New triterpenoid saponins from the herb hylomecon japonica

Article abstract of DOI:10.3390/molecules22101731

Background: Hylomecon japonica, a plant of the Papaveraceae family which is well-known for the alkaloids they produce, is a perennial plant widely distributed in the northeast, central and east regions of China. Although a variety of chemical constituents, including alkaloids, flavonoids, and megastigmoids, have been isolated from H. japonica, the investigation of saponins in H. japonica has not been reported until now. Methods: Various separation techniques, including polyporous resin column chromatography, silica gel column chromatography and hemi-preparative HPLC were applied to the isolation of triterpenoid saponins, and chemical methods such as acid hydrolysis and spectroscopic methods including HRESIMS and NMR were applied to their structure elucidation, and the XTT reduction method was used to assay cytotoxicity. Results: Two new triterpenoid saponins, named hylomeconoside A (1) and B (2) which were identified as 3-O-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl-gypsogenin-28-O-β-D-xylopyranosyl- (1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-quinovopyranoside (1) and 3-O-β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl-gypsogenin-28-O-β-D-xylopyranosyl- (1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (2), and two known triterpenoid saponins identified as dubioside C (3) and lucyoside P (4) on the basis of spectroscopic and chemical evidence, were isolated from H. japonica. Compound 1 exhibited moderate cytotoxicity on MGC-803 and HL-60 cells, with IC50 values of 43.8 and 32.4 μg.mL^-1, respectively. Conclusions: Compounds 1 and 2 are new saponins, and 1 is considered to be one of the antitumor principles in this plant. This is the first time that triterpenoid saponins have been isolated from plants of the Papaveraceae family.

Full text of DOI:10.3390/molecules22101731

molecules
Article
New Triterpenoid Saponins from the Herb
Hylomecon japonica
Yan-Fei Qu 1,2, Jing-Yu Gao 1,3, Jing Wang 1, Yan-Mei Geng 1, Yu Zhou 1, Cheng-Xin Sun 1,
Fei Li 1, Lei Feng 1, Meng-Juan Yu 1 and Guang-Shu Wang 1,
*
ID
1
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, Jilin, China;
cream0023@163.com (Y.-F.Q.); gaojingyu1989@126.com (J.-Y.G.); 13578752256@163.com (J.W.);
gym130123@163.com (Y.-M.G.); 17623233985@163.com (Y.Z.); 13124303229@163.com (C.-X.S.);
18844194997@163.com (F.L.); xiaoweiaw@163.com (L.F.); 13194312537@163.com (M.-J.Y.)
Logistics University of PAP, Tianjin 300162, China
2
3
Department of Pharmacy, Central Hospital of Yingkou Economic and Technological Development Zone,
Yingkou 115007, Liaoning, China
*
Correspondence: wgs@jlu.edu.cn; Tel.: +86-431-8561-9706
Received: 17 August 2017; Accepted: 12 October 2017; Published: 23 October 2017
Abstract: Background: Hylomecon japonica, a plant of the Papaveraceae family which is well-known
for the alkaloids they produce, is a perennial plant widely distributed in the northeast, central
and east regions of China. Although a variety of chemical constituents, including alkaloids,
flavonoids, and megastigmoids, have been isolated from H. japonica, the investigation of saponins
in H. japonica has not been reported until now. Methods: Various separation techniques, including
polyporous resin column chromatography, silica gel column chromatography and hemi-preparative
HPLC were applied to the isolation of triterpenoid saponins, and chemical methods such as
acid hydrolysis and spectroscopic methods including HRESIMS and NMR were applied to their
structure elucidation, and the XTT reduction method was used to assay cytotoxicity. Results:
Two new triterpenoid saponins, named hylomeconoside A (
3-O- -D-galactopyranosyl-(1 2)- -D-glucuronopyranosyl-gypsogenin-28-O-
(1 3)- -D-xylopyranosyl-(1 4)-
3-O- -D-galactopyranosyl-(1 2)-
(1 3)- -D-xylopyranosyl-(1 4)-
1
) and B (
2
) which were identified as
-D-xylopyranosyl-
-D-quinovopyranoside ( ) and
-D-xylopyranosyl-
-L-arabinopyranoside ( ), and
) on the basis
of spectroscopic and chemical evidence, were isolated from H. japonica. Compound exhibited
moderate cytotoxicity on MGC-803 and HL-60 cells, with IC50 values of 43.8 and 32.4
mL−1
respectively. Conclusions: Compounds and are new saponins, and is considered to be one of the
β
→
β
β
→
β
→
α
-L-rhamnopyranosyl-(1
→
2)-
β
1
β
→
β
-D-glucuronopyranosyl-gypsogenin-28-O-
β
→
β
→
α
-L-rhamnopyranosyl-(1
→
2)-
α
2
two known triterpenoid saponins identified as dubioside C (3) and lucyoside P (4
1
µ
g
·
,
1
2
1
antitumor principles in this plant. This is the first time that triterpenoid saponins have been isolated
from plants of the Papaveraceae family.
Keywords: Hylomecon japonica; Papaveraceae; triterpenoid saponin; gypsogenin; quillaic acid
1. Introduction
Hylomecon japonica (Thunb.) Prantl et Kundig (Papaveraceae) is a perennial plant widely distributed
in the northeast, central and east regions of China. Its roots have been used as a traditional Chinese
medicine for the treatment of rheumatism and injuries [1]. Although a variety of chemical constituents,
including alkaloids, flavonoids, and megastigmoids, were previously isolated from H. japonica, and
pharmacological studies on its anti-inflammatory properties, antibacterial activities, and antitumor
action have been reported [
2–7], the investigation of saponins in H. japonica has not been reported
until now. As part of our study on the plants of Papaveraceae family, we have been carrying out an
Molecules 2017, 22, 1731; doi:10.3390/molecules22101731
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1731
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investigation of saponins in H. japonica. In this paper, we now report the isolation and characterization
of two new triterpenoid saponins, named hylomeconoside A ( ) and B ( ), which were identified as
3-O- -D-galactopyranosyl-(1 2)- -D-glucuronopyranosyl-gypsogenin-28-O- -D-xylo-pyranosyl-
(1 3)- -D-xylopyranosyl-(1 4)- -L-rhamnopyranosyl-(1 2)- -D-quinovopyranoside and
3-O- -D-galactopyranosyl-(1 2)- -D-glucuronopyranosyl-gypsogenin-28-O- -D-xylopyranosyl-
(1 3)- -D-xylopyranosyl-(1 4)- -L-rhamnopyranosyl-(1 2)- -L-arabinopyranoside ( ), and two
], and lucyoside P (
1
2
β
→
β
β
→
β
→
α
→
β
(1)
β
→
β
β
→
β
→
α
→
α
2
known triterpenoid saponins identified as dubioside C (
3) [
8
4) [
9] (Figure 1) on the
basis of spectroscopic and chemical evidence.
COOR2
R3
O
HOOC
O
HO
H3
C
O
HO
R1O
HO
O
O
HO
CHO
HO
O
CHO
O
HO
O
OH
HO
1
2
3
4
R1 = S1 R2 = S2 R3 = H
R1 = S1 R2 = S3 R3 = H
R1 = S1 R2 = S3 R3 = OH
R1 = S1 R2 = S4 R3 = H
S1 = -GlcA(2→1)Gal
O
O
O
HO
H3C
S2 = -Qui(2→1)Rha(4→1)Xyl(3→1)Xyl
S3 = -Ara(2→1)Rha(4→1)Xyl(3→1)Xyl
S4 = -Ara(2→1)Rha[(4→1)Xyl](3→1)Glc
O
O
HO
OH
OH
OH
HO
OH
(a)
(b)
) Key HMBC correlations of compound 1
Figure 1.
(a
) Chemical structures of compounds
1–
4; (
b
(arrows point from proton to carbon).
2. Results and Discussion
The crude saponins prepared from the 70% EtOH extract of H. japonica herbs through a D101
polyporous resin column were subjected to silica gel chromatographies and semi-preparative HPLC
to yield two new triterpenoid saponins, named as hylomeconoside A (
1
) and B ( ), and two known
2
triterpenoid saponins identified as dubioside C (
spectroscopic and chemical evidence.
3) [
8
] and lucyoside P ( ) [9] on the basis of
4
Hylomeconoside A (
was determined as C64H100O31 by high-resolution HRESI-MS which indicated a [M + H]+ ion at m/z
1365.6318. Acid hydrolysis of afforded an aglycone which was identified as gypsogenin on the basis
1) was obtained as a white amorphous solid. The molecular formula of 1
1
of the 1H- and 13C-NMR data [10]. The sugars obtained from the saponin hydrolysate were identified
as D-xylose, L-rhamnose, D-quinovose, D-glucuronic acid and D-galactose based on GC analysis of
their chiral derivatives. The 1H-NMR spectrum revealed signals due to six quaternary methyls at δH
0.68, 0.76, 0.79, 0.90, 1.14, 1.28, an olefinic proton at δH 5.30 (br. s), an aldehyde proton at δH 9.83 (s),
and six anomeric protons at δH 6.34 (br s), 5.98 (d, J = 8.0 Hz), 5.11 (d, J = 7.5 Hz) , 5.08 (d, J = 7.4 Hz),
4.94 (d, J = 6.8 Hz), and 4.72 (d, J = 6.3 Hz). The 13C-NMR spectrum displayed signals due to six
quaternary carbon at δC 30.9, 36.4, 40.4, 42.4, 47.3, and 55.2, an oxygen-bearing methine carbon at δC
83.5, a set of olefinic carbons at δC 122.7 and 144.2, an ester carbonyl carbon at δC 176.7, an aldehyde
carbon at δC 210.2, and six anomeric carbons at δC 94.5, 101.4, 103.4, 105.9, 106.2, and 106.9. All the
above spectral information suggested that the aglycone moiety of
1 was gypsogenin and the chemical
shift of the carbonyl C-28 (δC 176.7) and the deshielded carbon C-3 (δC 83.5) of gypsogenin indicated
that 1 was the 3,28-bisdesmoside of gypsogenin, having six monosaccharide units.
The six anomeric proton signals at δH 6.34 (br s), 5.98 (d, J = 8.0 Hz), 5.11 (d, J = 7.5 Hz), 5.08
(d, J = 7.4 Hz), 4.94 (d, J = 6.8 Hz), and 4.72 (d, J = 6.3 Hz) were correlated with anomeric carbon
1
signals at δC 101.4, 94.5, 106.2,105.9, 106.9, 103.4, respectively, and the analysis of H-1H COSY,
HMQC, HMBC, DEPT NMR and spin-spin couplings in 1H-NMR of
1 allowed the identification of

Molecules 2017, 22, 1731
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one
(GlcA), one
-anomeric configurations for D-xylopyranosyl, D-quinovopyranosyl, D-glucuronopyranosyl and
α
-L-rhamnopyranosyl (Rha), one
β-D-quinovopyranosyl (Qui), one
β-D-glucuronopyranosyl
β-D-galactopyranosyl (Gal) and two
β-D-xylopyranosyl (Xyl) moieties.
The
β
3
D-galactopyranosyl moieties were determined by their large JH1-H2 coupling constants of 6–8 Hz
and the
-anomeric configuration for L-rhamnopyranosyl unit was determined by its small 3JH1-H2
coupling constant.
One Xyl unit, Xyla, identified starting from anomeric signals at δH 5.08 (H-1 of Xyla) and δC 105.9
α
(C-1 of Xyla), was identified to be in the terminal position, as observed by its 13C-NMR chemical
shifts. Another Xyl unit, Xylb, identified starting from anomeric signals at δH 4.94 (H-1 of Xylb)
and δC 106.9 (C-1 of Xylb), was substituted at the position of C-3 of Xylb based on the deshielding
of C-3 of Xylb (δC 87.2), and the Xyla unit was attached to this position based on the long-range
correlation observed in the HMBC experiment between signals at δH 5.08 (H-1 of Xyla) and δC 87.2
(C-3 of Xylb). The methyl doublet at δH 1.66 (3H, d, J = 6.4 Hz) and the typical broad single of anomeric
proton at δH 6.34 (br s) were characteristic of Rha unit, and the deshielding of C-4 of Rha (δC 85.3)
indicated a substitution at the position of C-4 of Rha, and the long-range correlation between signals
at δH 4.94 (H-1 of Xylb) and δC 85.3 (C-4 of Rha) indicated that Xylb was attached to the position
of C-4 of Rha. The Qui unit was identified starting from the deshielded anomeric proton at δH 5.98
(J = 8.0 Hz) and characterized by its methyl doublet at δH 1.42 (3H, d, J = 6.0 Hz). The deshielding
of anomeric proton and the chemical shift of anomeric carbon at δC 94.5 suggested that Qui was
attached by an ester linkage to the C-28 carboxylic group of the aglycone, which was further confirmed
by the long-range correlation observed in the HMBC experiment between signals at δH 5.98 (H-1
of Qui) and δC 176.7 (C-28 of gypsogenin). Qui was substituted at the position of C-2 of Qui as
observed by its deshielded H-2 of of Qui (δH 4.23) and the long-range correlation observed in the
HMBC experiment between signals of the Rha anomeric proton (δH 6.34) and C-2 of Qui (δC 76.6).
Therefore, the sequencing of the ester chain was obtained by analysis of HMBC experiment which
showed cross-peaks between H-1 of Rha (δH 6.34) and C-2 of Qui (δC 76.6), between H-1 of Xylb
(inner xylose) (δH 4.94) and C-4 of Rha (δC 85.3), and between H-1 of Xyla (the terminal xylose) (δH
5.08) and C-3 of Xylb (δC 87.2), and thus the ester chain was a tetrasaccharide,
β-D-xylopyranosyl-
(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-quinovopyranosyl unit.
The Gal unit, identified starting from anomeric signals at δH 5.11 (H-1 of Gal) and δC 106.2 (C-1 of
Gal), was identified to be in terminal position, as observed by its 13C-NMR chemical shifts. Starting
from the anomeric proton at δH 4.72 (d, J = 6.3 Hz), a GlcA unit was identified with its carbonyl
C-6 at δC 173.7. The deshielding of C-2 of GlcA (δC 82.1) indicated a substitution of glucuronic acid.
Observation of long-range proton-carbon correlations in the HMBC spectrum between the anomeric
proton of GlcA (δH 4.49) and C-3 of gypsogenin (δC 83.5) and between the anomeric proton of Gal
(
δH 5.11) and C-2 of GlcA (δC 82.1) indicated a disaccharide chain attached at C-3 of gypsogenin,
β-D-galactopyranosyl-(1→2)-β-D-glucuronopyranosyl unit.
The complete assignment of the signals of
was based on DEPT 13C-NMR and 2D NMR of 1H-1H
COSY, HMQC and HMBC. All the data of 1H-, 13C-NMR and 2D-NMR of
see Table 1, and the key
correlations in HMBC NMR and the structure of see Figure 1. In conclusion, compound was
identified as 3-O- -D-galactopyranosyl-(1 2)- -D-glucuronopyranosyl-gypsogenin-28-O- -D-xylo-
pyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-β-D-quinovopyranoside.
Hylomeconoside B ( ) was obtained as a white amorphous solid. The molecular formula of
was determined as C63H98O31 by high-resolution HRESI-MS which showed a [M + H]+ ion at m/z
1
1
1
1
β
→
β
β
2
2
1351.6151. Acid hydrolysis of
2 afforded an aglycone which was identified as gypsogenin on the
basis of the 1H- and 13C-NMR spectra [10]. The sugars obtained from the saponin hydrolysate were
identified as L-arabinose, D-xylose, L-rhamnose, D-glucuronic acid and D-galactose based on GC
analysis of their chiral derivatives. In comparative analysis of the 13C-NMR data of
2
with those of
are same as those of , and
3 (Table 2), which suggested
1
and
3, it was found that the spectra data of the aglycone moiety of
2
1
the spectra data of the sugar moiety of
2
are consistent with those of

Molecules 2017, 22, 1731
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that the aglycone of
2
is gypsogenin and the structure of the sugar moiety is same as those of
3.
By further analysis of spectral data with the similar method as above,
galactopyranosyl-(1 2)- -D-glucuronopyranosyl-gypsogenin-28-O- -D-xylopyranosyl-(1
xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside.
2
was identified as 3-O-
β
-D-
-D-
→
β
β
→
3)-
β
Table 1. The spectral data of 1H-NMR (400 MHz), 13C-NMR (100 MHz), and 2D-NMR of 1H-1H COSY
and HMQC of compound 1 in C5D5N.
Aglycone Moiety
Sugar Moiety
1H-1H COSY
1H-1H COSY
Position
δH (J in Hz)
δC HMQC
Position
δH (J in Hz)
δC HMQC
1α
1β
2α
2β
3
0.69 m
1.26 m
2.04 m
1.73 m
3.90 m
H-1β, H-2β
H-1α
H-2β, H-3
38.3
3-O-
GlcA-1
4.72 d (6.3)
4.06 m
4.12 m
4.28 m
4.27 m
H-2
H-1, H-3
H-2, H-4
103.4
82.1
77.0
73.2
77.9
173.7
106.2
74.4
75.0
70.3
77.0
62.2
25.2
2
3
4
5
H-2
α
, H-1
α
, H-3
H-2α, H-2β
83.5
55.2
48.7
20.8
4
5
1.25 m
1.26 m
1.01 m
1.91 m
1.65 m
H-6β
H-6β
H-6α, H-7α
H-6β, H-7β
H-7α
6
6α
6β
7α
7β
8
9
10
11α
11β
12β
13
Gal-1
2
3
4
5
6
28-O-
Qui-1
2
3
4
5
6
5.11 d (7.5)
4.42 m
3.99 m
4.45 m
3.98 m
H-2
H-1, H-3
H-2
32.8
40.4
48.0
36.4
23.8
H-6
H-5
1.52 m
H-11α, H-11β
4.40 m
1.71 m
1.79 m
5.30 br.s
H-9, H-11β
H-11α, H-9
H-11α, H-11β
5.98 d (8.0)
4.23 t (8.0)
4.05 t (8.4)
3.55 t (8.8)
3.71 m
1.42 d (6.0)
6.34 br. s
4.69 br. s
4.54 dd (9.2, 2.8)
4.23 dd (8.4, 9.2)
4.36 m
1.66 d (6.4)
4.94 d (6.8)
3.89 m
3.91 m
3.97 m
H-2
H-1, H-3
H-2, H-4
H-3, H-5
H-4, H-6
H-5
94.5
76.6
79.3
76.9
73.9
18.5
101.4
71.8
72.7
85.3
68.3
18.7
106.9
75.2
87.2
69.0
67.1
122.7
144.2
42.4
14
15α
15β
16α
16β
17
1.33 m
1.90 m
2.04 m
1.76 m
H-15β
H-15α
H-16β
H-16α
28.6
Rha-1
2
3
4
H-2
23.9
H-1, H-3
H-2, H-4
H-3, H-5
H-4, H-6
H-5
47.3
42.3
46.4
18
3.02 m
1.63 m
1.09m
H-19α, H-19β
H-18, H-19β
H-19α, H-18
5
6
19α
19β
20
21α
21β
22α
22β
23
24
25
26
27
Xylb-1
H-2
30.9
34.1
2
3
4
H-1, H-3
H-2, H-4
H-3, H-5α
H-4, H-5β
H-4, H-5α
H-2
1.20 m
1.03 m
1.44 m
1.42 m
9.83 s
1.28 s
0.68 s
0.90 s
1.14 s
32.5
5α
5β
Xyla-1
2
3
4
3.37 t (10.4)
4.10 m
5.08 (d, 7.4)
3.93 (m)
4.02 (m)
4.03 (m)
210.2
11.1
15.9
17.6
26.1
176.7
33.3
23.9
105.9
75.5
78.2
71.0
67.4
H-1
H-5α
H-4, H-5β
H-4, H-5α
5α
5β
3.54 t (9.6)
4.15 m
28
29
30
0.76 s
0.79 s
Dubioside C (
possess analgesic and anti-inflammatory effects [11
of Luffa cylindrica fructus, has beneficial effects on intelligence [14], therefore we have only carried
out an activity assay on the new saponins and The cytotoxic activities of compounds
and on MGC-803 (human gastric cancer), HL-60 (human promyelocytic leukemia), BEL-7404
3), one of active constituents of Thladianthae dubiae roots, has been reported to
–
13] and lucyoside P ( ), one of active constituntes
4
1
2
.
1
2
(humanhepatoma carcinoma), MCF-7 (human breast cancer), and SPC-A1 (lung adenocarcinoma) cells
were assessed by the XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)
reduction method.

Molecules 2017, 22, 1731
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Table 2. The 13C-NMR data of compound 2–4 in C5D5N.
C No.
2
3
4
C No.
2
3
4
1
2
3
4
5
6
7
8
38.2
25.0
83.5
55.2
48.4
20.6
32.8
40.2
47.9
36.3
23.3
122.8
144.3
42.2
28.4
23.9
47.4
41.8
46.3
31.0
34.2
32.6
209.9
11.1
15.8
17.5
26.2
176.4
33.3
23.8
38.3
24.9
83.3
55.2
48.5
20.6
32.9
40.4
47.1
36.4
23.9
122.6
144.6
42.2
36.2
74.1
49.6
41.4
47.1
31.1
36.1
32.3
209.6
11.0
15.9
17.6
27.3
176.0
33.4
24.9
38.3
25.1
84.7
55.9
48.3
20.8
32.8
40.3
48.0
36.3
23.4
123.0
144.4
42.4
28.4
23.9
47.7
41.9
46.5
31.0
34.2
32.6
211.8
10.8
15.9
17.6
26.3
177.6
33.4
23.9
C-3-O-sugars
GlcA-1
103.3
82.4
77.2
73.3
77.9
173.7
106.4
74.6
75.0
69.9
77.2
62.2
103.0
82.3
77.2
73.3
77.9
173.7
106.3
74.6
75.0
70.3
77.2
62.3
103.1
80.0
76.6
72.4
77.3
176.4
104.4
73.1
74.3
69.8
77.3
62.2
2
3
4
5
6
Gal-1
2
3
4
5
6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
C-28-O-sugars
Ara-1
2
3
4
93.6
75.1
69.9
66.2
63.1
101.0
71.9
72.8
84.0
68.6
18.5
106.6
75.2
87.1
69.1
67.0
106.4
75.4
78.3
71.0
67.4
93.6
75.2
69.9
66.1
63.1
101.1
72.0
72.8
83.5
68.6
18.5
106.3
75.1
87.2
69.1
67.0
106.1
75.4
78.3
71.0
67.4
93.6
75.9
69.9
66.4
63.1
101.2
71.2
82.2
78.6
69.0
18.7
5
Rha-1
2
3
4
5
6
Xyl(inner)-1
2
3
4
5
Xyl(terminal)-1
104.6
75.2
76.6
70.8
66.5
105.4
72.9
77.5
70.2
77.8
62.0
2
3
4
5
Glc(terminal)-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
The results (Table 3) showed that compound
HL-60, with IC50 values of 43.8 and 32.4
mL−1, respectively. Therefore, compound
to be one of the antitumor principles in this plant.
1
exhibited moderate cytotoxicity on MGC-803 and
µg
·
1
is considered

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Table 3. The cytotoxicity of compouds 1 and 2 assayed by XTT assay (n = 3).
Inhibitory Rate, Mean ± SD (%)
Concentration
Compds.
1
−l
(µg·mL
)
MGC-803
HL-60
BEL-7402
MCF-7
SPC-A1
9.3 ± 1.3 1
18.5 ± 1.1
43.2 ± 1.6
75.0 ± 2.3
92.1 ± 1.7
0
10
20
40
80
160
10
11.3 ± 1.6
19.7 ± 1.5
58.4 ± 1.3
91.7 ± 2.1
98.2 ± 1.9
0
0
0
0
0
0
0
0
0
0
0
0
25.1 ± 1.2
22.0 ± 1.7
25.2 ± 1.8
40.1 ± 2.2
0
0
0
20
0
0
0
0
0
40
80
160
10.2 ± 1.8
18.4 ± 1.6
44.3 ± 1.5
9.2 ± 1.3
20.3 ± 1.2
55.0 ± 2.2
0
0
0
0
0
0
2
22.0 ± 1.9
25.20 ± 1.8
is 43.8
23.1 ± 2.1
1
Results are expressed as mean
±
standard deviation. The IC50 values of compound
1
µg
mL−1 on MGC-803
·
and 32.4 µg·mL−1 on HL-60.
3. Materials and Methods
3.1. General Information
NMR spectra were recorded on an AV-400 spectrometer (Bruker Corporation, Faellanden,
Switzerland). HRESI-MS were recorded on a Bruker microOTOF-Q II mass spectrometer (Bruker
Corporation, Bremen, Germany). Optical rotations were measured with a HORIBA SEPA-300
high-sensitive polarimeter (Horiba Ltd, Kyoto, Japan). HPLC was performed on a Shimadzu LC-10A
system equipped with a SPD-10A detector (Shimadzu Corporation, Kyoto, Japan) and a Gemini
5
µ
m C18 110A column (250 mm
performed an Agilent 7820A gas chromatograph with a quartz capillary column (30 mm
0.25 m, Agilent Technologies Inc., Santa Clara, CA, USA); detection, FID. Column chromatography
×
10.00 mm, 5
µ
m, Phenomenex, Torrance, CA, USA). GC was
×
0.32 mm
×
µ
was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), D101
polyporous resin (Tianjin Pesticide Co., LTD., Resin Branch, Tianjin, China). TLC was performed
on glass precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd, Qingdao, China),
detection under UV light or by heating after spraying with 10% H2SO4 in 95% EtOH. Distilled
water was purchased from Hangzhou Wahaha Group Co., Ltd. (Hongzhou, China). Acetonitrile of
chromatographic grade for HPLC was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Other
chemicals and reagents of analytical grade were from Beijing Chemical Works (Beijing, China).
The bioactivities were measured on a DNM-9602 enzyme immunoassay spectrophotometer
(Beijing, China). RPMI 1640 medium was purchased from HyClone (Logan, UT, USA), PBS
from Gibco company (Carlsbad, CA, USA), fetal bovine serum (FBS) from Zhejiang Tianhang
Biotechnology Co., Ltd. (Hangzhou, China), XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide) and phenazine methosulphate from Sigma-Aldrich Shanghai Trading Co.
Ltd. (Shanghai, China).
The Hylomecon japonica herb was collected in the Changchun District of Jilin Province, China.
They were identified by Jing-min Zhang of the School of Pharmaceutical Sciences, Jilin University.
A voucher specimen (No. 2015062001) is deposited at the Herbarium of the School of Pharmaceutical
Sciences, Jilin University.
3.2. Extraction and Isolation
Two kg of air-dried whole Hylomecon japonica herbs were extracted twice with 20 L of 70%
aqueous ethanol solution (v/v) at room temperature. The extraction solution was concentrated under
reduced pressure to remove ethanol, and the water concentrate was filtered and then passed through
a D101 polyporous resin column eluting successively with H2O, 30% EtOH, 50% EtOH, 70% EtOH,
and 95% EtOH. The crude saponin extracts were obtained from 50% aqueous ethanol eluate by
vacuum distillation recovery and used for the next experiments. The crude saponin extracts were

Molecules 2017, 22, 1731
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chromatographed on silica gel columns repeatedly eluted with EtOAc–MeOH–H2O (7:3:0.5) and futher
purified by semi-preparative HPLC using acetonitrile and 0.1% formic acid solution in water as the
mobile phase and the eluate was monitored at 207 nm, to yield compounds
1 (60 mg), 2 (50 mg),
3 (30 mg), and 4 (30 mg).
3.3. Characterization
: White amorphous solid, [α]2D5 +5.0 (c 0.5, pyridine). HRESIMS, m/z: 1365.6318 [M + H]+
;
◦
Compound
1
calcd for C64H101O31, 1365.6327. H-NMR (400 MHz, C5D5N) and 13C-NMR (100 MHz, C5D5N): see
1
Table 1.
Compound
2
: White amorphous solid, [α]D25
−
15.0◦(c 0.5, pyridine). HRESIMS, m/z: 1351.6151
: 0.67 (3H, s, H-25), 0.80 (3H,
1
[M + H]+; calcd for C63H99O31, 1351.6165. H-NMR (400 MHz, C5D5N)
δ
s, H-26), 0.87 (6H, s, H-30, H-29), 1.12 (3H, s, H-27), 1.29 (3H, s, H-24), 1.61 (3H, d, J = 4.8, Rha-6), 3.13
(1H, m, H-18), 3.34 (1H, t-like, J = 10.0 Hz, Xyl(inner)-5 ), 3.55 (1H, t-like, J = 10.0 Hz, Xyl(terminal)-5 ),
α
α
4.71 (1H, d, J = 6.8 Hz, GluA-1), 5.01 (1H, d, J = 6.8 Hz, Xyl(inner)-1), 5.08 (1H, d, J = 7.2 Hz, Gal-1),
5.10 (1H, d, J = 7.6 Hz, Xyl(terminal)-1), 5.29 (1H, br. s, H-12), 5.66 (1H, br. s, Rha-1), 6.34 (1H, br. s,
Ara-1), 9.82 (1H, s, H-23). 13C-NMR (100 MHz, C5D5N): see Table 2.
Compound
3
: White amorphous solid, [α]D25
−
29.2◦(c 0.5, pyridine). HRESIMS, m/z: 1367.6101
: 0.70 (3H, s, H-25), 0.90 (6H,
1
[M + H]+; calcd for C63H99O32, 1367.6114. H-NMR (400 MHz, C5D5N)
δ
s, H-26, H-29), 1.02 (3H, s, H-30), 1.28 (3H, s, H-27), 1.57 (3H, s, H-24), 1.65 (3H, d, J = 4.8, Rha-6), 3.44
(1H, m, H-18), 3.31 (1H, t-like, J = 10.0 Hz, Xyl(inner)-5 ), 3.54 (1H, t-like, J = 10.0 Hz, Xyl(terminal)-5 ),
α
α
4.68 (1H, d, J = 6.8 Hz, GluA-1), 4.98 (1H, d, J = 6.8 Hz, Xyl(inner)-1), 5.05 (1H, d, J = 7.2 Hz, Gal-1),
5.10 (1H, d, J = 7.6 Hz, Xyl(terminal)-1), 5.47 (1H, br. s, H-12), 5.62 (1H, br. s, Rha-1), 6.35 (1H, br. s,
Ara-1), 9.76 (1H, s, H-23). 13C-NMR (100 MHz, C5D5N): see Table 2.
Compound
4
: White amorphous solid, [α]D25
−
10.0◦(c 0.5, pyridine). HRESIMS, m/z: 1381.6258
: 0.90 (3H, s, H-25), 0.98
1
[M + H]+; calcd for C64H101O32, 1381.6270. H-NMR (400 MHz, D2O-C5D5N)
δ
(3H, s, H-26), 1.03 (3H, s, H-29), 1.04 (3H, s, H-30), 1.26 (3H, s, H-27), 1.45 (3H, s, H-24), 1.67 (3H, d,
J = 4.8, Rha-6), 3.17 (1H, m, H-18), 3.57 (1H, t-like, J = 10.0 Hz, Xyl(terminal)-5 ), 4.82 (1H, d, J = 7.2 Hz,
α
GluA-1), 5.09 (1H, d, J = 8.0 Hz, Glu-1), 5.18 (1H, d, J = 7.6 Hz, Gal-1), 5.29 (1H, d, J = 8.0 Hz, Xyl-1),
5.45 (1H, br. s, H-12), 5.57 (1H, br. s, Rha-1), 6.23 z, Ara-1), 9.78 (1H, s, H-23). 13C-NMR (100 MHz,
D2O-C5D5N): See Table 2.
3.4. Acid Hydrolysis and Sterochemistry of Sugars of 1 and 2
◦
Compounds
The reaction mixture was extracted with EtOAc (3
dryness. The EtOAc extract was purified by silica gel column using a gradient of CHCl3-MeOH
(1:0–95:5) to the aglycones of and which were determined to be gypsogenin by comparison of
their spectral data with those of an authentic sample. Aglycone: White amorphous solid; 13C-NMR
(100 MHz, C5D5N) : 207.7 (C-23), 180.0 (C-28), 144.8 (C-13), 122.2 (C-12), 71.6 (C-3), 56.2(C-4), 48.0
1
and
2
(each 20 mg) were hydrolyzed with 2 M HCl (5 mL) at 90 C for 4 h.
×
5 mL), washed with H2O, and evaporated to
1
2
δ
(C-5), 47.9 (C-9), 46.6 (C-17), 46.5 (C-19), 42.2 (C-14), 41.9 (C-18), 40.0 (C-8), 38.4 (C-1), 36.2 (C-10),
34.2 (C-21), 33.2 (C-29), 32.8 (C-7), 32.5 (C-22), 30.9 (C-20), 28.3 (C-15), 26.1 (C-27), 27.0 (C-2), 23.8
(C-16, C-30), 23.7 (C-11), 21.0 (C-6), 17.3 (C-26), 15.7 (C-25), 9.6 (C-24); 1H-NMR (400 MHz, C5D5N)
δ
: 0.94 (3H, s), 0.97 (6H, s), 1.12 (6H, s), 1.23 (6H, s), 1.41 (6H, s), 3.02 (1H, m), 3.90(1H, m), 5.29 (1H,
brs), 9.66 (1H, m). The H2O layer was concentrated under reduced pressure to dryness, to give a
residue of the sugar fraction. The residue was dissolved in pyridine (2 mL), L-cysteine methyl ester
◦
hydrochloride (1.5 mg) was added, and the mixture was heated at 60 C for 1 h. Trimethylsilylimidazole
(1.5 mL) was added, and the mixture was heated at 60 ◦C for another 0.5 h. An aliquot (4
µL) of the
supernatant was subjected to GC analysis under the following conditions: c◦olumn temp 180–280 ◦C
at 3 deg/min, carrier gas N2 (1 mL/min), injector and detector temp 250 C, split ratio 1:50. The
configurations of monosugars for
1 and 2 were determined by comparison of the retentions times

Molecules 2017, 22, 1731
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(tR) of the corresponding derivatives with standard L-arabinose (tR 10.748 min), D-xylose (tR 11.496
min), L-rhamnose (tR 12.162 min), D-quinovose (tR 13.648 min), D-glucuronic acid (tR 15.814 min) and
D-galactose (tR 18.026 min).
1 yielded D-xylose, L-rhamnose, D-quinovose, D-glucuronic acid and
D-galactose, and 2 gave L-arabinose, D-xylose, L-rhamnose, D-glucuronic acid and D-galactose.
3.5. Cytotoxicity Assay
The procedure for the cytotoxic assay was performed according to the XTT (2,3-bis-(2-methoxy-
4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction method. In this study, MGC-803
(human gastric cancer), HL-60 (human promyelocytic leukemia), BEL-7402 (humanhepatoma
carcinoma), MCF-7 (human breast cancer), and SPC-A1 (lung adenocarcinoma ) cell lines were used.
Compounds
1
and
2
was dissolved at a concentration of 10 mg
·
mL−1 in phosphate-buffered saline
µm (Millipore, Bedford, MA, USA) and stored
(PBS) (as a stock solution), filtered through a filter 0.22
at 4 ◦C. The stock solution was diluted to 160, 80, 40, 20, 10 µg·mL−1 before the cytotoxic assay.
Logarithmic-phase growing cells were diluted into 5
×
104 cells mL−1, seeded in in 96-well plates
·
with 100
100 U
mL−1 penicillin, 100
Blank control wells were added 100
After 24 h, 100 L of the above culture medium containing different concentrations of
µ
L per well, and incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum,
mL−1 streptomycin at 37 ◦C in a humidified atmosphere with 5% CO2.
L of cultrure medium, and maintained under the same conditions.
and and
·
µg
·
µ
µ
1
2
normal culture medium (untreated control group) were added, and incubated for 72 h. For the analysis
−1
of cytotoxicity, 50
µL (1 mg
·
mL−1) XTT containing 0.15 mg
·
mL phenazine methosulphate was added
to each well, and cells were incubated for 3 h at 37 ◦C. The absorbances (A) of the produced formazan
were measured by a DNM-9602 enzyme immunoassay spectrophotometer at 450 nm. Three replicate
well were used for each analysis. The cell inhibitory rate (%) = (Asample
−
Ablank)/(Auntreated
−
Ablank)
×
100. The concentration of compound producing 50% of cell inhibitory rate (IC50) was calculated by
using SPSS version 20.0 (International Business Machines Corporation, Armonk, NY, USA).
4. Conclusions
Two new triterpenoid saponins, named hylomeconoside A (1) and B (2), along with two known
triterpenoid saponins identified as dubioside C ( ), and lucyoside P (
3
4
), have been isolated from an
ethanolic extract of Hylomecon japonica herbs. It is well known that alkaloids are main constituents of the
plants of Papaveraceae family and until now besides alkaloids, flavonoids, megastigmane derivatives,
volatile oil, furan derivatives, triterpenes and sterols have also reported, but saponins have not been
reported [15
family. Compound
considered to be one of the antitumor principles in this plant.
–17]. This is the first time that saponins have been isolated from plants of the Papaveraceae
−1
−1
µg mL on HL-60 is
1
with the IC50 values of 43.8
µg
·
mL on MGC-803 and 32.4
·
Acknowledgments: The authors gratefully thank Jing-Min Zhang (School of Pharmaceutical Sciences, Jilin
University) for the identification of plants.
Author Contributions: Guang-Shu Wang conceived and designed the experiments; Yan-Fei Qu, Jing-Yu Gao,
Jing Wang, Yan-Mei Geng, Yu Zhou, Cheng-Xin Sun, Fei Li, Lei Feng, and Meng-Juan Yu performed the
experiments; Guang-shu Wang analyzed the data; Guang-shu Wang wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
The Editorial Board of Zhong Hua Ben Cao of State Administration of Traditional Chinese Medicine of the
People’s Republic of China. Zhong Hua Ben Cao 3, 1st ed.; Shanghai Science and Technology Publishing
House: Shanghai, China, 1999; p. 653.
2.
3.
Cheng, H.Y.; Cheng, J.X.; Wei, H.; Bai, J.Q. The research progress of Hylomecon japonica. J. Shaanxi Coll. Tradit.
Chin. Med. 2011, 34, 94–95.
Lee, S.Y.; Kim, K.H.; Lee, I.K.; Lee, K.H.; Choi, S.Y.; Lee, K.R. A new flavonol glycoside from Hylomecon
vernalis. Arch. Pharm. Res. 2012, 35, 415–421. [CrossRef] [PubMed]

Molecules 2017, 22, 1731
9 of 9
4.
5.
Lee, S.Y.; Choi, S.U.; Lee, K.R. Three new megastigmane glycosides from Hylomecon vernalis. Bull. Korean
Chem. Soc. 2011, 32, 3813–3816. [CrossRef]
Choi, J.G.; Kang, O.H.; Chae, H.S.; Obiang-Obounou, B.; Lee, Y.S.; Oh, Y.C.; Kim, M.S.; Shin, D.W.;
Kim, J.A.; Kim, Y.H.; et al. Antibacterial activity of hylomecon hylomeconoides against methicillin-resistant
staphylococcus aureus. Appl. Biochem. Biotechnol. 2010, 160, 2467–2474. [CrossRef] [PubMed]
Chae, H.S.; Kang, O.H.; Keum, J.H.; Kim, S.B.; Mun, S.H.; Seo, Y.S.; Kim, M.R.; Choi, J.G.; Shin, D.W.; Oh, Y.C.;
et al. Anti-inflammatory effects of hylomecon hylomeconoides in RAW 264.7 cells. Eur. Rev. Med. Pharmaco.
2012, 16, 121–125.
Lee, Y.J.; Yin, H.Q.; Kim, Y.H.; Li, G.Y.; Lee, B.H. Apoptosis inducing effects of
6-methoxydihydrosanguinarine in HT29 colon carcinoma cells. Arch. Pharml. Res. 2004, 27, 1253–1257.
[CrossRef]
Nagao, T.; Okabe, H.; Mihashi, K.; Yamauchi, T. Studies on the constituents of Thladiantha dubia BUNGE.
I.: The structures of dubiosides A, B and C, the quillaic acid glucuronide saponins isolated from the tuber.
Chem. Pharm. Bull. 1989, 37, 925–929. [CrossRef]
Yoshikawa, K.; Arihara, S.; Wang, D.J.; Narui, T.; Okuyama, T. Structures of two new fibrinolytic saponins
from the seed of Luffa cylindrica Roem. Chem. Pharm. Bull. 1991, 39, 1185–1188. [CrossRef] [PubMed]
6.
7.
8.
9.
10. Ma, L.; Yu, A.H.; Sun, L.L.; Gao, W.; Zhang, M.M.; Su, Y.L.; Liu, H.; Ji, T.F. Two new bidesmoside triterpenoid
saponins from the seeds of Momordica charantia L. Molecules 2014, 19, 2238–2246. [CrossRef] [PubMed]
11. Tong, J.M.; Liu, Y.L.; Liu, Y.P.; Mao, X.X. Mechanism of analgesic effect of total saponin from the root of
Thladianthae dubiae Bunge. Lishizhen Med. Mater. Medica Res. 2012, 23, 1085–1087.
12. Song, L.P.; Zhao, C.Y.; Tong, J.M. Site of analgesic effect of total saponin from Thladianthae dubia root. China J.
Exp. Tradit. Med. Formulae 2013, 19, 200–202.
13. Chen, J.S.; Zhang, Y.L.; Zhao, B.; Tong, J.M.; Mao, X.X.; Liu, Y.P. Anti-inflammatory effect of the total saponins
from Thladianthae dubiae roots. China J. Exp. Tradit. Med. Formulae 2012, 18, 163–166.
14. Zhang, L.; Zhong, D.H.; Pu, X.R.; Qin, J. Studies on the beneficial effects of Luffa saponin to intelligence.
Lishizhen Med. Mater. Medica Res. 2000, 11, 676.
15. Qian, Y.C. Progress on the Study of alkaloids constituents and pharmacological action for the medicinal
plants of Papaveraceae. Primary J. Chin. Mater. Medica 2001, 15, 48–49.
16. Yu, R.M.; Wang, C.S.; Song, L.Y. Progress on the study of chemical constituents and pharmacological action
for the plants of Papaveraceae. Shanghai J. Tradit. Chin. Med. 2004, 38, 59–61.
17. Deng, A.J.; Zhang, Z.H.; Li, Q.; Ma, L.; Qin, H.L. Two new hopane-type triterpenes from the aerial part of
Chelidonium majus. Phytochem. Lett. 2016, 17, 75–78. [CrossRef]
Sample Availability: Samples of the compounds 1–4 are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).