Two new triterpenoid-chromone hybrids from the rhizomes of Actaea cimicifuga L. (syn. Cimicifuga foetida L.) and their cytotoxic activities

Article abstract of DOI:10.1080/14786419.2020.1775228

Two new triterpenoid-chromone hybrids, cimitriteromones H (1) and I (2), along with two known analogues (3, 4) were isolated from the phytochemical research on the n-butyl alcohol extracts of Actaea cimicifuga rhizomes. The new compounds were elucidated by spectroscopic experiments and chemical method. The cytotoxic activities of the isolated compounds were tested on A-549/Taxol cell line. Cimitriteromone I (2) showed cytotoxicity with IC₅₀ value of 27.14 ± 1.38 μM comparable to positive control group cisplatin (IC₅₀ value of 25.80 ± 1.15 μM).

Full text of DOI:10.1080/14786419.2020.1775228

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Two new triterpenoid-chromone hybrids from the
rhizomes of Actaea cimicifuga L. (syn. Cimicifuga
foetida L.) and their cytotoxic activities
Qiang-Qiang Shi, Ya Gao, Jing Lu, Lin Zhou & Ming-Hua Qiu
To cite this article: Qiang-Qiang Shi, Ya Gao, Jing Lu, Lin Zhou & Ming-Hua Qiu (2020): Two new
triterpenoid-chromone hybrids from the rhizomes of Actaeaꢀcimicifuga L. (syn. Cimicifugaꢀfoetida L.)
and their cytotoxic activities, Natural Product Research, DOI: 10.1080/14786419.2020.1775228
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1775228
Two new triterpenoid-chromone hybrids from the
rhizomes of Actaea cimicifuga L. (syn. Cimicifuga foetida
L.) and their cytotoxic activities
a,b,c
a,b,c
a,b,c
a,b
a,b,c
Qiang-Qiang Shi , Ya Gao , Jing Lu , Lin Zhou and Ming-Hua Qiu
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of
b
Botany, Chinese Academy of Sciences, Yunnan, China; University of the Chinese Academy of
Sciences, Beijing, China; Yunnan Key Laboratory of Natural Medicinal Chemistry Chinese Academy
c
of Sciences, Yunnan, China
ABSTRACT
ARTICLE HISTORY
Two new triterpenoid-chromone hybrids, cimitriteromones H (1)
and I (2), along with two known analogues (3, 4) were isolated
from the phytochemical research on the n-butyl alcohol extracts
of Actaea cimicifuga rhizomes. The new compounds were eluci-
dated by spectroscopic experiments and chemical method. The
cytotoxic activities of the isolated compounds were tested on A-
Received 14 August 2019
Accepted 17 May 2020
KEYWORDS
Actaea cimicifuga;
Triterpenoid-chromone
hybrid; Cytotoxicity
549/Taxol cell line. Cimitriteromone I (2) showed cytotoxicity with
IC50 value of 27.14 ± 1.38 lM comparable to positive control group
cisplatin (IC50 value of 25.80 ± 1.15 lM).
1. Introduction
Plants of the genus Actaea comprise about 18 species of perennial herbs distributed
among the temperate zone of the Northern Hemisphere (Editorial Committee of Flora
of China CAS 1979). Phytochemical and biological studies over the past several deca-
des revealed that triterpenoid glycosides, chromones and cinnamic acid derivatives
were the major constituents of this genus (Li and Yu 2006). Those of them were
thought to be the major bioactive constituents of functional foods (Agretti et al. 2018)
and alternative medicines (Qiu et al. 2012; Sun et al. 2017), including anti-proliferation
CONTACT Ming-Hua Qiu
mhchiu@mail.kib.ac.cn
State Key Laboratory of Phytochemistry and Plant Resources
in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan, China.
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1775228.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
Q.-Q. SHI ET AL.
Figure 1. Chemical structures of compounds 1ꢀ4.
(
Nian et al. 2010; Ahn et al. 2013), anti-inflammatory (Thao, Kim, et al. 2017; Thao,
Luyen, et al. 2017), anti-osteoporotic (Li et al. 2007; Qiu et al. 2007; Kolios et al.
010; Ahn et al. 2012), anti-angiogenic (Nian et al. 2015) and relieving the symptoms
2
of the menopause (Mahady et al. 2002, 2010; Wuttke et al. 2003; He et al. 2006;
Byron 2007). In our previous work, LC-UV/MS guided separation method was used to
analyse the extraction of Actaea cimicifuga and seven undescribed hybrid dimers of
cycloartane triterpenoid and chromone fused via a glucose moiety, cimitriteromones
A ꢀ G were isolated from the ethyl acetate extracts (Shi et al. 2018). And, cimitritero-
mones C and E showed potential anti-proliferative activities against taxol-resistant
human lung cancer A-549/taxol with IC50 values of 15.73 and 24.21 lM, respectively.
So, further phytochemical investigation on the n-butyl alcohol extracts of A. cimici-
fuga led to the isolation of three more hybrid dimers of cycloartane triterpenoid and
chromone, including two new ones (Figure 1). Herein, we report the structure deter-
mination by 1D/2D NMR analyses and cytotoxic activities against A-549/Taxol of the
isolated compounds.
2. Results and discussion
Cimitriteromone H (1) was obtained to be a white, amorphous powder, and its
molecular formula was C H O determined by HRESIMS ion signal at m/z
5
9 86 21
ꢀ
1
129.5588 [M ꢀ H] (calcd for 1129.5589) with 17 degrees of unsaturation. Clearly,
1 13
the H NMR and C-DEPT spectra of 1 displayed the characteristic signals of 9,19-
cycloartane-type triterpene moiety and chromone moiety (Shi et al. 2018). Direct

NATURAL PRODUCT RESEARCH
3
comparison of its NMR data (Tables 1S and Tables 2S, Supplementary material) with
compound 3 (Shi et al. 2018) suggested compound 1 resemble 3 except for the
signals for the side-chain and glucose moiety change a lot. The acid hydrolysis and
GC analysis experiments suggested that the D-glucose and D-xylose units occurred
1
1
in 1. The H- H COSY associations of d 5.94 (OH-24, 1H, d, J ¼ 9.7 Hz) with d 3.75
H
H
(
H-24, 1H, m), and the HMBC correlations of dH 6.27 (OH-23, 1H, s) to d 41.9 (C-
C
22), dC 101.9 (C-23) and dC 77.7 (C-24) indicated that the epoxy bond between C-
23 and C-24 was substituted with two hydroxyl groups. This elucidation was also
supported by 18 atomic units (H O) more of the molecular weight in compound 1.
2
The attachment of the two moieties could be observed by the HMBC correlation
between proton signal at dH 4.40 (glu-6b, 1H, m) and quaternary carbon at d 81.3
C
(
C-25). Thus, the planar structure of 1 was assigned and further verified by a com-
1
1
prehensive analysis of the HSQC, H- H COSY and HMBC spectra (Figure 1S,
Supplementary material).
The significant ROESY associations of H-3/H-5, H-12/H-17/CH -28, H-16/H-17/CH -28
3
3
suggested the b-orientation of the substituents at C-3, C-12, C-16. The a-oriented
hydroxyl group at C-23 was determined by the ROESY association of OH-23 (d 6.27,
H
1
H, s) with the H-16 (d 4.77, 1H, m) (Figure 1S, Supplementary material). In addition,
H
the stereo structure of C-24 was proposed as ‘R’ configuration on the basis of the
cleavage pattern of the epoxy ring catalysed by the epoxide hydrolases (Archer 1997)
(Figure 3S, Supplementary material). Accordingly, the structure of 1 was characterised
as shown (Figure 1).
Cimitriteromone I (2) was assigned to the molecular formula C H O with 18
59
84 20
ꢀ
degrees of unsaturation, as established by HRESIMS (m/z 1111.5486 [M ꢀ H] , calcd for
1
13
1
1
1
111.5483). Based on the H, C-DEPT, H- H COSY, HSQC, HMBC NMR data, the inte-
gral structure of 2 was closely related to cimifoetidanoside G (4) (the configuration of
C-24 was not confirmed) (Chen et al. 2014) and (16S,20S,24R)-12b-acetoxy-16,23-
epoxy-24,25-dihydroxy-3b-(b-D-xylopyranosyloxy)-9,19-cyclolanost-22(23)-ene (4) (the
configuration of C-24 was confirmed as R based on reduced density gradient calcula-
tion) (Fang et al. 2019) with an additional chromone moiety. The HMBC correlation of
glu-6b (d 4.36, 1H) with C-25 (d 78.2) indicated the connection of the two moieties
H
C
(Figure 2S, Supplementary material). The relative configuration of compound 2 was
determined as 3b, 12b, 16b, 24 R on the basis of ROESY experiment (Figure 2S,
Supplementary material) and comprehensive comparison of the chemical shifts of
compounds 2, 4, and cimifoetidanoside G with that of (16S,20S,24R)-12b-acetoxy-
16,23-epoxy-24,25-dihydroxy-3b-(b-D-xylopyranosyloxy)-9,19-cyclolanost-22(23)-ene
(Tables 3S).
The chromone glycoside moiety of compounds 1 and 2 was identical to that of
prim-O-glucosylcimifugin (Sasaki et al. 1982), as evidenced by comparison of the 1 D
1
1
NMR data, and further established on the basis of 2D NMR correlations ( H- H COSY,
HMBC, and HSQC) (Figures 1S and 2S, Supplementary material). The isolated com-
pounds were evaluated against taxol-resistant human lung cancer A-549/taxol for their
cytotoxicities, using the MTS methods with cisplatin and taxol as positive controls.
Compound 2 showed anti-proliferative activity with IC50 value of 27.14 ± 1.38 lM com-
parable to the positive control cisplatin (IC50 25.80 ± 1.15 lM).

4
Q.-Q. SHI ET AL.
3. Experimental
3
.1. General experimental procedures
All of the 1D and 2D NMR spectra were obtained on Bruker Ascend
00 MHz spectrometers in pyridine-d with TMS as an internal standard. HRESIMS data
TM
600 and
8
5
were measured on a Waters API QSTAR Pulsar spectrometer. Optical rotation was
obtained in methanol with a JASCO P-1020 digital polarimeter. UV spectra were meas-
ured using a Shimadzu UV-2401PC spectrometer. A Bruker Tensor-27 Fourier trans-
formed infrared spectrometer was used to scan IR spectra with KBr pellets. Column
chromatography (CC) was performed on Silica gel (200 ꢀ 300 mesh, Qingdao Marine
Chemical, Ltd.), Lichroprep RP-18 (40 ꢀ 75 lm, Merck), and Sephadex LH-20
(
20 ꢀ 150 lm, Pharmacia). Semi-preparative high performance liquid chromatography
was performed on an Agilent 1260 liquid chromatography system equipped with an
SB-PHENYL column (5 lm, 9.4 mm ꢁ 250 mm, 3 mL/min) and a DAD detector.
3.2. Plant material
Rhizomes of Actaea cimicifuga L. (syn. Cimicifuga foetida L.) were collected from Lijiang
County, Yunnan Province, China, in August 2014. The material was identified by Prof.
Shengji Pei, Kunming Institute of Botany, Chinese Academy of Science. A Voucher spe-
cimen (KUN No. 20140828) has been deposited at the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Kunming, China.
3.3. Extraction and isolation
The air-dried rhizomes of A.cimicifuga L. (15 kg) were milled and extracted with 70%
ꢂ
aqueous methanol (3 ꢁ 50 L, 2 hours each) at 60 C. The solvent was evaporated in
vacuo to afford a crude extracts (1 kg), which was suspended in H O, and then
2
extracted successively with EtOAc and n-BuOH. The n-BuOH extracts (50 g) were frac-
tionated by RP-18 (MeOH/H O 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) to yield five frac-
2
tions, Fr.A ꢀ E. Fr.C (MeOH/H O 70:30, 5 g) was further fragmented by a silica gel
2
column (CHCl /MeOH/H O 9:1:0.1, 8:2:0.2, 7:3:0.5 and 6:4:1) and yield fractions C1 ꢀ C4.
3
2
Fraction C2 (2 g) was separated by RP-18 with a gradient elution of MeOH/H O (60:40
2
to 80:20) to give five sub-fractions, Fr.C2.1 ꢀ C2.5. Fraction C2.2 (500 mg) was purified
by Sephadex LH-20 (MeOH), followed by semi-preparative HPLC (3 mL/min, detector
UV kmax 210, 254, 285 and 360 nm, MeCN/H O 37:63) to afford 3 (2 mg with purity
2
over 95%, 15.7 min). Fraction C3 (1.3 g) was fragmented by Sephadex LH-20 to give
two parts (Fr.C3.1 and C3.2), Fr.C3.2 was further subjected on a silica gel column
(
CHCl /MeOH/H O 8.5:1.5:0.15) to obtain fractions C3.2.1 to C3.2.3. Fraction C3.2.1 was
3 2
further purified by HPLC (3 mL/min, detector UV kmax 210, 254, 285 and 360 nm,
MeCN/H O 60:40)to obtain compound 4 (17.7 mg with purity over 95%, 15.3 min).
2
Compounds 1 (7.7 mg with purity over 95%, 27.3 min) and 2 (3.3 mg with purity over
95%, 30.5 min) were yield from Fraction C3.2.2 using semi-preparative HPLC (3 mL/min,
detector UV kmax 210, 254, 285 and 360 nm, MeCN/H O 40:60).
2

NATURAL PRODUCT RESEARCH
5
3.3.1. Cimitriteromone H (1)
White, amorphous powder; [a] þ 32.5 (c 0.38, MeOH); UV (MeOH) kmax (log e) 215.0
(
1
0.65), 243.5 (0.49), 294.0 (0.31); IR (KBr) ꢀ
383, 1075, 1047, 565 cm ; H and C NMR data (pyridine-d ) (see Supplementary
3429, 2966, 2935, 1717, 1656, 1629, 1469,
max
ꢀ
1
1
13
5
ꢀ
material Tables 1S and Tables 2S); HRESIMS m/z [M ꢀ H] calcd for C H O
59 85 21
1
129.5589, found at 1129.5588.
3.3.2. Cimiriteromone I (2)
White, amorphous powder; [a] ꢀ 81.1 (c 0.10, MeOH); UV (MeOH) kmax (log e) 211.0
(
1
0.54), 241.5 (0.30), 293.0 (0.18); IR (KBr) ꢀ
384, 1077, 1046, 566 cm ; H and C NMR data (pyridine-d ) (see Tables 1S and
3440, 2966, 2934, 1725, 1656, 1630, 1468,
max
ꢀ
1
1
13
5
ꢀ
Tables 2S, Supplementary material); HRESIMS m/z [M ꢀ H] calcd for C H O
59 83 20
1
111.5483, found at 1111.5486.
3.4. Cytotoxicity bioassay
Drug-resistant human lung cancer A-549/Taxol was used in the cytotoxic assay.
The cell viability was assessed by MTS method in 96-well micro plates (Monks
et al. 1991). 100 lL of adherent cells was seeded in 96-well plate and allowed to
be adhere for 12 h before test compounds addition. The tumor cell line was
exposed to the tested compounds at various concentrations of 0.064, 0.32, 1.6, 8,
and 40 lM in triplicate for 48 h, with cisplatin (Sigma) as positive control. After
the incubation, cell viability was detected and a cell growth curve was graphed.
IC50 values were calculated by Reed and Muench’s method (Reed and
Muench 1938).
3.5. Hydrolysis and identification of the sugar moieties in compound 1
The hydrolysis experiment was conducted as we described previously (Shi et al.
ꢂ
2
018). Briefly, compound 1 (2.5 mg) was hydrolysed with 1.0 N HCl (2 mL) at 90 C
for 2 h. After neutralising with 1.0 N NaOH, the reaction mixture was extracted by
CHCl , and the aqueous layer was evaporated under reduced pressure to give the
3
residue of monosaccharide. The residue was dissolved in pyridine (150 ll) contain-
ing (L)-cysteine methyl ester hydro chloride (1.5 mg), and the mixture was heated
ꢂ
at 80 C for 1.5 h. Then, the trimethylsilylation reagents HMDS (100 mL) and TMCS
ꢂ
(
50 mL) were added successively, and the mixture was heated at 60 C for 30 min.
The reaction product was extracted by n-hexane (1 mL), and the upper layer was
subjected to gas chromatography (GC) analysis under the following conditions:
ꢂ
carrier gas, N ; constant flow 1.2 mL/min; The GC oven was set at 120 C to
2
2
ꢂ ꢂ ꢂ ꢂ
70 C, 8 C/min. Transfer line temperature: 200 C; injector temperature, 250 C;
injector volume, 1 mL; and split ratio, 1/30. The sugar moieties of compound 1
corresponding to those of D-xylose (tR 6.45, 7.31 min) and D-glucose (tR
1
1.28, 12.46 min).

6
Q.-Q. SHI ET AL.
4. Conclusions
Further phytochemical investigation on the rhizomes of Actaea cimicifuga led to the
acquisition of three triterpenoid-chromone hybrids, including two new ones. The new
compound 2 showed anti-proliferative activity against taxol-resistant human lung can-
cer A-549/Taxol with IC50 value of 27.14 lM comparable to positive control group cis-
platin (IC50 value of 25.80 lM).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by Autonomous Deployment Project of Kunming Institute of Botany,
CAS under Grant No. KIB2017010; Programs of National Natural Science Foundation of China
under Grant No. U1132604 and 81302670); and The Major Program of CAS under Grant No.
KSZD-EW-Z-004-01.
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