranosyl-3,5,8-trihydroxyxanthone (8) [10],
Gentianella acuta (Michx.) Hulten belongs to the family Gentianaceae,
norswertianolin (9) [11], swertianolin (10) [12], 1,8-dihydroxy-3,4-
dimethoxyxanthone 5-O-β-D-glucopyranoside (11) [13], 3,8-dihydroxy-
4,5-dimethoxyxanthone 1-O-β-D-glucopyranoside (12) [14], 1-O-
[β-D-glucopyranosyl(1 → 6)-β-D-glucopyranosyl]-3,8-dihydroxy-
4,5-dimethoxyxanthone (13) [15], triptexanthoside A (14) [16], 1-
hydroxy-3,4-dimethoxyxanthone 7-O-β-D-glucopyranoside (15) [13],
1-O-[β-D-xylopyranosyl(1 → 6)-β-D-glucopranosyl]-7-hydroxyl-3,8-
dimethoxyxanthone (16) [17], 3,7,8-trimethoxyxanthone 1-O-β-D-
glucopyranoside (17) [18], and mangiferin (18) [19] were obtained
(Chart 1). In this paper, we report the isolation and identification of
these compounds, along with their inhibitory effect evaluations on
motility of mouse isolated intestine tissue.
mainly distributes in the north of China, Mongolia plateau, Siberia, and Far
East areas of Russia [1]. G. acuta has been traditionally used as a Mongolia
native medicine, mainly to cure hepatitis, jaundice, fever, and headache.
According to pharmacological studies, it could exhibit bioactivity
in protecting cardiomyocytes of arrhythmia induced by aconitine in
mice [2]. Although xanthones were reported as major constituents of
Gentianella sp., the systemic phytochemistry and activity evaluation of
G. acuta were remained unclear. Xanthones analogues isolated from
other natural medicines were found to possess antiarrhythmic, antihy-
pertensive, antihypoglycemic, antitumor, antibacterial, and antioxidant
activities [3–6]. Methoxyphenylpiperazine xanthone derivatives showed
spasmolytic activity on the isolated rabbit small intestine tissue [7].
In the course of our investigation on bioactive constituents from the
whole plants of G. acuta, we found its 70% EtOH extract had inhibitory
effect on motility of mouse isolated intestine tissue. On the basis of liter-
ature report, we suggested xanthones were the effective ingredients for
inhibiting motility of mouse isolated intestine tissue in the plant. As
2. Results and discussion
The 70% EtOH extract from the whole plants of G. acuta was
partitioned with H
macroporous resin column chromatography (CC) and eluted with
2
H O, 95% EtOH, and acetone, successively. Then 95% EtOH eluted
2 3 2
O and CHCl , the H O layer was subjected to D101
results, five new compounds, gentixanthones A
1
(1), A
2
(2), and
gentichromones A -A (3–5), along with thirteen known xanthones,
1
3
fraction was isolated by Silica gel, Sephadex LH-20 and preparative
HPLC to obtain compounds 1–18.
Gentixanthone A
11 as its molecular formula deduced from [M−H] quasi-
molecular ion at m/z 421.0796 (calcd for C19 11, 421.0776) in the
negative-ion HRESI-TOF-MS spectrum. The UV spectrum displayed
1
(1) was isolated to be yellow powder with
⁎
Corresponding authors at: Tianjin State Key Laboratory of Modern Chinese Medicine,
−
19 18
C H O
Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District,
Tianjin 300193, China.
17
H O
E-mail addresses: zhwwxzh@263.net (Y. Zhang), wangtao@tjutcm.edu.cn (T. Wang).
http://dx.doi.org/10.1016/j.fitote.2016.08.001
0367-326X/© 2016 Elsevier B.V. All rights reserved.

Y. Liu et al. / Fitoterapia 113 (2016) 164–169
165
Chart 1. Structures of compounds 1–18.
three absorption bands at 252, 274, and 331 nm, respectively, which
indicated the presence of xanthone skeleton. The IR spectrum showed
absorption bands ascribable to hydroxyl (3362 cm ), unsaturated
(H-1′) to δ
the structure of gentixanthone A
tetrahydroxyxanthone 3-O-β-D-glucopyranoside (Fig. 1).
C
164.8 (C-3). On the basis of above mentioned evidence,
1
(1) was elucidated to be 1,3,5,8-
−
1
−
1
−1
carbonyl (1635 cm ), aromatic ring (1612, 1587, and 1499 cm ),
2
Gentixanthone A (2) was obtained as yellow powder with negative
−
1
25
and O-glycosidic linkage (1073 cm ). Acid hydrolysis of 1 with 1 M
HCl afforded D-glucose, whose absolute configuration was determined
D
optical rotation [[α] −55.7° (c 0.23, MeOH)]. It had the molecular
formula, C25
(m/z 583.1325 [M−H] , calcd for C25
NMR (DMSO-d , Table 2) and kinds of 2D NMR ( H H COSY, HSQC
and HMBC) spectra suggested 2 had the same aglycon, 1,3,5,8-
tetrahydroxyxanthone [δ 6.46 (1H, br. s, H-4), 6.54 (1H, d, J = 8.5 Hz,
H-7), 6.69 (1H, br. s, H-2), 7.15 (1H, d, J = 8.5 Hz, H-6);δ 180.2 (C-9)]
28
H O16 determined by the negative-ion HRESI-TOF-MS
by HPLC analysis [20]. The 13C NMR (125 MHz, DMSO-d
) of it displayed
9 signals, among them, two group signals for aromatic ring [δ 94.6
C-4), 99.0 (C-2), 102.8 (C-8b), 157.1 (C-4a), 161.9 (C-1), 164.8 (C-3);
−
H
O
16, 583.1305). The H,
1
13
C
6
27
1
1
1
C
6
(
1
1
07.5 (C-8a), 109.5 (C-7), 123.9 (C-6), 137.3 (C-5), 143.4 (C-4b),
51.7 (C-8)], and a carbonyl signal [δ 184.1 (C-9)] were characteristic
) of 1 suggested
H
C
C
1
signals for xanthone. The H NMR (500 MHz, DMSO-d
6
as 1. Moreover, two β-D-glucopyranosyl moieties [δ 4.25 (1H, d, J =
7.5 Hz, H-1″), 4.94 (1H, d, J = 7.5 Hz, H-1′)] were determined in 2. Linkage
positions between sugars and between sugar and aglycon were elucidat-
the presences of a 1,2,3,5-tetrasubstituted phenyl [δ 6.47 (1H, br. s,
H-2), 6.68 (1H, br. s, H-4)], a 1,2,3,4-tetrasubstituted phenyl [δ 6.66
(
1H, d, J = 8.0 Hz, H-7), 7.29 (1H, d, J = 8.0 Hz, H-6)], together
ed by the long-range correlations from δ
and δ 4.25 (H-1″) to δ 68.2 (C-6′) observed in the HMBC spectrum.
Treated it with 1 M HCl to give D-glucose [20]. Consequently, the structure
of gentixanthone A (2) was clarified to be 1,3,5,8-tetrahydroxyxanthone
1-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside.
Gentichromone A (3), white powder. Its molecular formula was
12 7
established as C13H O based on the [M−H] quasi-molecular ion at
H C
4.94 (H-1′) to δ 159.6 (C-1)
with a β-D-glucopyranosyl [δ 5.14 (1H, d, J = 7.0 Hz, H-1′)]. In the
HMBC spectrum, the long-range correlations were observed from δ
6
6
7
H
C
H
H
H
.47 (H-2) to δ
.68 (H-4) to δ
.29 (H-6) to δ
107.5 (C-8a), 137.3 (C-5), 151.7 (C-8), 184.1 (C-9); δ
C
94.6 (C-4), 102.8 (C-8b), 161.9 (C-1), 164.8 (C-3); δ
99.0 (C-2), 102.8 (C-8b), 157.1 (C-4a), 164.8 (C-3); δ
2
C
C
137.3 (C-5), 143.4 (C-4b), 151.7 (C-8); δ
H
6.66 (H-7)
5.14
1
−
to δ
C
H
Fig. 1. The main 1H 1H COSY and HMBC correlations of 1–5.

166
Y. Liu et al. / Fitoterapia 113 (2016) 164–169
m/z 279.0522 (calcd for C13
H
11
O
7
, 279.0510) in the HRESI-TOF-MS
which gained from those of Gentiana genus were usually with 1,2,6,8-
substitution pattern. Moreover, the xanthone dimmers were only
found in the plants of Gentianella genus. The above mentioned evi-
dences indicated that we can facilitate classification of Gentianaceae
using phytochemical data. Which was confirmed by the structures of
xanthones (mainly with 1,3,5,8-, and 1,3,4,5,8-substitution) reported
in this paper and the literature reported by Urbain et al. [27].
spectrum. UV (λmax 229, 248, 254 sh and 292 nm) and IR spectra
−
1
−1
−1
(
1
3362 cm , OH; 1698 cm , COOH; 1663 cm , unsaturated CO;
622, 1591, 1455 cm−1, aromatic ring) were consistent with the pres-
1
ence of a chromone ring. The H NMR (500 MHz, DMSO-d
indicated the presence of 1,2,3,5-tetrasubstituted phenyl [δ 6.23 (1H,
br. s, H-6), 6.35 (1H, br. s, H-8)]; Meanwhile, a group carbon signals
6
) spectrum
[
δ
C
93.5 (C-8), 98.7 (C-6), 102.9 (C-10), 157.3 (C-9), 161.3 (C-5), 164.2
Our research suggested that xanthones were the active constituents
on intestinal motility, which indicated they may have potential as lead-
ing compounds for diarrhea in gastroenteritis new drug development.
Our preliminary results are encouraging, but further studies are needed
to clarify the activities in vivo and mechanism behind the inhibitory
effects on intestinal motility of these compounds.
13
(
C-7), 180.5 (C-4)] displayed in C NMR (125 MHz, DMSO-d
6
) furtherly
2.86 (2H, t,
3.78 (2H, t, J = 6.5 Hz) in the H H COSY experiment
suggested the presence of “\\CH \\CH \\O” moiety. Moreover, there
were a methene [δ 3.49 (2H, s, H -1″); δ 29.2 (C-1″)] and an ester
carbonyl [δ 171.9 (C-2″)] in 3. Finally, according to the long-range
correlations observed from δ 2.86 (H -1′) to δ 58.5 (C-2′), 114.5
C-3), 165.7 (C-2); δ 3.49 (H -1″) to δ 114.5 (C-3), 165.7 (C-2),
71.9 (C-2″), 180.5 (C-4); δ 6.23 (H-6) to δ 93.5 (C-8), 102.9 (C-10),
61.3 (C-5), 164.2 (C-7), 180.5 (C-4); δ 6.35 (H-8) to δ 98.7 (C-6),
02.9 (C-10), 157.3 (C-9), 164.2 (C-7), 180.5 (C-4) in the HMBC
(3) was identified as
-hydroxyethyl-3-carboxymethyl-5,7-dihydroxychromone.
Both of gentichromones A (4) and A (5) were also obtained as
white powder. The molecular formula, C14
proved it was a chromone. The correlation found between δ
H
1
1
J = 6.5 Hz) and δ
H
2
2
H
2
C
C
H
2
C
3. Experimental
(
H
2
C
1
1
1
H
C
3.1. General
H
C
UV and IR spectra were recorded on a Varian Cary 50 UV–Vis and
Varian 640-IR FT-IR spectrophotometer, respectively. Optical rotations
were determined on a Rudolph Autopol® IV automatic polarimeter.
NMR spectra were run on a Bruker 500 MHz NMR spectrometer at
spectrum, the structure of gentichromone A
1
2
2
3
1
13
H
14
O
7
of 4 and C19
H
22
O
12 of
500 MHz for H and 125 MHz for C NMR (internal standard: TMS).
Negative-ion HRESI-TOF-MS were made on an Agilent Technologies
6520 Accurate-Mass Q-Tof LC/MS spectrometer. Column chromatogra-
phies (CC) were performed on macroporous resin D101 (Haiguang
Chemical Co., Ltd., Tianjin, China), Silica gel (74–149 μm, Qingdao
Haiyang Chemical Co., Ltd., Qingdao, China), and Sephadex LH-20
(Ge Healthcare Bio-Sciences, Uppsala, Sweden). Preparative high
1
5
were determined based on the negative-ion HRESI-TOF-MS. The H
13
and C NMR (DMSO-d
had the same aglycon, 2-hydroxyethyl-3-carboxymethyl-5,7-
dihydroxychromone as 3. The major difference between 4 and 3 was
the presence of methoxy [δ 3.62 (3H, s, 2″-OCH )] in 4. On the other
hand, the difference between 5 and 3 was lie in the β-D-glucopyranosyl
δ 5.06 (1H, d, J = 7.5 Hz, H-1′)] presented in 5. Meanwhile, in the HMBC
spectrum, the long-rang correlations were observed from δ 3.62 (2″-
OCH ) to δ 170.7 (C-2″) in gentichromone A (4), and δ 5.06 (H-1′)
to δ 162.9 (C-7) in gentichromone A (5). Furthermore, treated 5
with 1 M HCl, D-glucose was identified [20]. Consequently, the
structures of gentichromones A (4) and A (5) were clarified.
6
, Table 4) spectra suggested that both of 4 and
5
3
[
performance liquid chromatography (PHPLC) column, Cosmosil 5C18-
MS-II (20 mm i.d. × 250 mm, Nakalai Tesque, Inc., Tokyo, Japan) were
used to isolate the compounds.
H
3
C
2
H
C
3
3.2. Plant material
2
3
As a continuing study of natural constituents on gastrointestinal
disorder [21,22], we screened the inhibitory of activities of compounds
isolated from G. acuta on intestine motility. Loperamide hydrochloride
is a piperidine derivative used to decrease the tension and frequency
of diarrhea in gastroenteritis, inflammatory bowel disease, and short
bowel syndrome [23]. In test on motility of mouse isolated intestine
tissue, loperamide hydrochloride showed significantly inhibitory effects
on intestinal tension and frequency at concentration of 10 μM.
The whole plants of Gentianella acuta (Michx.) Hulten were collected
from Alxa Youqi, Inner Mongolia Autonomous region, China, and identi-
fied by Dr. Li Tianxiang (Experiment Teaching Department, Tianjin
University of Traditional Chinese Medicine). The voucher specimen
was deposited at the Academy of Traditional Chinese Medicine of
Tianjin University of TCM.
3.3. Extraction and isolation
All of test samples 1–18 showed no significant changing on isolated
intestinal tissue contraction frequency, but compounds 1, 2, 6, 7, 9, 10
and 14 showed significant reduce effect on contraction tension. Struc-
The whole plants of G. acuta (3.0 kg) were cut and refluxed with 70%
ethanol-water. Then the 70% EtOH extract (868.5 g) was partitioned in a
ture-activity relationship analysis revealed that R
is essential factor (strong activity of 1, 2, 6, 7, 9, 10), but the activity can
be quenched by R glucose moiety substitution (no activity of 8, 13, 16,
7). R methoxy substitution weaken the reduce activity on contraction
4
hydroxy substitution
CHCl
subjected to D101 macroporous resin CC (H
As a result, H O (332.4 g) 95% EtOH (294.9 g), and acetone (5.1 g)
3
-H
2
O mixture (1:1, v/v). Furthermore, the H
2
O layer (670.0 g) was
2
O → 95% EtOH → acetone).
1
2
1
3
eluates were obtained.
tension (no activity of 11, 12, 13, 15) (Chart 1, Table 5).
2
The 95% EtOH eluate (200.0 g) was isolated by SiO gel CC
In conclusion, we obtained three new xanthones, two new
chromones, together with thirteen known xanthones from the whole
plants of G. acuta collected from Alxa Youqi, Inner Mongolia Autono-
mous region, China. As one of the main constituents in the plants
belonging to Gentianaceae family, xanthones are widely distributed
among plants from two genera, Gentiana and Gentianella, which were
segregated into two subtribes, subtribes Gentianinae and Swertiinae
by Struwe et al. based on phylogenic and phenotypic considerations,
respectively [24]. Until 2014, about 400 species were found to belong
to Gentiana genus, and 57 kinds of xanthones were isolated from them
3 3 3 2
[CHCl → CHCl –MeOH (100:1 → 100:5, v/v) → CHCl –MeOH–H O
(10:3:1 → 7:3:1 → 6:4:1, v/v/v, lower layer)], and sixteen fractions (Fr.
1–16) were given. Fraction 7 (25.7 g) was separated by PHPLC
3 2
[CH CN–H O (18:82 → 35:65 → 42:58, v/v) + 1% HAc] to afford twenty
seven fractions (Fr. 7-2-1–7-2-27). Fraction 7-2-22 (54.4 mg) was
further purified by Sephadex LH-20 CC (MeOH), and 1,3,5-
trihydroxyxanthone (6, 25.6 mg) was yielded. Fraction 9 (15.0 g) was
subjected to Sephadex LH-20 CC [CHCl
seven fractions (Fr. 9-1–9-7). Fraction 9-4 (7.3 g) was isolated by
PHPLC [CH CN–H O (22:78 → 30:70 → 45:55, v/v) + 1% HAc], and
fourteen fractions (Fr. 9-4-1–9-4-14) were yielded. Fraction 9-4-8
(381.4 mg) was separated by [CH CN–H O (21:79, v/v) + 1% HAc] to af-
3
–MeOH (1:1, v/v)] to give
3
2
[25]; And 24 xanthones were obtained from 125 species belonging to
Gentianella genus [26]. Comparing the structure diversity of the
reported xanthones obtained from the above mentioned genera, we
found that the xanthones in plants of Gentianella genus were with
3
2
ford 1,8-dihydroxy-3,4-dimethoxyxanthone 5-O-β-D-glucopyranoside
(11, 27.1 mg), 3,8-dihydroxy-4,5-dimethoxyxanthone 1-O-β-D-
glucopyranoside (12, 39.6 mg), along with 1-hydroxy-3,4-
1,3,5,8-, 1,3,4,5,8-, and 1,3,4,7,8-substitution patterns mainly; meanwhile,

Y. Liu et al. / Fitoterapia 113 (2016) 164–169
167
dimethoxyxanthone 7-O-β-D-glucopyranoside (15, 14.2 mg). Fraction
-4-9 (277.1 mg) was further purified by PHPLC [MeOH–H
45:55, v/v) + 1% HAc] to give 3,7,8-trimethoxyxanthone 1-O-β-D-
glucopyranoside (17, 11.4 mg). Fraction 9-6 (3.0 g) was subjected to
PHPLC [MeOH–H O (40:60 → 60:40 → 80:20, v/v) + 1% HAc], as result,
gentichromones A (3, 241.0 mg), A (4, 21.0 mg), together with
,3,5,8-tetrahydroxyxanthone (7, 76.9 mg) were obtained. Fraction 11
20.0 g) was isolated by PHPLC [CH CN–H O (15:85 → 25:75 → 42:58,
Table 1
1
H and 13C NMR data for 1 in DMSO-d
6
.
9
(
2
O
No.
δC
δH (J in Hz)
No.
δC
δH (J in Hz)
1
2
3
4
161.9
99.0
–
8b
9
102.8
184.1
99.6
73.0
76.2
69.4
77.1
60.5
–
–
2
6.47 (br. s)
1
2
164.8
94.6
–
1′
2′
3′
4′
5′
6′
5.14 (d, 7.0)
3.28 (dd, 7.0, 8.0)
3.30 (dd, 7.0, 8.0)
3.19 (dd, 7.0, 8.0)
3.48 (m, overlapped)
3.48 (m, overlapped)
3.71 (br. d, ca. 10)
11.09 (br. s)
6.68 (br. s)
1
(
4
4
5
6
7
8
8
a
b
157.1
143.4
137.3
123.9
109.5
151.7
107.5
–
–
–
3
2
v/v) + 1% HAc], and twenty nine fractions (Fr. 11-1–11-29) were
yielded. Fraction 11-17 (790.7 mg) was purified by PHPLC [MeOH–
7.29 (d, 8.0)
6.66 (d, 8.0)
–
–
2
H O (42:58, v/v) + 1% HAc] to give triptexanthoside A (14, 14.6 mg).
1-OH
–
Fraction 13 (20.0 g) was subjected to PHPLC [MeOH–H
2
O
a
(
35:65 → 45:55 → 55:45, v/v) + 1% HAc] to afford twenty fractions
Fr. 13-1–13-20). Fraction 13-3 (1.0 g) isolated by PHPLC [CH CN–
O (9:91, v/v) + 1% HAc], and nine fractions (Fr. 13-3-1–Fr. 13-3-9)
were given. Fraction 13-3-5 (52.4 mg) was purified by Sephadex LH-
0 CC [MeOH–H O (1:1, v/v)] to yield gentichromone A (5, 13.8 mg).
Fraction 13-7 (480.7 mg) was subjected to PHPLC [CH CN–H O (9:91,
(
3
H
2
Table 2
1
H and 13C NMR data for 2 in DMSO-d
6
.
2
2
3
3
2
No.
δ
C
δ
H
(J in Hz)
No.
δ
H
δ (J in Hz)
C
v/v) + 1% HAc], and eight fractions (Fr. 13-7-1–Fr. 13-7-8) were
yielded. Fraction 13-7-4 (22.2 mg) was separated by Sephadex LH-20
1
2
3
4
159.6
101.5
167.1
97.0
–
2′
3′
4′
5′
6′
73.3
76.0
69.4
75.9
68.2
3.42 (dd, 7.5, 8.5)
3.33 (dd, 8.5, 8.5)
3.28 (dd, 8.5, 8.5)
3.62 (m)
3.66 (m, overlapped)
4.03 (br. d, ca. 11)
4.25 (d, 7.5)
3.03 (dd, 7.5, 8.5)
3.17 (dd, 8.5, 8.5)
3.08 (m, overlapped)
3.08 (m, overlapped)
3.43 (dd, 5.0, 11.5)
3.67 (br. d, ca. 12)
12.54 (br. s)
6.69 (br. s)
–
6.46 (br. s)
–
–
–
7.15 (d, 8.5)
6.54 (d, 8.5)
–
–
–
–
CC [MeOH–H
3–10 (1.1 g) was isolated by PHPLC [CH
HAc] to yield 1-O-β-D-glucopyranosyl-3,5,8-trihydroxyxanthone
8, 15.6 mg). Fraction 13-12 (501.4 mg) was separated by PHPLC
CH CN–H O (18:82, v/v) + 1% HAc], and nine fractions (Fr. 13-12-
–Fr. 13-12-9) were afforded. Fraction 13-12-7 (49.3 mg) was fur-
O (40:60, v/v) + 1% HAc] to give
-O-[β-D-glucopyranosyl(1 → 6)-β-D-glucopyranosyl]-3,8-dihy-
droxy-4,5-dimethoxyxanthone (13, 6.7 mg). Fraction 13-14 (1.6 g)
was isolated by PHPLC [CH CN–H O (18:82, v/v) + 1% HAc], and 1-
2
O (1:1, v/v)] to gain mangiferin (18, 5.2 mg). Fraction
1
3
CN–H O (14:86, v/v) + 1%
2
4
4
5
6
7
8
8
8
9
a
b
158.6
142.7
136.5
122.3
108.7
152.6
108.6
103.3
180.2
101.6
(
1″
2″
3″
4″
5″
6″
103.4
73.5
76.5
70.0
76.8
60.9
[
3
2
1
ther purified by PHPLC [MeOH–H
2
a
b
1
3
2
1′
4.94 (d, 7.5)
8-OH
–
O-[β-D-xylopyranosyl(1 → 6)-β-D-glucopranosyl]-7-hydroxyl-3,8-
dimethoxyxanthone (16, 32.0 mg) was yielded. Fraction 13-17
(
504.0 mg) was subjected to PHPLC [CH
HAc] to afford nine fractions (Fr. 13-17-1–Fr. 13-17-9). Fraction 13-
2
7-5 (35.4 mg) was purified by Sephadex LH-20 CC [MeOH–H O
3 2
CN–H O (22:78, v/v) + 1%
ν
max 3362, 2925, 2852, 1698, 1663, 1622, 1591, 1455, 1398, 1355,
−
1
1
13
1
296, 1038 cm ; H and C NMR data, see Table 3; HRESI-TOF-MS
1
(
−
11 7
Negative-ion mode m/z 279.0522 [M−H] (calcd for C13H O ,
1:1, v/v)] to give gentixathone A
1
(1, 7.1 mg). Fraction 14 (15.3 g)
279.0510).
was subjected to PHPLC [CH
3
2
CN–H O (15:85 → 25:75, v/v) + 1%
HAc], and twenty fractions (Fr. 14-1–Fr. 14-20) were given. Fraction
4-17 (72.6 mg) was further purified by PHPLC [MeOH–H O (50:50,
v/v) + 1% HAc] to afford swertianolin (10, 27.9 mg). Fraction 15
14.4 g) was subjected to Sephadex LH-20 CC [MeOH–H O (1:1, v/v)]
to yield nine fractions (Fr. 15-1–Fr. 15-9). Fraction 15-4 (2.4 g) was iso-
lated by PHPLC [MeOH–H O (40:60, v/v) + 1% HAc], and seventeen
fractions (Fr. 15-4-1–Fr. 15-4-17) were afforded. Fraction 15-4-6
215.7 mg) was separated by PHPLC [CH CN–H O (13:87, v/v) + 1%
HAc] to give gentixathone A (2, 19.4 mg). Fraction 15-4-7 (284.5 mg)
was purified by PHPLC [CH CN–H O (15:85, v/v) + 1% HAc] to obtain
norswertianolin (9, 139.8 mg).
1
2
3
.3.4. Gentichromone A
White powder; [α]
2
(4)
2
5
D
−5.7° (c 0.14, MeOH); UV λmax (MeOH) nm
(
2
(
log ε): 228 (4.26), 248 (4.33), 254 (4.32, sh), 292 (3.89); IR (KBr)
ν
1
max 3358, 2922, 2851, 1724, 1655, 1622, 1591, 1506, 1453, 1361,
2
−
1
1
13
292, 1168, 1045 cm
;
H and C NMR data, see Table 4; HRESI-
−
TOF-MS Negative-ion mode m/z 293.0678 [M−H] (calcd for
C H O , 293.0667).
14 13 7
(
3
2
2
3
2
3
3.3.5. Gentichromone A (5)
2
5
White powder; [α]
(log ε): 240 (4.38), 285 (3.83), 313 (3.72); IR (KBr) νmax 3351, 2920,
850, 1700, 1655, 1618, 1590, 1497, 1456, 1397, 1302, 1176, 1073,
D
−58.6° (c 0.14, MeOH); UV λmax (MeOH) nm
3
.3.1. Gentixathone A
Yellow powder; [α]
log ε): 252 (4.41), 274 (4.30), 331 (4.07); IR (KBr) νmax 3362, 2922,
1
(1)
2
5
D
−89.5° (c 0.11, MeOH); UV λmax (MeOH) nm
2
(
2
−
1
1
13
−
1
1
1030 cm ; H and C NMR data, see Table 4; HRESI-TOF-MS Nega-
tive-ion mode m/z 441.1056 [M−H] (calcd for C19H O12, 441.1038).
21
852, 1635, 1612, 1587, 1499, 1289, 1243, 1209, 1173, 1073 cm ; H
−
13
and C NMR data, see Table 1; HRESI-TOF-MS Negative-ion mode m/z
−
4
17
21.0796 [M−H] (calcd for C19H O11, 421.0776).
3
.3.2. Gentixathone A
Yellow powder; [α]
log ε): 252 (4.41), 274 (4.30), 331 (4.07); IR (KBr) νmax 3356, 2922,
2
(2)
Table 3
2
5
1
H and 13CNMR data for 3 in DMSO-d
D
−55.7° (c 0.23, MeOH); UV λmax (MeOH) nm
6
.
(
2
No.
δ
C
δ
H
(J in Hz)
No.
δ
C
H
δ (J in Hz)
−
1 1
852, 1648, 1610, 1585, 1490, 1457, 1294, 1243, 1209, 1073 cm ; H
13
2
3
4
5
6
7
8
165.7
114.5
180.5
161.3
98.7
–
–
–
–
9
10
1′
2′
1″
157.3
102.9
35.4
58.5
29.2
171.9
–
–
–
and C NMR data, see Table 2; HRESI-TOF-MS Negative-ion mode m/z
5
−
27
83.1325 [M−H] (calcd for C25H O16, 583.1305).
2.86 (t, 6.5)
3.78 (t, 6.5)
3.49 (s)
3
.3.3. Gentichromone A
White powder; [α]
1
(3)
6.23 (br. s)
–
6.35 (br. s)
2
5
164.2
93.5
2″
5-OH
–
D
−3.2° (c 0.13, MeOH); UV λmax (MeOH) nm
12.75 (br. s)
(
log ε): 229 (4.19), 248 (4.27), 254 (4.25, sh), 292 (3.83); IR (KBr)

168
Y. Liu et al. / Fitoterapia 113 (2016) 164–169
Table 4
3.5. Inhibitory effects of 1–18 on motility of mouse isolated intestine tissue
1
H and 13CNMR data for 4 and 5 in DMSO-d
6
.
No.
4
5
Mice were fasted for 12 h before experiments, intestinal tissue were
collected immediately. The Maxwell bath was filled with 10 mL of
δ
C
δ
H
(J in Hz)
δ
C
H
δ (J in Hz)
Tyrode's solution (1 L contains: NaCl 8.0 g, CaCl
MgCl 0.1 g, NaHCO 1.0 g, KH PO 0.05 g, glucose 1.0 g, pH .4) and
maintained at a constant temperature (37.0 ± 0.5 °C), and bubbled
with 95% O and 5% CO gas. The intestinal tissue was fixed on bottom
2
0.2 g, KCl 0.2 g,
2
3
4
5
6
7
8
9
166.0
114.0
180.3
161.2
98.7
164.2
93.5
157.3
102.7
35.3
58.4
28.9
170.7
–
51.7
–
–
–
–
166.4
115.1
180.8
160.8
99.3
162.9
94.2
156.9
104.4
35.4
–
–
–
–
2
3
2
4
2
2
6.20 (d, 1.5)
–
6.34 (d, 1.5)
–
–
2.84 (t, 6.5)
3.73 (t, 6.5)
3.55 (s)
–
12.65 (br. s)
3.62 (s)
6.44 (d, 2.0)
–
6.67 (d, 2.0)
–
hook in and the other end was connected to an isometric tension trans-
ducer. Samples in DMSO solution were added after 15 min equilibrate
incubation, the final DMSO concentration was 0.1% and final concentra-
tion of samples were 40 μM. The mean tension and frequency of intes-
tine muscle contractions were recorded for 1 min before and 4 min
after drug additions using isolated tissue bath systems (Radnoti Glass
Technology Inc., Monrovia, CA, 159901A). Loperamide hydrochloride
1
1
2
1
2
5
2
1
2
3
4
5
6
0
′
′
″
–
2.87 (t, 6.5)
3.75 (t, 6.5)
3.48 (s)
–
58.4
29.2
171.6
–
″
(
Xi'an Janssen Pharmaceutical Ltd., Xi'an, China) was used as positive
-OH
″-OCH
12.74 (br. s)
control, and the final concentration was 10 μM.
3
‴
‴
‴
‴
‴
‴
99.9
73.0
76.3
69.5
77.1
60.6
5.06 (d, 7.5)
3.25 (dd, 7.5, 9.0)
3.30 (dd, 9.0, 9.0)
3.17 (dd, 9.0, 9.0)
3.43 (m)
Conflict of interest
We declare that we have no conflict of interest.
3.45 (m, overlapped)
3
.70 (br. d, ca. 11)
Acknowledgments
This research was supported by Program for National Natural
Science Foundation of China (81202995), New Century Excellent
Talents in University (NCET-12-1069), and Program for Tianjin Innova-
tive Research Team in University (TD12-5033).
3
.4. Acid hydrolysis for 1, 2 and 5
Compounds 1, 2 and 5 (each 2.0 mg) were treated by using the
similar method as reference [20]: were refluxed on a water bath for
h in 1 M HCl, respectively. The reaction mixture was neutralized and
removed by filtration. The aqueous layer was subjected to HPLC
analysis: HPLC column, Kaseisorb LC NH -60-5, 4.6 mm i.d. × 250 mm
Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation [Shodex
OR-2 (Showa Denko Co., Ltd., Tokyo, Japan); mobile phase, CH CN–H
(75:25, v/v; flow rate 1.0 mL/min)]. and D-glucose (16.8 min, positive
3
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74.1 ± 9.3*
85.2 ± 4.1*
82.6 ± 5.8*
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