Phytochemistry 190 (2021) 112870

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Bioactive oleanane-type saponins from Hylomecon Japonica

Fei Li, Si-Tong Wu, Ming-Hui Qu, Yi-Xiao Wang, Chun-Liu Ma, Bai-Hong Yu, Guang-Shu Wang *
School of Pharmaceutical Sciences, Jilin University, Changchun, 130021, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: Six undescribed oleanane-type saponins, named as Hylomeconosides L-Q, were isolated from the whole herb of
Hylomecon japonica Hylomecon Japonica, their structures were determined by analysis of 1D and 2D-NMR (1H–1H COSY, HSQC, and
Papaveraceae HMBC) spectroscopic data, mass spectrometry (HRESI-MS) and chromatographic data (GC and LC). Their
Triterpenoid saponins
structures were identified as 3-O-β-D-galactopyranosyl-(1 → 2)-β-D-glucuronopyranosyl gypsogenin 28-O-β-D-
Gypsogenin
Quillaic acid
galactopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-β-L-arabinopyranoside; 3-O-β-D-galactopyranosyl-(1 →
Cytotoxic activities 2)-β-D-glucuronopyranosyl gypsogenin 28-O-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-qui­
novopyranoside; 3-O-β-D-glucuronopyranosyl gypsogenin 28-O-β-D-xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1
→ 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside; 3-O-β-D-xylopyranosyl-(1 → 3)-β-D-glucuronopyr­
anosyl gypsogenin 28-O-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside; 3-O-
β-D-galactopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl quillaic acid 28-O-β-D-
xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside; 3-O-
β-D-galactopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl quillaic acid 28-O-β-D-
xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside. Hylo­
meconosides L-Q showed selective cytotoxicities against human cancer cell lines A549, AGS, HeLa, Huh 7, HT29
and K562. These results represent a contribution to the chemotaxonomy of the saponins of Hylomecon Japonica
and their bioactivities.

1. Introduction 2. Results and discussion

Hylomecon Japonica (Thunb.) Prantl & Kundig (Papaveraceae) is
well-known in the traditional Chinese medicine widely distributed in the
northeast, east and central province of China as the Chinese drug Guai-
Zao-Qi (root or rhizome of Hylomecon Japonica) and is utilized to treat
rheumatism, weakness of limbs, stomachache and bruises (The Editorial
Board of Zhong Hua Ben Cao of State Administration of Traditional
Chinese Medicine of the People’s Republic of China, 1999; Cheng et al.,
2011). In the continuation of our study on saponin constituents of this
plant (Qu et al., 2017; Li et al., 2020), we have further examined the
saponin fraction of Hylomecon Japonica. There are some saponin con­
stituents from Hylomecon Japonica exhibit selective anti-tumor activities
(Qu et al., 2017; Li et al., 2020). In this paper, we describe the isolation,
structure elucidation and cytotoxic activity analysis of six undescribed
oleanane-type saponins designated as Hylomeconosides L-Q.
Six undescribed oleanane-type saponins, named as Hylomeconosides
L-Q were identified by comparison of the NMR data with that in the
literature.
Hylomeconosides L-Q (1–6) were obtained as white, amorphous
powder. The HRESI-MS indicated [M + H]+ ion at m/z 1249.5818,
1233.5877, 1203.5745, 1203.5732, 1527.6770, 1543.6696, indicating
an empirical molecular formula of C59H92O28, C59H92O27, C58H90O26,
C58H90O26, C70H110O36, C70H110O37, correspondingly. Acid hydrolysis
of 1–4 provided an aglycone which was identified as gypsogenin, while
acid hydrolysis of 5–6 provided an aglycone which was identified as
quillaic acid according to the 1H-NMR and 13C-NMR data (Yoshikawa
et al., 1991), correspondingly. The sugars obtained from the hydrolysate
of 1–6 were identified as follows: 1 has D-galactose (Gal), L-rhamnose
(Rha), L-arabinose (Ara) and D-glucuronic acid (GlcA); 2 has D-xylose
(Xyl), L-rhamnose, D-quinovose (Qui), D-galactose, and D-glucuronic
acid; 3 has D-xylose, L-rhamnose, D-quinovose and D-glucuronic acid; 4
has D-xylose, L-rhamnose, D-quinovose and D-glucuronic acid; 5 has

* Corresponding author.
E-mail address: wgs@jlu.edu.cn (G.-S. Wang).

https://doi.org/10.1016/j.phytochem.2021.112870
Received 16 April 2021; Received in revised form 2 July 2021; Accepted 4 July 2021
Available online 14 July 2021
0031-9422/© 2021 Elsevier Ltd. All rights reserved.
F. Li et al.
D-xylose, L-rhamnose, D-galactose D-fucose (Fuc) and D-glucuronic
acid; 6 has D-xylose, L-rhamnose, D-galactose and D-glucuronic acid
based on GC analysis of their chiral derivatives (Yoshikawa et al., 1991;
Hikaru et al., 1989; Kim et al., 1992; Ghezala et al., 2000, 2004; Ivone
et al., 2011; Voutquenne-Nazabadioko et al., 2013). The all above acid
hydrolysis data combining with HRESI-MS information indicated that 1
has two D-galactosyl, one L-rhamnosyl, one L-arabinosyl and one
D-glucuronosyl moieties; 2 has one D-xylosyl, one L-rhamnosyl, one
D-quinovosyl, one D-galactosyl and one D-glucuronosyl moieties; 3 has
two D-xylosyl, one L-rhamnosyl, one D-quinovosyl and one D-glucur­
onosyl moieties; the variety and number of suger moiety of 4 were as
same as 3; 5 has two D-xylosyl, two L-rhamnosyl, one D-galactose, one
D-fucosyl and one D-glucuronosyl moieties; 6 has two D-xylosyl, two
L-rhamnosyl, two D-galactose and one D-glucuronosyl moieties,
correspondingly.
As for the spectroscopic data of 1, the 1H-NMR spectrum revealed signals
due to six quaternary methyl group protons at δH 0.67, 0.88, 0.88, 0.90, 1.07,
1.11, an olefinic proton at δH 5.22 (br. s), an aldehyde proton at δH 9.47 (s),
and five anomeric protons at δH 4.10 (d, J = 7.2 Hz), 4.23 (d, J = 6.1 Hz),
4.32 (d, J = 7.6 Hz), 4.86 (s) and 5.57 (s). The 13C-NMR spectrum displayed
signals due to six quaternary methyl group carbons at δC 10.2, 15.3, 16.6,
23.4, 25.4, and 32.7, an oxygen-bearing methine carbon at δC 81.9, a set of
olefinic carbons at δC 121.6 and 143.4, an ester carbonyl carbon at δC 175.1,
an aldehyde carbon at δC 209.5, and five anomeric carbons at δC 91.7, 99.4,
101.5, 104.7 and 105.4. Observation of long-range proton-carbon correla­
tions in the HMBC spectrum between the H-24 (δH 1.07) and C-3 (δC 81.9),
H-24 (δH 1.07) and C-4 (δC 53.9), H-24 (δH 1.07) and C-5 (δC 47.2), H-24 (δH
1.07) and C-23 (δC 209.5); H-25 (δH 0.90) and C-1 (δC 37.5), H-25 (δH 0.90)
and C-5 (δC 47.2), H-25 (δH 0.90) and C-9 (δC 46.8); H-26 (δH 0.67) and C-7
(δC 31.6), H-26 (δH 0.67) and C-9 (δC 46.8), H-26 (δH 0.67) and C-14 (δC
41.3); H-27 (δH 1.11) and C-8 (δC 39.5), H-27 (δH 1.11) and C-13 (δC 143.4),
H-27 (δH 1.11) and C-14 (δC 41.3); H-29 (δH 0.88) and C-19 (δC 45.3), H-29
(δH 0.88) and C-20 (δC 30.4), H-29 (δH 0.88) and C-21 (δC 33.2); H-30 (δH
0.88) and C-19 (δC 45.3), H-30 (δH 0.88) and C-20 (δC 30.4), H-30 (δH 0.88)
and C-21 (δC 33.2), and proton-proton signals in the 1H–1H COSY spectrum
between H-2 (δH 1.62) and H-3 (δH 3.71), and between H-9 (δH 1.58) and H-
11 (δH 1.83), and between H-11 (δH 1.83) and H-12 (δH 5.22), and between
H-18 (δH 2.81) and H-19 (δH 1.65), suggested that the aglycone section of 1
was gypsogenin, combining analysis of the all above spectroscopic data of 1.
Comparing of the spectroscopic data of 1 with gypsogenin, that the signal
due to C-3 of the aglycone section downfielded to δC 81.9 and the signal due
to C-28 upfielded to δC 175.1 suggested that this aglycone was substituted at
positions C-3 and C-28, having five monosaccharide units. The five anomeric
proton signals at δH 5.57 (s), 4.86 (s), 4.32 (d J = 7.6 Hz), 4.23 (d, J = 6.1 Hz),
4.10 (d, J = 7.2 Hz) were correlated with anomeric carbon signals at δC 91.7,
99.4, 105.4, 104.7, 101.5, respectively, in the HSQC experiment, and they
were assigned to L-arabinpyranosyl, L-rhamnopyranosyl, D-galactopyr­
anosyl, D-galactopyranosyl and D-glucuronopyranosyl moieties, respec­
tively, based on the comprehensive analysis of the 1D and 2D-NMR data of 1.
The small 3JH1-H2 coupling constant (H-1 of Ara, s) of the anomeric protons
indicated the β-configurations for L-arabinpyranosyl unit, and the large 3JH1-
H2 coupling constants (H-1 of Gal1, d, J = 6.1; H-1 of Gal2, d, J = 7.6; H-1 of
GlcA, d, J = 7.2) of the anomeric protons indicated the β-configurations for
D-galactopyranosyl and D-glucuronopyranosyl moieties, which were
further confirmed by the 13C-NMR data of 1. Integrated assignments of each
glycosidic unit were achieved by analysis of the 1H–1H COSY, HSQC and
HMBC spectra. One Gal uint, Gal2, identified starting from anomeric signals
at δH 4.32 (H-1 of Gal2) and δC 105.4 (C-1 of Gal2) with the methylene signals
at δH 3.46 and 3.59 (2H, H-6 of Gal2) and δC 60.2 (C-6 of Gal2), was identified
to be in terminal position, as observed by their 13C-NMR chemical shifts. The
Rha unit, characteristic of a methyl doublet at δH 1.12 (3H, d, J = 6.1 Hz) and
a typical broad singlet of anomeric proton at δH 4.86 (s), was identified to be
a rhamnopyranosyl unit. The signals at δC 67.9, δC 81.6, δC 71.3, δC 68.3 was
confirmed as C-2, C-3, C-4, C-5 of Rha based on the long-range proton-car­
bon correlations in the HMBC spectrum between the H-6 (δH 1.12) and C-4
(δC 71.3), H-6 (δH 1.12) and C-5 (δC 68.3); the H-1 (δH 4.86) and C-3 (δC
2
Phytochemistry 190 (2021) 112870
81.6), H-1 (δH 4.86) and C-5 (δC 68.3), and the proton-proton signals in the
1
H–1H COSY spectrum between H-1 (δH 4.82) and H-2 (δH 3.83), and the
proton-carbon signals in the HSQC spectrum between H-2 (δH 3.83) and C-2
(δH 69.7). Comparing of the spectroscopic data of Rha with methyl-
α-rhamnoside and methyl-β-rhamnoside, δC 102.6,72.1, 72.7, 73.8, 69.5,
18.6; δC 102.6, 72.1, 75.3, 73.7, 73.4, 18.5, respectively, in book of Natural
Medicinal Chemistry (Wu et al., 2012), it was found that the carbon signals of
C-3 and C-5 were upfielded when it was α-configurations. Thus, the carbon
signals of Rha-C-3 and Rha-C-5 in compound 1 were more in line with
methyl-α-rhamnoside. The Rha unit was substituted at C-3 based on the
deshielding of C-3 (δC 81.6), which was identified as C-3 of the Rha based on
the long-range correlations observed in the HMBC experiment between
signals at δH 4.86 (H-1 of Rha) and δC 81.6 (C-3 of Rha). And the terminal
Gal2 uint was attached to C-3 of the Rha uint based on the long-range cor­
relations observed in the HMBC experiment between signals at δH 4.32 (H-1
of Gal2) and δC 81.6 (C-3 of Rha). The Ara unit, identified starting from
anomeric signals at δH 5.57 (s) and δC 91.7 (C-1 of Ara), was substituted at
the position of C-2 of Ara based on the deshielding of C-2 of Ara (δC 73.1), and
the Rha unit was attached to this position based on the long-range correla­
tion observed in the HMBC experiment between signals at δH 4.86 (H-1 of
Rha) and δC 73.1 (C-2 of Ara). The Ara unit was attached to C-28 of gypso­
genin based on the long-range correlation observed in the HMBC experiment
between signals at δH 5.57 (H-1 of Ara) and δC 175.1 (C-28 of gypsogenin).
Therefore, the ester chain of trisaccharide attached to C-28 of gypsogenin
was identified as β-D-galactopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 →
2)-β-L-arabinpyranosyl unit. The Gal1 unit, identified starting from
anomeric signals at δH 4.23 (H-1 of Gal1) and δC 104.7 (C-1 of Gal1) with the
methylene signals at δH 3.36 and 3.50 (2H, H-6 of Gal1) and δC 59.8 (C-6 of
Gal1), was identified to be in terminal position, as observed by its 13C-NMR
chemical shifts. Starting from the anomeric signals at δH 4.10 (d, J = 7.2 Hz)
and δC 101.5, a GlcA unit was identified with its carbonyl C-6 at δC 172.7.
Observation of proton-proton correlations in the 1H–1H COSY spectrum
between the anomeric proton of GlcA (δH 4.10) and H-2 of GlcA (δH 3.15),
and the proton-carbon correlations in the HSQC spectrum between H-2 of
GlcA (δH 3.15) and C-2 of GlcA (δC 81.6) suggested the signal at δC 81.6 was
identified as C-2 of GlcA, and the deshielding of C-2 of GlcA (δC 81.6) indi­
cated the glucuronic acid was substituted at C-2. Observation of long-range
proton-carbon correlations in the HMBC spectrum between the anomeric
proton of GlcA (δH 4.10) and C-3 of gypsogenin (δC 81.9) and between the
anomeric proton of Gal1 (δH 4.23) and C-2 of GlcA (δC 81.6) 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 1 was based on 13C-NMR and 2D NMR of 1H–1H COSY, HSQC and HMBC.
All the data of 1H, 13C-NMR and 2D-NMR of 1 see Tables 1 and 3, and the key
correlations in HMBC NMR with 1H–1H COSY of 1 see Fig. 3. In conclusion,
compound 1 was elucidated as 3-O-β-D-galactopyranosyl-(1 → 2)-β-D
-glucuronopyranosyl gypsogenin 28-O-β-D-galactopyranosyl-(1 → 3)-α-L-
rhamnopyranosyl-(1 → 2)-β-L-arabinopyranoside.
In the spectroscopic data of 2, the large 3JH1-H2 coupling constants
(H-1 of Xyl, d, J = 6.8 Hz; H-1 of Qui, d, J = 6.8 Hz) of the anomeric
protons indicated the β-configurations for D-xylopyranosyl and D-qui­
novopyranosyl moieties, which were further confirmed by the 13C-NMR
data of 2. In comparative analysis of the 1H-NMR and 13C-NMR data of 2
with 1, it was found that 2 has the same aglycone moiety as that of 1,
that is gypsogenin. The Xly unit, identified starting from anomeric sig­
nals at δH 4.30 (H-1 of Xyl) and δC 105.7 (C-1 of Xyl), were identified to
be in terminal position. The Rha unit, characteristic of a methyl doublet
at δH 1.11 (3H, d, J = 5.2 Hz) and a typical broad singlet of anomeric
proton at δH 5.23 (s) was identified to be a rhamnopyranosyl unit with
substitutions at C-4 based on the deshielding of C-4 (δC 82.9), and the
signals at δC 82.9 was identified as C-4 of the Rha, based on the long-
range correlations observed in the HMBC experiment between signals
at δH 1.11 (3H of methyl) and δC 82.9 (C-4 of Rha). And the terminal Xyl
uint were attached tom C-4 of the Rha uint, based on the long-range
correlations observed in the HMBC experiment between signals at δH
4.30 (H-1 of Xyl) and δC 82.9 (C-4 of Rha) as observed by their 13C-NMR
F. Li et al. Phytochemistry 190 (2021) 112870

Table 1
13
C (100 MHz) and 1H (400 MHz) NMR spectroscopic data for the aglycone moieties of 1–3 in DMSO.a
NO. 1 2 3

Position δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz)

1α 37.5, CH2 0.97 m 38.0, CH2 0.97 m 37.6, CH2 1.01 m

1β 1.53 m 1.52 m 1.58 m
2α 23.9, CH2 1.96 m 23.9, CH2 1.96 m 24.2, CH2 1.96 m
2β 1.62 m 1.53 m 1.59 m
3 81.9, CH 3.71 dd (12.5, 4.5) 82.9, CH 3.79 dd (11.4, 3.8) 79.4, CH 3.83 dd (12.7, 4.2)
4 53.9, C 53.9, C 54.3, C
5 47.2, CH 1.28 overlapped 47.3, CH 1.26 overlapped 46.8, CH 1.29 overlapped
6α 19.7, CH2 1.37 m 19.7, CH2 1.37 m 19.6, CH2 1.37 m
6β 0.84 m 0.82 m 0.79 m
7α 31.6, CH2 1.37 m 31.6, CH2 1.38 m 31.5, CH2 1.37 m
7β 1.18 m 1.18 m 1.17 m
8 39.5, C 39.5, C 39.5, C
9 46.8, CH 1.58 d (11.7, 3.0) 45.9, CH 1.56 d (11.3, 3.2) 46.4, CH 1.59 d (11.6, 3.2)
10 35.5, C 35.5, C 35.4, C
11α 22.1, CH2 1.57 m 22.0, CH2 1.53 m 22.3, CH2 1.59 m
11β 1.83 m 1.81 m 1.83 m
12β 121.6, CH 5.22 br.s 121.6, CH 5.18 br.s 121.5, CH 5.18 br.s
13 143.4, C 143.0, C 143.1, C
14 41.3, C 41.3, C 41.3, C
15α 27.3, CH2 1.00 m 27.2, CH2 1.06 m 27.2, CH2 1.07 m
15β 1.52 m 1.52 m 1.50 m
16α 22.4, CH2 1.95 m 22.4, CH2 1.92 m 22.9, CH2 1.96 m
16β 1.60 m 1.56 m 1.47 m
17 46.2, C 45.9, C 46.0, C
18 40.7, CH 2.81 dd (12.4, 4.2) 41.2, CH 2.70 dd (12.9, 3.9) 41.1, CH 2.73 dd (13.2, 3.3)
19α 45.3, CH2 1.65 t (12.7) 45.4, CH2 1.66 t (13.1) 45.4, CH2 1.66 t (12.3)
19β 1.09 m 1.05 m 1.06 m
20 30.4, C 30.3, C 30.3, C
21α 33.2, CH2 1.34 m 33.2, CH2 1.33 m 33.0, CH2 1.39 m
21β 1.15 m 1.16 m 1.17 m
22α 31.6, CH2 1.62 m 31.4, CH2 1.62 m 31.6, CH2 1.64 m
22β 1.48 m 1.45 m 1.39 m
23 209.5, CHO 9.47 s 209.3, CHO 9.48 s 207.0, CHO 9.34 s
24 10.2, CH3 1.07 s 10.8, CH3 1.07 s 9.67, CH3 0.97 s
25 15.3, CH3 0.90 s 15.4, CH3 0.90 s 15.3, CH3 0.89 s
26 16.6, CH3 0.67 s 16.7, CH3 0.67 s 16.7, CH3 0.67 s
27 25.4, CH3 1.11 s 25.4, CH3 1.10 s 25.4, CH3 1.10 s
28 175.1, C 175.3, C 175.4, C
29 32.7, CH3 0.88 s 32.7, CH3 0.87 s 32.7, CH3 0.88 s
30 23.4, CH3 0.88 s 23.5, CH3 0.86 s 23.5, CH3 0.86 s
a
Overlapped signals are reported without designated multiplicity.

chemical shifts. The Qui unit, identified from anomeric signals at δH 5.32
and δC 92.8 (C-1 of Qui) with the methyl doublet at δH 1.05 (3H, d, J =
6.4 Hz), was identified as β-D-quinovopyranosyl unit (Qui) which was
substituted by Rha unit at C-2 based on the deshielding of C-2 (δC 75.1)
and the long-range proton-carbon correlations between the signals at δH
5.23 (H-1 of Rha) and δC 75.1 (C-2 of Qui) in the HMBC spectrum. And
the Qui uint was attached to C-28 of gypsogenin based the long-range
correlation observed in the HMBC experiment between signals at δH
5.32 (H-1 of Qui) and δC 175.3 (C-28 of gypsogenin). Therefore, the ester
chain of trisaccharide attached to C-28 of gypsogenin was identified as
β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovo­
pyranosyl unit. That the spectra data of disaccharide chain attached at C-
3 of gypsogenin were consistent with those of 1 indicated that the rest
structures of the sugar chains of 2 were same as those of 1, which was
also confirmed by the long-range proton-carbon correlations in the
HMBC spectrum between the signals at δH 4.09 (H-1 of GlcA) and δC 82.9
(C-3 of gypsogenin), δH 4.24 (H-1 of Gal) and δC 81.8 (C-2 of GlcA).
Compound 2 was elucidated as 3-O-β-D-galactopyranosyl-(1 → 2)-β-D-
glucuronopyranosyl gypsogenin 28-O-β-D-xylopyranosyl-(1 → 4)-α-L-
rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside.
In comparative analysis of the 1H-NMR and 13C-NMR data of 3 with
2, it was found that 3 has the same aglycone moiety as that of 2, that is
gypsogenin. One Xyl unit, Xyl2, starting from anomeric signals at δH 4.41
(H-1 of Xyl2) and δC 104.2 (C-1 of Xyl2), were identified to be in terminal
position. Another Xyl unit, Xyl1, starting from anomeric signals at δH
4.40 (H-1 of Xyl1) and δC 104.9 (C-1 of Xyl1), was substituted at C-3
based on the deshielding of C-3 of Xyl1 (δC 85.6) and Xyl2 was attached
to C-3 of the Xyl1 uint, based on the long-range correlations observed in
the HMBC experiment between signals at δH 4.41 (H-1 of Xyl2) and δC
85.6 (C-3 of Xyl1) as observed by their 13C-NMR chemical shifts. The
spectra data of Rha and Qui of 3 were consistent with those of 2 indi­
cated that the rest structures of the sugar chains of C-28 of gypsogenin of
3 were same as those of 2, which was also confirmed by the long-range
proton-carbon correlations in the HMBC spectrum between the signals at
δH 4.40 (H-1 of Xyl1) and δC 82.7 (C-4 of Rha), δH 5.26 (H-1 of Rha) and
δC 74.8 (C-2 of Qui), δH 5.31 (H-1 of Qui) and δC 175.4 (C-28 of gyp­
sogenin). Therefore, the tetrasccharide chain attached at C-28 of gyp­
sogenin was β-D-xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-
rhamnopyranosyl-(1 → 2)-β-D-quinovopyranosyl unit. The GlcA unit,
identified starting from anomeric signals at δH 4.04 (H-1 of GlcA) and δC
102.5 (C-1 of GlcA), were identified to be in terminal position and the
terminal GlcA was attached to C-3 of the gypsogenin, based on the long-
range correlations observed in the HMBC experiment between signals at
δH 4.04 (H-1 of GlcA) and δC 79.4 (C-3 of gypsogenin) as observed by
their 13C-NMR chemical shifts. Compound 3 was elucidated as 3-O-β-D-
glucuronopyranosyl gypsogenin 28-O-β-D-xylopyranosyl-(1 → 3)-β-D-
xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyra
noside.
In comparative analysis of the 1H-NMR and 13C-NMR data of 4 with
3, it was found that 4 has the same aglycone moiety as that of 3, that is

3
F. Li et al. Phytochemistry 190 (2021) 112870

Table 2
13
C (100 MHz) and 1H (400 MHz) NMR spectroscopic data for the aglycone moieties of 4–6 in DMSO.a
NO. 4 5 6

Position δC, type δH (J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz)

1α 37.6, CH2 1.01 m 37.6, CH2 0.97 m 37.6, CH2 1.02 m

1β 1.55 m 1.53 m 1.52 m
2α 24.1, CH2 1.90 m 24.1, CH2 1.96 m 24.1, CH2 1.91 m
2β 1.61 m 1.48 m 1.47 m
3 78.6, CH 3.86 dd (11.6, 4.0) 82.2, CH 3.68 dd (13.4, 4.2) 81.4, CH 3.72 dd (11.7, 4.4)
4 54.3, C 54.0, C 54.3, C
5 46.8, CH 1.26 overlapped 47.3, CH 1.26 overlapped 46.7, CH 1.28 overlapped
6α 19.6, CH2 1.37 m 19.7, CH2 1.37 m 19.0, CH2 1.37 m
6β 0.78 m 0.80 m 0.92 m
7α 31.4, CH2 1.40 m 31.8, CH2 1.38 m 31.7, CH2 1.39 m
7β 1.18 m 1.19 m 1.18 m
8 39.5, C 39.5, C 39.5, C
9 46.3, CH 1.56 d (12.2, 3.4) 45.9, CH 1.59 overlapped 44.9, CH 1.59 d (11.5, 3.8)
10 35.4, C 35.4, C 35.6, C
11α 22.4, CH2 1.53 m 22.8, CH2 1.59 m 22.4, CH2 1.63 m
11β 1.82 m 1.80 m 1.82 m
12β 121.5, CH 5.17 br.s 121.4, CH 5.21 br.s 121.3 CH 5.51 br.s
13 143.2, C 143.7, C 143.7, C
14 41.3, C 40.9, C 41.3, C
15α 27.2, CH2 1.09 m 34.6, CH2 1.10 m 34.7, CH2 1.13 m
15β 1.48 m 1.26 m 1.27 m
16α 22.4, CH2 1.96 m 73.6, CH 4.31 d (7.2) 73.5, CH 4.36 d (8.0)
16β 1.47 m
17 46.0, C 47.7, C 47.7, C
18 41.1, CH 2.73 dd (12.3, 4.1) 40.5, CH 2.83 dd (12.7, 3.4) 40.7, CH 2.96 dd (11.7, 4.4)
19α 45.4, CH2 1.66 t (13.5) 46.3, CH2 2.26 t (13.2) 45.1, CH2 2.28 t (12.1)
19β 1.06 m 0.98 m 1.05 m
20 30.3, C 30.1, C 30.0, C
21α 33.2, CH2 1.34 m 30.5, CH2 1.73 m 30.8, CH2 1.66 m
21β 1.17 m 1.29 m 1.24 m
22α 31.4, CH2 1.64 m 31.8, CH2 1.37 m 31.7, CH2 1.38 m
22β 1.34 m 1.20 m 1.24 m
23 207.0, CHO 9.35 s 209.5, CHO 9.51 s 209.5, CHO 9.45 s
24 9.6, CH3 0.97 s 10.1, CH3 1.10 s 10.0, CH3 1.07 s
25 15.3, CH3 0.89 s 15.4, CH3 0.90 s 14.9, CH3 0.86 s
26 16.7, CH3 0.66 s 16.6, CH3 0.64 s 16.3, CH3 0.88 s
27 25.5, CH3 1.10 s 26.3, CH3 1.31 s 26.6, CH3 1.29 s
28 175.4, C 175.1, C 174.7, C
29 32.7, CH3 0.87 s 32.8, CH3 0.82 s 32.5, CH3 0.86 s
30 23.5, CH3 0.86 s 24.1, CH3 0.90 s 23.9, CH3 0.91 s
a
Overlapped signals are reported without designated multiplicity.

gypsogenin. And for the spectra data of sugar moiety, the signal due to C-
3 of Xyl1 upfielded 8.7 ppm to δC 76.9, the signal due to C-3 of GlcA
downfielded 8.4 ppm to δC 85.7, and the rest of spectra data of the sugar
moiety of 4 are consistent with those of 3. One Xyl unit, Xyl1, starting
from anomeric signals at δH 4.40 (H-1 of Xyl1) and δC 104.9 (C-1 of Xyl1),
were identified to be in terminal position. The Rha unit, characteristic of
a methyl doublet at δH 1.11 (3H, d, J = 5.2 Hz) and a typical broad
singlet of anomeric proton at δH 5.29 (s) was identified to be a rham­
nopyranosyl unit with substitutions at C-4 based on the deshielding of C-
4 (δC 82.7), and Xyl1 was attached to C-4 of the Rha uint, based on the
long-range correlations observed in the HMBC experiment between
signals at δH 4.40 (H-1 of Xyl1) and δC 82.7 (C-3 of Rha) as observed by
their 13C-NMR chemical shifts. Another Xyl unit, Xyl2, starting from
anomeric signals at δH 4.42 (H-1 of Xyl2) and δC 104.3 (C-1 of Xyl2),
were identified to be in terminal position. The GlcA unit, starting from
anomeric signals at δH 3.99 (H-1 of GlcA) and δC 101.9 (C-1 of GlcA),
was substituted at C-3 based on the deshielding of C-3 of GlcA (δC 85.7)
and Xyl2 was attached to C-3 of the GlcA uint, based on the long-range
correlations observed in the HMBC experiment between signals at δH
4.42 (H-1 of Xyl2) and δC 85.7 (C-3 of GlcA) as observed by their 13C-
NMR chemical shifts. That the rest of spectra data of 4 were consistent
with those of 3 indicated that the rest structures of the sugar chains of 4
were same as those of 3, which was also confirmed by the long-range
proton-carbon correlations in the HMBC spectrum between the signals
at δH 3.99 (H-1 of GlcA) and δC 78.6 (C-3 of gypsogenin), δH 5.29 (H-1 of
Rha) and δC 75.1 (C-2 of Qui), δH 5.32 (H-1 of Qui) and δ C 175.4 (C-28
of gypsogenin). Compound 4 was elucidated as 3-O-β-D-xylopyranosyl-
(1 → 3)-β-D-glucuronopyranosyl gypsogenin 28-O-β-D-xylopyranosyl-
(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside.
In comparative analysis of the 1H-NMR and 13C-NMR data of agly­
cone moiety of 5 with 1, it was found that the signals assigned to C-15
(δC 27.3) and C-16 (δC 22.4) of gypsogenin disappeared; instead, two
signals assigned to C-15 (δC 34.6) and C-16 (δC 73.6) of quillaic acid
appeared in the 13C-NMR spectrum of 5, and the rest of spectra data of
the aglycone moiety of 5 are consistent with those of 1, indicated that
the aglycone section of 5 was quillaic acid. The comparison of the
spectroscopic data of 5 with quillaic acid indicated that this aglycone is
substituted at positions C-3 (δC 82.2) and C-28 (δC 175.1). The large
3
JH1-H2 coupling constants (H-1 of Fuc, d, J = 7.4 Hz) of the anomeric
protons indicated the β-configurations for β-D-fucopyranosyl moieties,
which were further confirmed by the 13C-NMR data of 5. One Xyl unit,
Xyl2, starting from anomeric signals at δH 4.40 (H-1 of Xyl2) and δC 104.2
(C-1 of Xyl2), were identified to be in terminal position. Another Xyl
unit, Xyl1, starting from anomeric signals at δH 4.43 (H-1 of Xyl1) and δC
104.7 (C-1 of Xyl1), was substituted at C-3 based on the deshielding of C-
3 of Xyl1 (δC 85.5) and Xyl2 was attached to C-3 of the Xyl1 uint, based on
the long-range correlations observed in the HMBC experiment between
signals at δH 4.40 (H-1 of Xyl2) and δC 85.5 (C-3 of Xyl1) as observed by
their 13C-NMR chemical shifts. One Rha unit, Rha1 characteristic of a
methyl doublet at δH 1.14 (3H, d, J = 6.1 Hz) and a typical broad singlet

4
F. Li et al. Phytochemistry 190 (2021) 112870

Table 3 Table 3 (continued )

13
C (100 MHz) and 1H (400 MHz) NMR spectroscopic data for the sugar moieties NO. 1 2 3
of compounds 1–3 in DMSO.a
60.2, α 3.46
NO. 1 2 3 CH2 m
Position δ C, δH(J in δC, δH (J in δC, δH (J in Hz) β 3.59
type Hz) type Hz) type m
3-O- GlcA GlcA GlcA Xyl2
sugers 104.2, 4.41 d (6.4)
1 101.5, 4.10 101.4, 4.09 102.5, 4.04 d (6.9) CH
CH d (7.2) CH d (7.6) CH 73.9, 3.08 m
2 81.6, 3.15 t 81.8, 3.15 t 74.6, 2.87 d (6.9) CH
CH (7.2) CH (7.6) CH 77.3, 3.36 m
3 76.5, 3.28 m 76.6, 3.33 m 77.3, 3.36 m CH
CH CH CH 67.6, 3.35 m
4 69.8, 3.51 m 69.7, 3.51 m 69.5, 3.51 m CH
CH CH CH 65.7, α 3.08
5 73.1, 3.28 m 73.1, 3.27 m 73.4, 3.41 m CH2 overlapped
CH CH CH β 3.74 m
6 172.7, 172.6, 171.8, a
Overlapped signals are reported without designated multiplicity.
C C C
Gal1 Gal
1 104.7, 4.23 104.7, 4.24 of anomeric proton at δH 5.10 (s) was identified to be a rhamnopyranosyl
CH d (6.1) CH d (6.3) unit with substitutions at C-4 based on the deshielding of C-4 (δC 82.2),
2 72.5, 3.29 t 72.5, 3.29 t
and the signals at δC 82.2 was identified as C-4 of the Rha1, based on the
CH (6.1) CH (6.3)
3 72.8, 3.29 m 72.8, 3.27 m long-range correlations observed in the HMBC experiment between
CH CH signals at δH 1.14 (3H of methyl) and δC 82.2 (C-4 of Rha1). And the Xyl1
4 67.7, 3.64 m 67.4, 3.64 m uint were attached to C-4 of the Rha1 uint, based on the long-range
CH CH correlations observed in the HMBC experiment between signals at δH
5 75.1, 3.36 m 75.1, 3.33 m
CH CH
4.43 (H-1 of Xyl1) and δC 82.2 (C-4 of Rha1) as observed by their 13C-
6 59.8, α 3.50 59.8, α 3.55 m NMR chemical shifts. The Fuc unit, identified from anomeric signals at
CH2 m CH2 δH 5.24 and δC 93.2 (C-1 of Fuc) with the methyl doublet at δH 1.05 (3H,
β 3.54 β 3.59 m d, J = 6.2 Hz), was identified as β-D-fucopyranosyl unit which was
m
substituted by Rha1 unit at C-2 based on the deshielding of C-2 (δC 73.5)
28-O- Ara Qui Qui
sugers and the long-range proton-carbon correlations between the signals at δH
1 91.7, 5.57 s 92.8, 5.32 92.8, 5.31 d (6.4) 5.10 (H-1 of Rha1) and δC 73.5 (C-2 of Fuc) in the HMBC spectrum. The
CH CH d (6.8) CH Fuc uint was attached to C-28 of quillaic acid based the long-range
2 73.1, 3.65 75.1, 2.89 t 74.8, 2.90 t (6.4) correlation observed in the HMBC experiment between signals at δH
CH d (7.2) CH (6.8) CH
3 65.0, 3.64 m 76.7, 3.26 m 76.2, 3.16 m
5.24 (H-1 of Fuc) and δC 175.1 (C-28 of quillaic acid). Therefore, the
CH CH CH ester chain of trisaccharide attached to C-28 of quillaic acid was iden­
4 68.3, 3.79 m 72.5, 3.42 m 73.3, 3.24 m tified as β-D-xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-
CH CH CH rhamnopyranosyl-(1 → 2)-β-D-fucopyranosyl unit. The Gal unit and
5 61.6, 3.37 m 72.1, 3.23 m 72.1, 3.24 m
Rha2 unit, identified starting from anomeric signals at δH 4.27 (H-1 of
CH2 CH CH
3.73 m 18.6, 1.05 18.5, 1.09 d (6.4) Gal) and δC 102.3 (C-1 of Gal), δH 4.98 (H-1 of Rha2) and δC 100.1 (C-1 of
CH3 d (6.4) CH3 Rha2) were identified to be in terminal position, respectively, as
Rha Rha Rha observed by its 13C-NMR chemical shifts. Starting from the anomeric
1 99.4, 4.86 s 99.3, 5.23 s 99.3, 5.26 s signals at δH 4.18 (d, J = 6.8 Hz) and δC 101.2, the GlcA unit was
CH CH CH
2 69.7, 3.83 dd 70.3, 3.72 dd 70.4, 3.72 dd
identified with its carbonyl C-6 at δC 172.8. Observation of proton-
CH (6.4, CH (5.7, CH (7.0, 1.6) proton correlations in the 1H–1H COSY spectrum between the anome­
1.8) 1.7) ric proton of GlcA (δH 4.18) and H-2 of GlcA (δH 3.40), and the proton-
3 81.6, 3.46 m 71.0, 3.62 m 71.8, 3.62 carbon correlations in the HSQC spectrum between H-2 of GlcA (δH
CH CH CH
3.40) and C-2 of GlcA (δC 81.7) suggested the signal at δC 81.7 was
4 71.3, 3.41 m 82.9, 3.35 m 82.7, 3.38
CH CH CH identified as C-2 of GlcA, and the signals at δC 81.2 was identified as C-3
5 68.3, 3.51 m 66.8, 3.60 m 66.7, 3.60m of the GlcA, based on the long-range correlations observed in the HMBC
CH CH CH experiment between signals at δH 4.18 (H-1 of GlcA) and δC 81.2 (C-3 of
6 17.6, 1.12 17.7, 1.11 17.7, 1.15 d (6.1) GlcA), and the proton-carbon correlations in the HSQC spectrum be­
CH3 d (6.1) CH3 d (5.2) CH3
Gal2 Xyl Xyl1
tween H-3 of GlcA (δH 3.43) and C-3 of GlcA (δC 81.2) suggested the
1 105.4, 4.32 105.7, 4.30 104.9, 4.40 d (6.8) signal at δC 81.2 was identified as C-3 of GlcA, and the deshielding of C-2
CH d (7.6) CH d (6.8) CH of GlcA (δC 81.7) and C-3 of GlcA (δC 81.2) indicated the glucuronic acid
2 71.8, 3.41 t 74.6, 3.00 t 74.8, 3.09 t (6.8) was substituted at C-2 and C-3. Observation of long-range proton-carbon
CH (7.6) CH (6.8) CH
correlations in the HMBC spectrum between the anomeric proton of
3 73.1, 3.15 m 77.3, 3.10 m 85.6, 3.35 m
CH CH CH GlcA (δH 4.18) and C-3 of quillaic acid (δC 82.2), between the anomeric
4 67.9, 3.64 m 69.3, 3.24 m 69.7, 3.30 m proton of Gal (δH 4.27) and C-2 of GlcA (δC 81.7) and between the
CH CH CH anomeric proton of Rha2 (δH 4.98) and C-3 of GlcA (δC 81.2) indicated a
5 65.1, 3.36 m 65.7, α 3.02 65.5, α 3.08 dd trisaccharide chain attached at C-3 of quillaic acid, β-D-galactopyr­
CH CH2 dd CH2 (14.4, 6.2)
anosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl
(14.2,
6.7) unit. Compound 5 was elucidated as 3-O-β-D-galactopyranosyl-(1 → 2)-
6 β 3.68 m β 3.74 m [α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl quillaic acid
28-O-β-D-xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-

5
F. Li et al.
Fig. 1. Chemical structures of Hylomeconoside L-O.
rhamnopyranosyl-(1 → 2)-β-D-fucopyranoside.
In comparative analysis of the 1H-NMR and 13C-NMR data of 6 with 5, it
was found that the spectra data of the aglycone moiety of 6 were same as
those of 5, which suggested that the aglycone of 6 is quillaic acid. And for
the spectra data of suger moiety, the signals belonging to the trisaccharide
chain attached at C-3 of quillaic acid (β-D-galactopyranosyl-(1 → 2)-[α-L-
rhamnopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl unit), the terminal
Xyl2 unit, the Xyl1 unit with substitutions at C-3 and the Rha unit with
substitutions at C-4 also appeared in the 13C-NMR spectrum of 6, but the
signals assigned to Fuc unit disappeared in the 13C-NMR spectrum of 6;
instead, one anomeric carbon signals (δC 93.2), the methylene carbon
6
Phytochemistry 190 (2021) 112870
signals (δC 59.9), and four oxygen-bearing methine carbon signals (δC 68.0,
71.8, 74.9, 76.0) appeared in the 13C-NMR spectrum of 6, which were
assigned to one Gal units together with the above conclusion. The addi­
tional Gal units (Gal2), identified starting from anomeric signals at δH 5.31
(H-1 of Gal2) and δC 93.2 (C-1 of Gal2) with the methylene signals at δH
3.42 and 3.48 (2H, H-6 of Gal2) and δC 59.9 (C-6 of Gal2), was substituted at
C-2 based on the deshielding of C-2 of Gal2 (δC 74.9), and the Rha1 unit was
attached to this position based on the long-range correlation observed in
the HMBC experiment between signals at δH 5.11 (H-1 of Rha1) and δC 74.9
(C-2 of Gal2). And the long-range correlation observed in the HMBC
experiment between signals at δH 5.31 (H-1 of Gal2) and δC 174.7 (C-28 of
F. Li et al.
Fig. 2. Chemical structures of Hylomeconoside P-Q.
quillaic acid) indicated that the Gal2 uint was attached to C-28 of quillaic
acid and the upperfielding anomeric carbon signal (δC 93.2) and down­
fielding anomeric proton signal (δH 5.31) suggested that the Gal2 uint was
attached to C-28 of quillaic acid through a ester glycosidic linkage. That the
rest of spectra data of 6 were consistent with those of 5 indicated that the
rest structures of the sugar chains of 6 were same as those of 5, which was
also confirmed by the long-range proton-carbon correlations in the HMBC
spectrum between the signals at δH 3.99 (H-1 of GlcA) and δC 81.4 (C-3 of
quillaic acid), δH 4.25 (H-1 of Gal1) and δC 81.7 (C-2 of GlcA), δH 5.00 (H-1
7
Phytochemistry 190 (2021) 112870
of Rha2) and δC 80.4 (C-3 of GlcA), δH 4.47 (H-1 of Xyl1) and δC 81.2 (C-4 of
Rha1), δH 4.42 (H-1 of Xyl2) and δC 85.5 (C-3 of Xyl1). Compound 6 was
elucidated as 3-O-β-D-galactopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1
→ 3)]-β-D-glucuronopyranosyl quillaic acid 28-O-β-D-xylopyranosyl-(1 →
3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-
galactopyranoside.
We have tested compounds 1–6 for cytotoxicity against six human
cancer cell lines (human lung cancer cell line (A549), human gastric cancer
cell line (AGS), human cervical cancer cell line (HeLa), human liver cancer
F. Li et al.
Fig. 3. Key HMBC and COSY correlations of aglycon and compounds 1–6.
cell line (Huh7), human colon cancer cell line (HT29) and human leukemic
cancer cell line (K562)), by using the MTT assay with topotecan as the
positive control. And cytotoxicity against those cell lines for Saponins 1–6
were selective (Table 5), among them, compound 1 was found to be the
most active on the HT29 cell lines (IC50 14.30 μM), and compound 5 was
found to be the most active on the Huh7 cell lines (IC50 18.23 μM).
8
Phytochemistry 190 (2021) 112870
3. Conclusions
Further phytochemical investigation of Hylomecon japonica afforded
six previously undescribed oleanane-type saponins (1–6). One distinct
feature of all these compounds is that they have two diverse chain
containing 2–4 pentoses or hexoses attached to C-3 and C-28. In addi­
tion, the cytotoxic activities of the isolated compounds were tested
F. Li et al. Phytochemistry 190 (2021) 112870

Table 4
13
C (100 MHz) and 1H (400 MHz) NMR spectroscopic data for the sugar moieties of compounds 4–6 in DMSO.a
NO. 4 5 6

position δC, type δH(J in Hz) δC, type δH (J in Hz) δC, type δH (J in Hz)
3-O-sugers GlcA GlcA GlcA
1 101.9, CH 3.99 d (7.2) 101.2, CH 4.18 d (6.8) 101.1, CH 3.99 d (6.4)
2 74.0, CH 2.85 t (7.2) 81.7, CH 3.40 t (6.8) 81.7, CH 3.42 t (6.4)
3 85.7, CH 3.55 m 81.2, CH 3.43 m 80.4, CH 3.40 m
4 69.5, CH 3.51 m 72.1, CH 3.14 m 73.0, CH 3.15 m
5 73.4, CH 3.42 m 77.8, CH 3.39 m 77.9, CH 3.40 m
6 172.6, C 172.8, C 172.6, C
Xyl2 Gal Gal1
1 104.3, CH 4.42 d (6.8) 102.3, CH 4.27 d (7.6) 102.4, CH 4.25 d (7.2)
2 73.7, CH 3.08 t (6.8) 72.3, CH 3.24 t (7.6) 72.1, CH 3.25 t (7.2)
3 76.2, CH 3.17 m 73.2, CH 3.24 m 72.3, CH 3.25 m
4 67.5, CH 3.35 m 67.7, CH 3.64 m 68.0, CH 3.66 m
5 75.7, CH2 α 3.08 m 75.1, CH 3.27 m 76.1, CH 3.35 m
6 β 3.74 m 59.8, CH2 α 3.47 m 59.6, CH2 α 3.48 m
β 3.57 m β 3.59 m
Rha2 Rha2
1 100.1, CH 4.98 s 100.1, CH 5.00 s
2 69.7, CH 3.75 d (5.9) 70.4, CH 3.72 d (6.4)
3 71.2, CH 3.71 m 71.4, CH 3.63 m
4 73.6, CH 3.17 m 73.2, CH 3.19 m
5 68.0, CH 3.60 m 68.0, CH 3.66 m
6 17.7, CH3 1.05 d (6.1) 17.7, CH3 1.05 d (6.3)
28-O-sugers Qui Fuc Gal2
1 92.8, CH 5.32 d (6.2) 93.2, CH 5.24 d (7.4) 93.2, CH 5.31 d (6.8)
2 75.1, CH 2.88 t (6.2) 73.5, CH 3.55 t (7.4) 74.9, CH 3.25 t (6.8)
3 77.4, CH 3.37 m 72.5, CH 3.28 m 71.8, CH 3.25 m
4 72.3, CH 3.24 m 71.3, CH 3.40 m 68.0, CH 3.96 m
5 72.1, CH 3.24 m 70.6, CH 3.60 m 76.0, CH 3.35 m
6 18.6, CH3 1.05 d (6.4) 16.2, CH3 1.05 d (6.2) 59.9, CH2 α 3.42 m
β 3.48 m
Rha Rha1 Rha1
1 99.2, CH 5.29 s 99.8, CH 5.10 s 99.6, CH 5.11 s
2 70.5, CH 3.72 dd (6.4, 1.7) 69.7, CH 3.75 dd (5.8, 1.9) 70.6, CH 3.72 dd (6.8, 1.7)
3 69.7, CH 3.62 m 70.3, CH 3.71 m 70.7, CH 3.63 m
4 82.7, CH 3.38 m 82.2, CH 3.38 m 81.2, CH 3.42 m
5 66.7, CH 3.61 m 67.0, CH 3.56 m 67.1, CH 3.61 m
6 17.7, CH3 1.11 d (5.2) 17.8, CH3 1.14 d (6.1) 17.9, CH3 1.11 d (5.7)
Xyl1 Xyl1 Xyl1
1 104.9, CH 4.40 d (6.8) 104.7, CH 4.43 d (7.8) 104.5, CH 4.47 d (7.2)
2 74.6, CH 3.11 t (6.8) 74.2, CH 3.07 t (7.8) 74.1, CH 3.09 t (7.2)
3 76.9, CH 3.04 m 85.5, CH 3.35 m 85.5, CH 3.36 m
4 69.7, CH 3.30 m 69.3, CH 3.30 m 69.8, CH 3.31 m
5 65.7, CH2 α 3.08 dd (14.3, 6.4) 65.5, CH2 α 3.09 dd (13.8, 6.7) 65.5, CH2 α 3.08 dd (13.2, 6.8)
β 3.74 m β 3.74 m β 3.73 m
Gal2 Xyl2 Xyl2
1 104.1, CH 4.35 d (7.6) 104.2, CH 4.40 d (6.8) 104.3, CH 4.42 d (7.1)
2 71.1, CH 3.40 t (7.6) 73.5, CH 3.07 t (6.8) 73.7, CH 3.09 t (7.1)
3 73.5, CH 3.20 m 76.1, CH 3.27 m 76.9, CH 3.15 m
4 67.9, CH 3.65 m 68.0, CH 3.65 m 67.1, CH 3.36 m
5 75.0, CH 3.32 m 65.7, CH2 α 3.09 overlapped 65.7, CH2 α 3.08 overlapped
β 3.74 m β 3.73 m
6 60.0, CH2 α 3.50 m
β 3.54 m
a
Overlapped signals are reported without designated multiplicity.

against A549, AGS, HeLa, Huh7, HT29 and K562 cell lines, and some of
these isolates showed selective inhibition on proliferation of tumor cells.
As a continuous study on saponin constituents of Hylomecon
Japonica, it was found that the preparative separation of oleanane-type
saponins from Hylomecon Japonica on macroporous resins, silica gel
columns and HPLC was achieved successfully, and shown to be very
favourable process. Comparing the cytotoxicity against K562 cell lines in
this reaserch with the previous results (Li et al., 2020), it was found that
Hylomeconoside H (Li et al., 2020) and Hylomeconoside M showed
apparent inhibition against K562 cell lines, which was because of the
structure of β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 →
2)-β-D-quinovopyranosyl on 28-C of sapogenin of them potentially. On
the other hands, it was found that compounds with structures that
β-L-arabinopyranosyl unit connected with 28-C of sapogenin was inac­
tive against K562 cell lines’ proliferation.
This reaserch represents an addition to the ongoing research on the
herb of Hylomecon Japonica, which may be helpful to understand the
constituents and pharmacological use of this plant and should continue
to clarify its mechanism.
4. Experimental
4.1. General experimental procedures
Optical rotations were recorded with a HORIBA SEPA-300 high-
sensitive polarimeter (Horiba Ltd, Kyoto, Japan). HRESI-MS were
recorded on a Bruker micro OTOF-Q II mass spectrometer (Bruker
Corporation, Bremen, Germany). NMR spectra were performed on an
AV-400 spectrometer (Bruker Corporation, Faellanden, Switzerland).
HPLC was carried on a Shi-madzu LC-10 A system equipped with SPD-10

9
F. Li et al. Phytochemistry 190 (2021) 112870

Table 5 EtOH, 40%EtOH, 45%EtOH) to obtain three main fractions represent H-

Cytotoxic Activity of Compound 1–6 against six human cancer cell lines. 1, H-2, H-3, respectively. H-1 was chromatographed on silica gel col­
compound IC50(μM), Mean ± RSD (%) umns eluted with CHCl3–MeOH-EtOAc–H2O (2:2.0:4:1) and two frac­
tions (H-1-1, H-1-2) were collected in sequential order. H-2 was
A549 AGS HeLa Huh7 HT29 K562
chromatographed on silica gel columns eluted with CHCl3–MeOH-
1 >50 21.52 >50 23.10 14.30 >50 EtOAc–H2O (2:1.8:4:1) and one main fractions (H-2-1) was collected. H-
± 2.2 ± 1.4 ± 1.9
2 >50 38.69 >50 >50 >50 24.04
3 was chromatographed on silica gel columns eluted with CHCl3–MeOH-
± 1.5 ± 1.2 EtOAc–H2O (2:1.5:4:1) and two fractions (H-3-1, H-3-2) were collected
3 >50 >50 >50 >50 >50 >50 in sequential order. After dissolving the fractions in MeOH–H2O (1:1 v/
4 >50 >50 >50 >50 >50 >50 v) (10 mL), separately, fractionation was carried out using a semi­
5 18.23
>50 >50 >50 >50 >50
preparative HPLC C18 column with a flow rate of 3 mL/min and solvents
± 2.4
6 >50 >50 >50 >50 >50 >50 consisting of CH3CN as solvent A and 0.1% formic acid in H2O (v/v) as
topotecan 76.95 >100 71.90 23.81 >100 5.95 ± solvent B. All semipreparative HPLC analysis was carried out by UV
± 1.5 ± 2.3 ± 1.2 1.2 detection at a wavelength of 206 nm. Separation of H-1-1 was conducted
A549 = human lung cancer cell line; AGS = human gastric cancer cell line; HeLa with the following solvent gradient: 0 min, 67% B; 40 min, 67% B;
= human cervical cancer cell line; Huh7 = human liver cancer cell line; HT29 = Accordingly, three subfractions, H-1-1-1 to H-1-1-3, were collected, in
human colon cancer cell line; K562 = human leukemic cancer cell line. which 1 (24.20 mg, tR 26.8 min) was afforded in H-1-1-3. Further sep­
aration of H-1-2 using the solvent gradient: 0 min, 67% B; 40 min, 67% B
A detector (Shimadzu Corporation, Kyoto, Japan) and a Gemini 5 μm was carried out, yielding in subfractions H-1-2-1 to H-1-2-2, saponin 2
C18 110 A column (250 mm × 10.00 mm, 5 μ m, Phenomenex, Torrance, (17.00 mg, tR 33.9 min) in fraction H-1-2-2. Chromatography of fraction
CA, USA). GC were measured on an Agilent 7820 A gas chromatograph, H-2-1 was achieved by applying the following gradient: 0 min, 64% B;
with a quartz capillary column (30 mm × 0.32 mm × 0.25 μm, Agilent 30 min, 63% B, obtaining fractions H-2-1-1 to H-2-1-4 with 3 (23.70 mg,
Technologies Inc, Santa Clara, CA, USA); detection, FID. Column chro­ tR 18.1 min) in H-2-1-1 and 4 (17.50 mg, tR 25.3 min). Separation of H-3-
matography was performed with silica gel (200–300 mesh, Qingdao 1 was analyzed with the following solvent gradient: 0 min, 62% B; 30
Marine Chemical Inc., Qingdao, China), D101 porous polymer resin min, 62% B. Accordingly, subfractions, H-3-1-1 to H-3-1-2, were
(Tianjin Pesticide Co., LTD., Resin Branch, Tianjin, China), HP20 porous collected, in which 5 (22.40 mg, tR 27.9 min) was afforded in H-3-1-2.
polymer resin (Tianjin Pesticide Co., LTD., Resin Branch, Tianjin, Separation of H-3-2 was analyzed with the following solvent gradient: 0
China). TLC analysis were carried out using precoated silica gel GF254 min, 62% B; 25 min, 62% B. Accordingly, subfractions, H-3-2-1 to H-3-2-
plates (Qingdao Haiyang Chemical Inc., Qingdao, China), and spots 2, were collected, in which 6 (24.50 mg, tR 18.9 min) was afforded in H-
were observed under UV254 light and sprayed by 10% H2SO4–EtOH re­ 3-2-1.
agent. HPLC grade solvents used for HPLC analysis and preparation were
purchased from Fisher Scientific International (Fair Lawn, NJ, USA). 4.3.1. Saponin 1
Other chemicals and reagents of analytical grade were from Beijing White amorphous powder; [α]D25 = +3◦ (c 0.5, pyridine); 1H-NMR
Chemical Works (Beijing, China). The cytotoxic activity were measured (400 MHz, DMSO) and 13C-NMR (100 MHz, DMSO); see Tables 1 and 3
on a DNM-9602 enzyme immunoassay spectrophotometer (Beijing, HRESI-MS, m/z: 1249.5818 [M + H]+ (calcd for C59H93O28,
China). DMEM medium was purchased from HyClone (Logan, UT, USA), 1249.5848).
RPMI-1640 medium was purchased from HyClone (Logan, UT, USA),
IMDM medium was purchased from Gibco (Invitrogen, UT, USA), F12–K 4.3.2. Saponin 2
medium was purchased from Gibco (Invitrogen, UT, USA), Mccoy’s 5A White amorphous powder; [α]D25 = +22◦ (c 0.5, pyridine); 1H-NMR
medium was purchased from Gibco (Invitrogen, UT, USA), PBS from (400 MHz, DMSO) and 13C-NMR (100 MHz, DMSO); see Tables 1 and 3
HyClone (Logan, UT, USA), FBS from BI (Biological Industries, Israel), HRESI-MS, m/z: 1233.5877 [M + H]+ (calcd for C59H93O27,
XTT (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-car­ 1233.5899).
boxanilide) and phenazine metho-sulphate from Sigma-Aldrich
Shanghai Trading Co. Ltd. (Shanghai, China). 4.3.3. Saponin 3
White amorphous powder; [α]D25 = − 15◦ (c 0.5, pyridine); 1H-NMR
(400 MHz, DMSO) and 13C-NMR (100 MHz, DMSO); see Tables 1 and 3
4.2. Plant material HRESI-MS, m/z: 1203.5745 [M + H]+ (calcd for C58H91O26,
1203.5793).
Plants of Hylomecon Japonica (Thunb.) Prantl & Kundig (Papaver­
aceae) were collected in the Changchun District of Jilin Province, China, 4.3.4. Saponin 4
(43 48′ 49′′ N 125 19′ 02′′ E) during May to July 2016. They were iden­ White amorphous powder; [α]D25 = − 8◦ (c 0.5, pyridine); 1H-NMR
tified by Jing-min Zhang of the School of Pharmaceutical Sciences, Jilin (400 MHz, DMSO) and 13C-NMR (100 MHz, DMSO); see Tables 2 and 4
University. A voucher specimen (No.2015062001) is deposited at the HRESI-MS, m/z: 1203.5732 [M + H]+ (calcd for C58H91O26,
Herbarium of the School of Pharma-ceutical Sciences, Jilin University. 1203.5793).

4.3. Extraction and isolation 4.3.5. Saponin 5

The air-dried whole Hylomecon japonica plants (10 kg) were extrac­
ted with 50% EtOH (3 × 120 L, 48 h each) at room temperature. The
extraction solution was concentrated under reduced pressure at 50 ◦ C to
give residue, which was dissolved in water and then filtered and passed
through a D101 polyporous resin column chromatography and eluted
successively with H2O, 30% EtOH, 50% EtOH, 70% EtOH and 95%
EtOH. The fraction obtained by elution with 50% EtOH (the crude
saponin extracts) was chromatographed on HP20 macroporous absor­
bent resin, eluting with gradient EtOH/H2O (25%EtOH, 30%EtOH, 35%
White amorphous powder; [α]D25 = +18◦ (c 0.5, pyridine); 1H-NMR
(400 MHz, DMSO) and 13C-NMR (100 MHz, DMSO); see Tables 2 and 4
HRESI-MS, m/z: 1527.6770 [M + H]+ (calcd for C70H111O36,
1527.6850).
4.3.6. Saponin 6
White amorphous powder; [α]D25 = +12◦ (c 0.5, pyridine); 1H-NMR
(400 MHz, DMSO) and 13C-NMR (100 MHz, DMSO); see Tables 2 and 4
HRESI-MS, m/z: 1543.6696 [M + H]+ (calcd for C70H111O37,
1543.6673).

10
F. Li et al.
4.4. Acid hydrolysis of saponins
Compounds 1–6 (5 mg) were hydrolyzed in 2 N CF3COOH (10 mL) at
100 ◦ C for 8 h. After extraction with EtOAc (3 × 10 mL), the aqueous
layer and EtOAc layer were collected separately (Shi et al., 2006). The
aqueous layer was evaporated with MeOH repeatedly under vacuum to
remove the solvent completely, the residue was dissolved in anhydrous
pyridine (0.100 mL), and a pyridine solution with L-cysteine methyl
ester hydrochloride (2 mg/mL, 0.100 mL) mixed. After stirring at 60 ◦ C
for 1 h. Hexamethyldisilazine (0.100 mL) and trimethylsilyl chloride
(0.040 mL) were added, and the mixture was heated at 60 ◦ C for another
0.5 h. The supernatant was dried under a stream of nitrogen and
resolved in n-hexane and water (0.1 mL each), and the hexane layer (1
μL) was analyzed by GC analysis under the following conditions: column
temp 50–280 ◦ C at 3 deg/min, carrier gas N2 (1 mL/min), injector temp
280 ◦ C, detector temp 300 ◦ C, split ratio 1:10 (Wulf et al., 1996; Stefan
et al., 2009). The following retention times were observed for the cor­
responding derivatives: D-galactose 19.691 min, D-glucuronic acid
21.157 min, L-rhamnose 17.589 min, L-arabinose 17.449 min, D-xylose
18.104 min, D-quinovose 17.970 min, D-fucose 18.145 min.
4.5. MTT cytotoxicity assay
Cytotoxic activity assessment was carried out for human cancer cell
lines (A549, AGS, HeLa, Huh7, HT29 and K562) by the MTT method
described in previous literatures (Spiro et al., 1999; Matthew et al.,
2009; Xu et al., 2013; Sara et al., 2018; Zhang et al., 2018). Topotecan
was used as the positive control. The experiments were repeated three
times. IC50 > 50 μM was considered to be inactive.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the National Key R&D Program of China
(No. 2018YFC1706105). The HRESI-MS were measured by the College
of Chemistry, Jilin University. The 1D-NMR and 2D-NMR were
measured by Changchun Institute of Chemistry, Chinese Academy of
Sciences.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2021.112870.
Author agreement
All authors have seen and approved the final version of the manu­
script being submitted. They warrant that the article is the authors’
original work, hasn’t received prior publication and isn’t under
consideration for publication elsewhere.
Funding source declaration
This research was funded by the National Key R&D Program of
11
Phytochemistry 190 (2021) 112870
China, grant number 2018YFC1706105.
Permission note
Permission has been received to use any material in the manuscript.
References
Cheng, H.Y., Cheng, J.X., Wei, H., Bai, J.Q., 2011. The research progress of Hylomecon
japonica. J. ShaanxiColl. Tradit. Chin. Med. 34, 94–95.
Ghezala, G., Tomofumi, M., Abdolhossein, R., Véronique, L., Marie-Aleth, L.D., 2000.
Two new biologically active triterpene saponins from acanthophyllum squarrosum.
J. Nat. Prod. 63, 1497–1502. https://doi.org/10.1021/np000212+.
Ghezala, G., Tomofumi, M., Mohammad, R., Marie-Aleth, L.D., 2004. Glandulosides A-D,
triterpene saponins from acanthophyllum glandulosum. J. Nat. Prod. 67,
1114–1118. https://doi.org/10.1021/np040001v.
Hikaru, O., Tsuneatsu, N., Shizuko, H., Tatatsuo, Y., 1989. Studies on the constituents of
Luffa operculata Cogn. II. Isolation and structure elucidation of saponins in the herb.
Chem. Pharm. Bull. 37, 895–900. https://doi.org/10.1248/cpb.37.18.
Ivone, L.A.C., Laurence, V.N., Huong, D.T.M., Nathalie, R., Naima, B., Dima, M.,
ElisabetH, L.M.D., Sophie, C.G., Marc, L., Thierry, S., Nguyen, V.H., Catherine, L.,
2011. Triterpenoid saponins from symplocos lancifolia. J. Nat. Prod. 74, 163–168.
https://doi.org/10.1021/np100502y.
Kim, Y.C., Higuchi, R., Komori, T., 1992. Application of hydrothermolysis to the studies
on the constituents of the merck saponin. Liebigs Ann. Chem. 92, 941–946. https://
doi.org/10.1002/jlac.1992199201155.
Li, F., Wu, S.T., Qu, M.H., Wang, Y.X., Ma, C.L., Yu, B.H., Wang, G.S., 2020. Triterpenoid
saponins from the herb Hylomecon japonica. Phytochemistry 181, 112542–112553.
https://doi.org/10.1016/j.phytochem.2020.112542.
Matthew, J.K., Mary, L.T., Mac, M., Stacey, L.T., Roxanne, P., John, T., Jiang, Z.H., 2009.
Structural analogues of diosgenyl saponins: synthesis and anticancer activity. Bioorg.
Med. Chem. 17, 7670–7679. https://doi.org/10.1016/j.bmc.2009.09.046.
Qu, Y.F., Gao, J.Y., Wang, J., Geng, Y.M., Zhou, Y., Sun, C.X., Li, F., Feng, L., Yu, M.J.,
Wang, G.S., 2017. New triterpenoid saponins from the herb Hylomecon japonica.
Molecules 22, 1731–1740. https://doi.org/10.3390/molecules22101731.
Sara, J.C., Rafael, G.B., Ana, J.A., Rocío, R.A., Sergio, L., 2018. In vitro toxicity of
Asparagus saponins in distinct multidrug-resistant colon cancer cells. Chem.
Biodivers. 15, e1800282 https://doi.org/10.1002/cbdv.201800282.
Shi, S., Jiang, D., Dong, C.X., Tu, P.F., 2006. Triterpene saponins from clematis
mandshurica. J. Nat. Prod. 69, 1591–1595. https://doi.org/10.1021/np060287z.
Spiro, M.K., HansjÖrg, E., Martin, R.B., 1999. BCR-ABL influences the antileukaemic
efficacy of alkylphosphocholines. Br. J. Haematol. 107, 365–374. https://doi.org/
10.1046/j.1365-2141.1999.01700.x.
Stefan, W., Andrey, P., Tarja, T., Juergen, P., Christiane, L., Anna, S., Bodo, S., Kati, U.,
Helena, S., Jarl, H., Bjarne, H., 2009. Carbohydrate analysis of plant materials with
uronic acid-containing polysaccharides–A comparison between different hydrolysis
and subsequent chromatographic analytical techniques. Ind. Crop. Prod. 29,
571–580. https://doi.org/10.1016/j.indcrop.2008.11.003.
The Editorial Board of Zhong Hua Ben Cao of State Administration of Traditional Chinese
Medicine of the People’s Republic of China, 1999. Zhong Hua Ben Cao, first ed., vol.
3. Shanghai Science and Technology Publishing House, Shanghai, China, p. 653.
Voutquenne-Nazabadioko, L., Gevrenova, R., Borie, N., Harakat, D., Sayagh, C.,
Weng, A., Thakur, M., Zaharieva, M., Henry, M., 2013. Triterpenoid saponins from
the roots of Gypsophila trichotoma Wender. Phytochemistry 90, 114–127. https://
doi.org/10.1016/j.phytochem.2013.03.001.
Wu, L.J., Lou, H.X., Zhou, J., 2012. Natural Medicinal Chemistry, sixth ed. People’s
Medical Publishing House, Beking, China, p. p99.
Wulf, A., Martin, V.C., Georg, G., 1996. Determinaion of neutral and acidic sugars in soil
by capillary gas-liquid chromatography after trifluoroacetic acid hydrolysis. Soil
Biol. Biochem. 28, 1631.
Xu, M.Y., Lee, D.H., Joo, E.J., Son, K.H., Kim, Y.S., 2013. Akebia saponin PA induces
autophagic and apoptotic cell death in AGS human gastric cancer cells. Food Chem.
Toxicol. 59, 703–708. https://doi.org/10.1016/j.fct.2013.06.059.
Yoshikawa, K., Arihara, S., Wang, D.J., Narui, T., Okuyama, T., 1991. Structures of two
new fibrinolytic saponins from the seed of Luffa cylindrica. Roem. Chem. Pharm.
Bull. 39, 1185–1188. https://doi.org/10.1248/cpb.39.1185.
Zhang, X., Zou, L.H., He, Y.L., Peng, C., Guo, L., Xiong, L., 2018. Triterpenoid saponins
from the buds of Lonicera similis. Nat. Prod. Res. 32, 2282–2290. https://doi.org/
10.1080/14786419.2017.1408092.