New 23, 27-dihydroxy-oleanane-type triterpenoid saponins from Anemone Raddeana Regel

Article abstract of DOI:10.1080/14786419.2019.1634708

Two new 23, 27-dihydroxy-oleanane-type triterpenoid saponins named Raddeanoside Rf and Raddeanoside Rg (1 and 2), along with thirteen known triterpenoid saponins (3–15) were isolated from the rhizome of Anemone raddeana Regel. Their structures were determined by chemical and spectral analysis, including 1 D, 2 D NMR data and HRESIMS. The type of aglycone 23, 27-dihydroxy oleanolic acid is extremely rare in natural products. In addition, the anti-cancer activity for all the compounds were evaluated. Compounds 9 and 10 exhibited significant cytotoxicity with IC₅₀ values of 4.47 and 8.97 μM against human pancreatic cancer lines (PANC-1), while compound 6 with IC₅₀ value of 8.19 μM against human lung lines (A549). The possible structure-activity relationships of these triterpenoid saponins were also tentatively discussed.

Full text of DOI:10.1080/14786419.2019.1634708

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
New 23, 27-dihydroxy-oleanane-type triterpenoid
saponins from Anemone Raddeana Regel
Chongning Lv, Ying Zhao, Bin Zhao, Ling Han & Jincai Lu
To cite this article: Chongning Lv, Ying Zhao, Bin Zhao, Ling Han & Jincai Lu (2019): New 23, 27-
dihydroxy-oleanane-type triterpenoid saponins from AnemoneꢀRaddeana Regel, Natural Product
Research, DOI: 10.1080/14786419.2019.1634708
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1634708
New 23, 27-dihydroxy-oleanane-type triterpenoid
saponins from Anemone Raddeana Regel
a
a
a
b
a
Chongning Lv , Ying Zhao , Bin Zhao , Ling Han and Jincai Lu
a
Department of Pharmaceutical Botany and Authentication of TCM, School of Traditional Chinese
b
Materia Medica, Shenyang Pharmaceutical University, Shenyang, China; Research and Development
Center, NERC for the Pharmaceutics of Traditional Chinese Medicines, Benxi, China
ABSTRACT
ARTICLE HISTORY
Two new 23, 27-dihydroxy-oleanane-type triterpenoid saponins
named Raddeanoside Rf and Raddeanoside Rg (1 and 2), along
with thirteen known triterpenoid saponins (3–15) were isolated
from the rhizome of Anemone raddeana Regel. Their structures
were determined by chemical and spectral analysis, including 1 D,
Received 21 February 2019
Accepted 18 June 2019
KEYWORDS
Anemone raddeana Regel;
triterpenoid saponins;
cytotoxicity
2
D NMR data and HRESIMS. The type of aglycone 23, 27-dihy-
droxy oleanolic acid is extremely rare in natural products. In add-
ition, the anti-cancer activity for all the compounds were
evaluated. Compounds 9 and 10 exhibited significant cytotoxicity
with IC50 values of 4.47 and 8.97 lM against human pancreatic
cancer lines (PANC-1), while compound 6 with IC50 value of
8
.19 lM against human lung lines (A549). The possible structure-
activity relationships of these triterpenoid saponins were also ten-
tatively discussed.
1. Introduction
The genus Anemone consists about 150 species distributed throughout the world.
About 17 species of Anemone in China are traditional herbal medicines (Editorial
Committee of the Flora of China 1980; Zhang et al. 2019; Zhou et al. 2007).
CONTACT Jincai Lu
jincailu@126.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1634708.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
C. LV ET AL.
Liangtoujian, which is recorded in pharmacopeia of the People’s Republic of China, is a
well-known traditional Chinese medicine for the treatment of rheumatism and pain. It
is derived from the dried rhizome of Anemone raddeana Regel (National
Pharmacopoeia Committee 2015). Modern research has proved that A. raddeana has
various pharmacological action such as anti-inflammatory, antipyretic and antitumor
(Guan et al. 2015; Lu et al. 2009). Phytochemical research showed triterpenoid saponin
was the major bioactive components of A. raddeana (Zhang et al. 2017). Some of
them have marked antitumor activity, such as Pulsatilla Saponin D (Jang et al. 2014)
and Pulsatilla saponin A (Tong et al. 2017).
Therefore, it is necessary to conduct a more in-depth study of the anticancer activ-
ities of triterpenoid saponin in A. raddeana. As part of a continuous investigation, two
new 23,27-dihydroxy oleanolic acid-type compounds named Raddeanoside Rf and
Raddeanoside Rg (1 and 2), together with thirteen known ones (3–15) were isolated
and elucidated. It is worth noting that the type of aglycone 23, 27-dihydroxy oleanolic
acid is extremely rare in natural products and the methyl group at C-27 oxidation to
hydroxymethyl group is considered as an impossible reaction due to steric hindrance
and reactive selectivity. The anti-cancer activity for all the compounds were evaluated.
Compounds 6, 9 and 10 exhibited significant cytotoxicity (Table S1).
2. Results and discussion
The rhizome of A. raddeana afforded two new compounds (1–2) and thirteen known
compounds (3–15) (Figure 1) by repeated column chromatography and preparative
reversed-phase high-performance liquid chromatography, as described in experimental
section. The known compounds were identified by comparison of experimental and
reported spectroscopic data as Mateglycoside D (3) (Tiwari and Singh 1978),
Eleutheroside K (4) (Liao et al. 2001), Hederacholichiside E (5) (Liao et al. 1999),
Hederacolchiside A1 (6) (Li et al. 2018), oleanolic acid 3-O-b-D-glucopyranosyl(1!4)-
a-L-arabinopyranoside (7) (Li et al. 1990), Raddeanoside 22 (8) (Fan et al. 2010),
Raddeanoside 23 (9) (Fan et al. 2010), oleanolic acid 3-O-b-D-glucopyranosyl(1!3)-a-L-
rhamnopyranosyl(1!2)[b-D-glucopyranosyl(1!4)]-a-L-arabinopyranoside (10) (Lv et al.
2
016), Pulsatiloside D (11) (Liao et al. 1999), Leontoside B (12) (Li et al. 1990),
Leontoside A (13) (Li et al. 1990), Raddeanoside 20 (14) (Lu et al. 2009), Raddeanoside
2 (15) (Lu et al. 2002). The new triterpenoids were identified by comprehensive spec-
1
troscopic analyses (1 D and 2 D NMR, HRESIMS). The sugar residues were identified by
HPLC analysis after hydrolysis.
Compound 1 was isolated as a white, amorphous powder. The positive results of
Molish and Liebermann-Burchard test suggested that 1 might be a triterpenoid glyco-
side or a steroidal glycoside. The HRESIMS (negative ion mode) showed the protonated
ꢀ
pseudo-molecular ion peak at m/z 827.4470 [M þ HCOOH-H] (calcd. for C H O ,
42 67 16
1
8
27.4429), corresponding to the molecular formula C H O . The H NMR spectrum
41 66 14
(
Table S2) exhibited five angular methyl groups at d 1.03, 1.02, 0.91, 0.89 and 0.88. The
H
1
3
C NMR spectrum (Table S2) showed 41 carbon signals, of which 30 were the aglycone.
Compared with hederagenin (23-hydroxy oleanolic acid), the angular methyl single C-27
d 26.0) disappeared and a single at d 65.2 appeared (Wang et al. 2014). The data
(
C
C

NATURAL PRODUCT RESEARCH
3
Figure 1. Structures of compounds 1–15.
hinted that C-27 may be a hydroxymethyl group. The above data showed that the agly-
cone of compound 1 maybe 23, 27-dihydroxy oleanolic acid. The structure was further
confirmed in HMBC spectrum by the correlation (Figure S1). The long-range correlations
were observed between H -23 (d 4.24, 3.66, d, J ¼ 10.9Hz) of aglycone and C-3 (d 83.
2
H
C
2
), C-4 (d 44.2), C-5 (d 48.2) of the aglycone. Meanwhile, the correlations were
C C
observed between H -27 (d 3.82, 4.09, d, J ¼ 12.1 Hz) of aglycone and C-8 (d 41.2), C-
2
H
C
1
3 (d 141.0), C-14 (d 48.7), and C-15 (d 24.7) of the aglycone. The above data con-
C C C
firmed the aglycone of compound 1 was 23,27-dihydroxy oleanolic acid.
On acid hydrolysis of compound 1, L-arabinose (L-Ara) and D-glucose (D-Glc) were
identified by comparison with retention time of their standard sugar derivatives on
1
1
HPLC. Meanwhile, according to the 2 D NMR ( H- H COSY, HSQC, HMBC and TOCSY)
spectra, an arabinose unit (d 107.1, 74.0,75.0, 80.4 and 67.1) and a glucose unit (d
C
C

4
C. LV ET AL.
1
07.1, 76.2, 78.7, 72.0 79.1 and 63.2) were confirmed. On the base of terminal proton
signals and anomeric carbons, the sugar moieties were determined as b for D-gluco-
pyranose and a-configuration for L-arabinopyranose (Pei et al. 2011).
The correlations between the anomeric signal at d 4.94 (Ara H-1) and d 83.2 (C-3) of
H
C
the aglycone, revealed a substitution at C-3 of the aglycone by an arabinose unit.
Meanwhile, a long-range correlation between d 5.17 (Glc H-1) and d 80.4 (Ara C-4) indi-
H
C
1
13
cated that the (1!4) linkage between these two sugars. Detailed analysis of the H, C,
1
1
H- H COSY, TOCSY, HSQC, HMBC and DEPT data indicated that Glc and Ara are unambigu-
ously supported the glyosidic linkages. Hence, compound 1 was elucidated as 23,27-dihy-
droxy oleanolic acid 3-O-b-D-glucopyranosyl(1!4)-a-L-arabinopyranoside, and it was
named Raddeanoside Rf.
Compound 2 was obtained as a white, amorphous powder. The negative ion mode
-
HRESIMS of 2 showed the pseudo-molecular ion at m/z 827.4479 [M þ HCOOH-H]
(
calcd. for C H O , 827.4429), in accordance with the molecular formula C H O .
42 67 16 41 66 14
13
By comparison of the C NMR signals of 2 with those of 1 (Table S2), the aglycone
was consistent with 23,27-dihydroxy oleanolic acid. It should be noted that there is a
13
missing of chemical shift in C NMR of C-28 for compound 2. It’s a common phenom-
13
enon that sometimes there’s no response with quaternary carbons in the C NMR.
However, the chemical shift of C-28(181.7) can be easily confirmed clearly with the
help of HMBC correlation. A long-range correlation between dH 1.75 (C-16)) and dC
1
81.7 (C-28) was observed. With the same method as compound 1, the sugar units
were confirmed as one a-L-arabinopyranose and one b-D-glucopyranose unit. In the
HMBC experiment, the long-range correlation between the anomeric proton at d 5.26
H
(
Glc H-1) and the carbon at d 80.5 (Ara C-2) indicated that the glucose (Glc) was
C
attached to position 2 of the arabinose. Detailed analysis of the data indicated that
Glucose and Arabinose are unambiguously supported glyosidic linkages (Figure S1).
Hence, compound 2 named Raddeanoside Rg was elucidated as 23,27-dihydroxy
oleanolic acid 3-O-b-D-glucopyranosyl(1!2)-a-L-arabinopyranoside.
All the isolated compounds in present word were tested for their inhibitory activity
against three human cancer cell lines by MTT method (Table S1). Among them, com-
pounds 9 and 10 exhibited significant cytotoxicity with IC50 values of 4.47 and 8.
97 lM against human pancreatic cancer lines (PANC-1), while compound 6 with IC50
value of 8.19 lM against human lung lines (A549). The inhibitory effects of all the
monomers on MDA-MB-231 cells were weaker than those of the other two tumor cells.
Only compounds 6, 9 and 10 showed moderate activity to MDA-MB-231 cells with the
values of 21.35, 28.52, and 26.65 lM, respectively. The results hinted that the sensitiv-
ity of different tumor cell lines to the above monomer was quite different.
The aglycone of all the isolated compounds could be divided into four types
according to hydroxymethyl substitution position (Figure 1), 23,27-dihydroxy oleanolic
acid (type A), oleanolic acid (type B), 23-hydroxy oleanolic acid (type C) and 27-
hydroxy oleanolic acid (type D). The saponins with the aglycone of type B displayed
stronger cytotoxic activity than those possessing the same sugar chain but the other
type of aglycone (Table S1 and Figure 1). The presence of hydroxyl group at C-23
(type C) or C-27 (type D) would reduce the activity, especially hydroxyl group at C-27.

NATURAL PRODUCT RESEARCH
5
When both existed at the same time (type A), activities were further weakened or
even disappeared.
The cytotoxicities of above saponin were also closely related to their sugar moiety.
Comparing the anti-proliferative activity of compounds 3 and 5 with 4 and 6, respect-
ively, we hypothesized that the free carboxyl groups at C-28 played an important role
for cytotoxic activity. The results are consistent with literature (Wang, et al. 2014).
Compounds 6 and 9 possessing the Rha(1!2)[Glc(1!4)]Ara had a better activity than
diglycosidic compounds 4 and 7. Meanwhile they showed stronger activity than com-
pound 8 which linked a Glc(1!2)[Glc(1!4)]Ara. These implied that the presence of
rhamnopyranose linked at C-2 or glucopyranose linked at C-4 of the arabinpyranosyl
group was propitious to enhance the cytotoxic activity. According to the data of 4, 6,
9
and 10, we confirmed that the extension of Rha(1!2)Ara group was important for
antitumor activity. It’s in accordance with the report of Raddeanin A (Ma et al. 2018)
and Pulsatilla Saponin D (Chen et al. 2018).
3. Experimental section
3.1. General
Optical rotations were measured on a MCP200 polarimeter (Anton Paar, Graz, Austria).
IR spectra were obtained on a Shimadzu ftir-8400s spectrophotometer (Shimadzu
Corporation, Tokyo, Japan). HRESIMS experiments were performed on a Micro TOF
spectrometer (Bruker Co., Karlsruhe, Germany). NMR spectra were recorded at 600 MHz
1
13
for H and 150 MHz for C on Bruker AV-600 instruments (Bruker Co., Billerica, MA,
USA). Preparative HPLC was conducted using a Shimadzu LC-10A instrument with an
SPD-10A detector (Shimadzu, Kyoto, Japan) and YMC-Pack ODS-A column (250 mm ꢁ
20 mm, 5 mm). HPLC method was conducted using an Agilent 1260 (Agilent
Technologies, Inc., Santa Clara, USA) with DAD detector. A Thermo ODS-2 Hypersil
analytical column (250 mm ꢁ 4.6 mm, 5 lm, Thermo Scientific Co., USA) was used.
2-Methylphenyl Isothiocyanate and L-cysteine methyl ester hydrochloride were pur-
chased from Sigma Co. (Shanghai, China). All of the cell lines, including human pancre-
atic cancer lines (PANC-1), human lung lines (A549) and human breast
adenocarcinoma (MDA-MB-231) cell lines were obtained from the American Type
Culture Collection (ATCC). All the medium was purchased from Gibco company
(Shanghai, China).
3.2. Plant material
The rhizome of Anemone raddeana Regel was collected in Liaoning Province (China)
and authenticated by Prof. Qishi Sun (Department of Medicinal Plant, Shenyang
Pharmaceutical University). A voucher specimen (No. 001875) was deposited in the
Herbarium of Shenyang Pharmaceutical University.

6
C. LV ET AL.
3.3. Extraction and isolation
Air-dried and powered rhizome of A. raddeana (4.0 kg) was refluxed with 75% EtOH
for three times. The combined alcohol extracts were concentrated under reduced pres-
sure to afford a residue (520 g), which was suspended in H O (10 L) and partitioned
2
successively by petroleum ether (10 L ꢁ 3), chloroform (10 L ꢁ 3), EtOAc (10 L ꢁ 3), and
n-BuOH (15 L ꢁ 3). The n-butanol fraction (210 g) was eluted with EtOAc-MeOH-H O
2
(10:1:0-30:10:5 gradient system) to afford eight fractions (B1–B8). Fr.B4 (38.2 g) was
separated by silica gel column chromatography (7.5 ꢁ 60 cm) with Dichlone-MeOH-H O
2
as the mobile phase (7:1:0.1-6:4:0.5) to afford eight subfractions Fr.B4-1-Fr.B4-8. Fr.B4-1
(
20 mg) was further purified by a preparative HPLC using MeOH: H O (82:18) to yield
2
compound 13 (4 mg, t 102.6 min). Fr.B4-2 was purified by a preparative HPLC using
R
MeOH: H O (85:15) to yield compounds 15 (10 mg, tR 47.0 min) and 4 (30 mg, tR
2
1
52.4 min). Compound 7 (22 mg, t 122.6 min) was obtained from Fr.B4-3 (52 mg) by a
R
preparative HPLC eluted with MeOH: H O (85:15). Fr.B4-4-1 (60 mg) was further puri-
2
fied by a preparative HPLC using MeOH: H O (74:26) to yield compounds 1 (8 mg, tR
2
6
1
7.1 min), 2 (7 mg, t 77.6 min) and 3 (5 mg, t 214.4 min). Compound 12 (106 mg, t
R R R
04.1 min) was obtained from Fr.B4-4-2 (300 mg) by a preparative HPLC eluted with
MeOH: H O (78:22). Compound 6 (400 mg, t 127.6 min) was obtained from Fr.B4-5
2
R
(
650 mg) by a preparative HPLC eluted with MeOH: H O (82:18). Fr.B4-6 (300 mg) was
2
purified by a preparative HPLC using MeOH: H O (82:18) to yield compound 14
2
(
171 mg, t 43.7 min). Fr.B4-7 (180 mg) was eluted by a preparative HPLC using MeOH:
R
H O (77:23) to yield compounds 8 (29 mg, t 214.9 min), 9 (45 mg, t 236.2 min), 10
2
R
R
(
7 mg, t 253.6 min), and 11 (25 mg, t 98.1 min). Fr.B4-8 (104 mg) was purified by a
R
R
preparative HPLC using MeOH: H O (72:28) to yield compound 5 (20 mg, t 77.1 min).
2
R
3
.3.1. Raddeanoside Rf (1)2
0
ꢀ1
White amorphous solid; ½aꢂ þ52.7 (c 0.125, MeOH); IR (KBr): v
(cm ): 3435, 2945,
max
D
1
13
1638, 1075; H and C NMR (C D N, 600 MHz and 150 MHz) data, see Table S2;
5 5
ꢀ
1
HRESIMS m/z 827.4470 [M þ HCOOH-H] (calcd. for C H O , 827.4429).
42 67 16
3
.3.2. Raddeanoside Rg (2)2
0
ꢀ1
White amorphous solid; ½aꢂ þ34.8 (c 0.125, MeOH); IR (KBr) v
(cm ): 3427, 2943,
D
max
1
13
1641, 1072. H and C NMR (C D N, 600 MHz and 150 MHz) data, see Table S2;
5 5
ꢀ
1
HRESIMS m/z 827.4479 [M þ HCOOH-H] (calcd. for C H O , 827.4429).
42 67 16
3.4. Determination of the absolute configuration of the sugars
Compounds 1 and 2 (2.0 mg) were hydrolyzed with 2 M HCl (5.0 mL), heated for 6 h at
ꢃ
9
0 C and extracted with CHCl (3 ꢁ 5.0 mL). Then, the aqueous layer was concentrated
3
in vacuo and concentrated to dryness to give a residue, which was dissolved in pyri-
dine (1.0 mL), and then L-cysteine methyl ester hydrochloride (3.0 mg) was added to
the solution. The mixture was heated at 60 C for 1 h, then 2-Methylphenyl
ꢃ
ꢃ
Isothiocyanate was added to the reaction mixture and further reacted at 60 C for
6
2
0 min. The reaction product was analyzed by HPLC and detected by UV detector (at
50 nm). The reaction mixture was analyzed with a Thermo ODS-2 Hypersil analytical

NATURAL PRODUCT RESEARCH
7
column. The elution was performed by a gradient system of water-phosphoric acid as
solvent A (1000:0.5) and acetonitrile as solvent B. The gradient program was carried
out as follows: 20–40% B in 0–50 min. The flow rate was kept 1 mL/min and the col-
ꢃ
umn temperature was at 30 C. The sugar parts from compounds 1 and 2 were com-
pared to an authentic sugar sample: 15.70 min (L-glucose); 16.62 min (D-glucose);
17.75 min (L-arabinose); 18.63 min (D-arabinose).
3.5. Cytotoxicity assay
Cytotoxic effects of all compounds against PANC-1, A549, and MDA-MB-231 cell lines
were measured by MTT assay (Han et al. 2017). The cells were seeded into 96-well cul-
3
ture plates (Costar, USA) at a density of 6 ꢁ 10 cells per well. After overnight incuba-
tion, the cells were treated with various concentrations of compounds for 72 h. Then
2
3
1
0 lL MTT solution was added to each well and incubated for an additional 4 h at
7 C. The supernatant was removed by centrifugation, followed by the addition of
50 lL DMSO. The absorbances at 570 nm were measured for the cells using Thermo
ꢃ
microplate reader. The IC50 values of the test compounds were calculated with SPSS
9.0 software.
1
4. Conclusion
In present work, two new 23, 27-dihydroxy-oleanane-type triterpenoid saponins
named Raddeanoside Rf and Raddeanoside Rg (1 and 2), along with 13 known olea-
nane-type triterpenoid saponins (3–15) were isolated and identified from the rhizome
of Anemone raddeana Regel. In addition, the anti-cancer activity for all the compounds
were evaluated. Compounds 6, 9 and 10 exhibited significant cytotoxicity. Meanwhile,
the possible structure-activity relationships of these triterpenoid saponin compounds
were also tentatively discussed.
Disclosure statement
The authors declare no conflicts of interest.
Funding
This work was supported by the National Natural Science Foundation of China (No. 81373900),
the Young and Middle-aged Key Talent Training Program of Shenyang Pharmaceutical
University (ZQN2018013) and the fourth national survey on Chinese materia medica resources in
Liaoning Province (2018018).
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