Two new steroidal saponins from the roots of Cynanchum limprichtii

Article abstract of DOI:10.1080/10286020.2017.1405939

As a part of our continuing research for bioactive constituents from Cynanchum limprichtii Schltr., two new C₂₁ steroidal glycosides limproside A (1) and limproside B (2) were isolated from the roots of Cynanchum limprichtii. Their structures were elucidated on the basis of 1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS analysis. The cytotoxicity of two compounds against two selected human cancer cell lines was assayed.

Full text of DOI:10.1080/10286020.2017.1405939

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Two new steroidal saponins from the roots of
Cynanchum limprichtii
Jia-Chuan Liu, Li-Li Yu, Ming-Xu Tang, Xiao-Jie Lu, Dan Zhao, Hai-Feng Wang,
Gang Chen, Guang-Yue Su & Yue-Hu Pei
To cite this article: Jia-Chuan Liu, Li-Li Yu, Ming-Xu Tang, Xiao-Jie Lu, Dan Zhao, Hai-
Feng Wang, Gang Chen, Guang-Yue Su & Yue-Hu Pei (2017): Two new steroidal saponins
from the roots of Cynanchum limprichtii, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2017.1405939
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Date: 02 December 2017, At: 20:46

Journal of asian natural Products research, 2017
https://doi.org/10.1080/10286020.2017.1405939
Two new steroidal saponins from the roots of Cynanchum
limprichtii
Jia-Chuan Liua,b,c, Li-Li Yuc, Ming-Xu Tanga,b, Xiao-Jie Lua,b, Dan Zhaoa,b
,
Hai-Feng Wanga,b, Gang Chena,b, Guang-Yue Sud and Yue-Hu Peia,b
aschool oftraditional chinese Materia Medica, shenyang Pharmaceutical university, shenyang 110016, china;
bKey laboratory of structure-Based drug design & discovery, Ministry of education, shenyang Pharmaceutical
c
university, shenyang 110016, china; college of Basic science, Jinzhou Medical university, Jinzhou 121001,
china; dschool of functional food and Wine, shenyang Pharmaceutical university, shenyang 110016, china
ABSTRACT
ARTICLE HISTORY
received 20 May 2017
accepted 13 november 2017
As a part of our continuing research for bioactive constituents from
Cynanchum limprichtii Schltr., two new C21 steroidal glycosides
limproside A (1) and limproside B (2) were isolated from the roots of
Cynanchum limprichtii. Their structures were elucidated on the basis of
1D- and 2D-NMR spectroscopic data as well as HR-ESI-MS analysis. The
cytotoxicity of two compounds against two selected human cancer
cell lines was assayed.
KEYWORDS
asclepiadaceae; Cynanchum
limprichtii; steroidal
glycoside; limproside;
cytotoxicity
1. Introduction
Plants of the genus Cynanchum belong to the family Asclepiadaceae, and their roots con-
tain steroidal glycosides with various biological activities such as anti-tumor, immunosup-
pressive, antidepressant, neuroprotective, antivirus, and appetite- suppressing effects [1].
CONTACT Guang-Yue su
suggyy@163.com; Yue-hu Pei
peiyueh@vip.163.com
supplemental data for this article can be accessed https://doi.org/10.1080/10286020.2017.1405939
© 2017 informa uK limited, trading as taylor & francis Group

2
J.-C. LIU ET AL.
In previous studies, essential oils [2], flavonoids, alkaloids, terpenes, and steroidals were
isolated from this genus [3]. Chemical investigation revealed that they contain charac-
teristic C21 steroidal glycosides, especially with the normal four-ring C21 steroid skeleton
or the aberrant 13, 14:14, 15-disecopregnane-type skeleton or 14, 15-secopregnane-type
skeleton [4]. As a kind of characteristic and bioactive constituents, more than 200 C21 ste-
roidal glycosides have been isolated from Cynanchum species [5]. As part of our continuing
chemical investigation of the steroidal constituents of Cynanchum plants, a phytochemical
screening of the roots of Cynanchum limprichtii was performed. We report the discovery
of two steroidal glycosides limproside A (1) and limproside B (2) (Figure 1). Extensive
spectroscopic studies were conducted to determine the structures of the new compounds,
including two-dimensional NMR, HR-ESI-MS, and hydrolytic cleavage followed by chro-
matographic and spectroscopic analysis. Additionally, the cytotoxicity of two compounds
was evaluated against selected human cancer cell lines, including HL-60 (human leukemic
promyelocytic cell) and Caco-2 (human Caco-2 colon cancer cell).
2. Results and discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula was deter-
mined as C40H58O15 by its pseudo molecular ion peak at m/z 801.3618 [M + Na]+ in the
positive HR-ESI-MS experiment, indicating 12 degrees of unsaturation. e IR spectrum
showed the absorption bands for hydroxyl (3439 cm−1), carbonyl (1655 cm−1) and olefinic
1
(1632 cm−1) groups. e H NMR spectrum of aglycone portion exhibited two tertiary
methyl protons at δH 1.18 (3H, s, H-19) and 1.61 (3H, s, H-21), one olefinic proton signal
at δH 6.31 (1H, brs, H-6), two oxygen-substituted methine protons at δH 3.80–3.84 (1H, m,
H-3) and 4.24 (1H, dd, J = 7.0, 3.4 Hz, H-16), and two methylene groups at δH 3.73 (1H, dd,
J = 10.4, 3.4 Hz, Hα-15) and 4.31 (1H, d, J = 10.4 Hz, Hβ-15), and at δH 3.89 (1H, d, J = 9.3 Hz,
Hα-18) and 5.22 (1H, d, J = 9.3 Hz, Hβ-18). e 13C NMR spectrum showed two oxygenated
tertiary carbon signals at δC 105.5 (C-14) and 118.3 (C-20), four olefinic carbon signals at
δC 164.7 (C-5), 126.3(C-6), 130.8 (C-8), and 162.1 (C-9), one conjugated ketonic carbonyl
carbon signal at δC 186.8 (C-7). Comparison of 1H and 13C NMR data (Table 1) with those
of stauntoside G [6], a known steroidal glycoside isolated from the roots of C. stauntonii,
revealed that compound 1 contained a same aglycone. ree anomeric protons and carbon
signals at δH 4.86 (1H, d, J = 9.6 Hz, H-1′)/98.1 (C-1′), 5.23 (1H, d, J = 9.3 Hz, H-1″)/99.6
Figure 1. the structures of compounds 1 and 2.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. 1h and 13c nMr spectral data of compounds 1 and 2 in c5d5n (at 400 and 100 Mhz, resp.).
1
2
Position
δC
δH(J Hz)
δC
δH(J Hz)
1
2
3
4
5
6
7
8
33.5
28.8
76.7
1.92–1.94 (m), 1.21–1.25 (m)
2.15–2.18 (m), 1.81–1.85 (m)
3.80–3.84 (m)
32.7
29.9
75.4
39.5
73.1
137.7
133.4
76.2
52.3
42.2
20.0
35.6
58.6
111.5
73.8
83.6
69.5
77.0
19.0
118.7
23.2
2.08–2.12 (m),1.60–1.63 (m)
2.21–2.25 (m),1.87–1.91 (m)
4.71–4.74 (m)
38.7
2.80 (brdd, 14.1, 2.2), 1.94–1.96 (m)
2.41–2.45 (m), 2.00–2.03 (m)
164.7
126.3
186.8
130.8
162.1
42.3
22.2
27.1
58.0
105.5
72.0
79.2
59.0
74.7
22.8
118.3
22.9
6.31 (brs)
6.01 (d, 9.8)
6.64 (d, 9.8)
9
2.89 (dd, 12.4, 6.0)
10
11
12
13
14
15
16
17
18
19
20
21
14-oh
2.40–2.45 (m), 1.54–1.56 (m)
1.88–1.91 (m), 2.05–2.08 (m)
1.82–1.84 (m), 1.68–1.71 (m)
2.23–2.28 (m)
4.31(d,10.4), 3.73(dd, 10.4, 3.4)
4.24 (dd, 7.0, 3.4)
3.00 (d, 7.0)
5.22 (d, 9.3), 3.89 (d, 9.3)
1.18 (s)
4.22 (o), 3.75 (dd, 9.8, 4.2)
5.14 (o)
3.12 (d, 7.8)
5.11 (o), 4.07 (o)
1.31 (s)
1.61 (s)
1.60 (s)
8.01 (s)
β-dcan
98.1
39.7
69.1
88.2
70.8
18.3
β-d-can
4.86 (d, 9.6)
2.51–2.55 (m), 1.97–2.02 (m)
4.17–4.21 (m)
β-d-the
102.8
75.0
86.2
83.6
72.0
18.9
60.6
β-d-the
4.77 (d, 7.7)
3.93–3.95 (m)
3.63–3.67 (m)
3.48–3.50 (m)
3.50–3.53 (m)
1.41 (d, 6.3)
1′
2′
3′
4′
5′
6′
3.29–3.32 (m)
3.54–3.56 (m)
1.32 (d, 6.2)
3-och3
3.93 (s)
β-d-digt
99.6
38.0
67.3
80.5
69.8
18.1
β-d-digt
5.23 (d, 9.3)
2.40–2.44 (m), 1.97–2.00 (m)
4.47–4.51 (m)
β-d-cym
99.9
35.3
77.8
83.2
69.7
19.0
β-d-cym
5.14 (d, 9.5)
2.39–2.42 (m), 1.68–1.71 (m)
3.92–3.96 (m)
3.69–3.73 (m)
4.19–4.25 (m)
1.31 (d, 6.2)
1″
2″
3″
4″
5″
6″
3.42–3.46 (m)
3.95–3.99 (m)
1.35 (d, 6.2)
3-och3
57.7
β-d-digt
99.2
40.1
69.2
82.6
68.1
19.0
3.55 (s)
β-d-digt
5.51 (d, 9.7)
α-l-cym
98.3
32.0
76.2
72.3
67.2
18.1
α-l-cym
5.03 (d, 3.8)
2.32–2.35 (m), 1.82–1.86 (m)
3.67–3.70 (m)
3.61–3.63 (m)
4.44–4.48 (m)
1.42 (d, 6.3)
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
2.38–2.40 (m), 1.97–2.00 (m)
4.28–4.33 (m)
3.40 (dd, 9.6, 2.6)
4.30–4.34 (m)
1.40 (d, 6.2)
3-och3
56.5
3.36 (s)
α-l-dign
101.5
31.3
76.2
68.1
68.0
18.1
α-l-dign
5.21 (d, 3.2)
2.40–2.43 (m), 2.07–2.10 (m)
3.84–3.87 (m)
4.62–4.66 (m)
4.06–4.09 (m)
1.57 (d, 6.2)
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
3-och3
55.4
3.32 (s)
(C-1″), 5.03 (1H, d, J = 3.8 Hz, H-1′′′)/98.3(C-1′′′) in the 1H and 13C NMR spectra, along
with the presence of three secondary methyl proton signals at δH 1.32 (3H, d, J = 6.2 Hz,
H-6′), 1.35 (3H, d, J = 6.2 Hz, H-6″) and 1.42 (3H, d, J = 6.3 Hz, H-6′′′), suggested that 1

4
J.-C. LIU ET AL.
contained three 6-deoxysugar. e sugar moieties were defined as digitoxose, cymarose,
and canaropyranose by their 13C NMR resonance data, which matched well with those of
stauntoside P [7]. e splitting patterns of anomeric proton signals indicated that 1 had
two sugar units with β-linkages and one with α-linkage. e linkage positions and sequence
of these sugars were ascertained by HMBC correlations (Figure 2) from δH 5.03 (H-1′′′ of
α-cymaropyranose) to δC 80.5 (C-4″), from δH 5.23 (H-1″ of β-digitoxopyranose) to δC 88.2
(C-4′), from δH 4.86 (H-1′ of β-canaropyranose) to δC 76.7 (C-3). Compound 1 was sub-
jected to acid hydrolysis and HPLC analysis, which gave one D-canarose, one L-diginose,
and one D-cymarose. erefore, 1 was characterized to be stauntogenin A 3-O-α-L- cym-
aropyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-canaropyranoside and was given
the trivial name limproside A.
Compound 2 was obtained as white amorphous powder. e positive mode HR-ESI-MS
showed a pseudomolecular ion peak at m/z 995.4910 [M + Na]+, revealing its molecu-
lar formula to be C48H76O20. e IR spectrum showed the absorption bands for hydroxy
1
(3425 cm−1) and olefinic (1632 cm−1) groups. e H NMR spectrum of 2 revealed the
diagnostic signals of steroidal glycoside, with a 14, 15-secopregnane-type skeleton aglycone
being exhibited typically by two tertiary methylic groups at δH 1.31 (3H, s, H-19) and 1.60
(3H, s, H-21) , two oxygen-substituted methine groups at δH 4.71–4.74 (1H, m, H-3) and
5.14 (1H, overlap, H-16), and two methylene groups at δH 3.75 (1H, dd, J = 9.8, 4.2 Hz,
Hα -15) and 4.22 (1H, overlap, Hβ-15), and at δH 4.07 (1H, overlap, Hα-18) and 5.11 (1H,
overlap, Hβ-18), and with four 6-deoxypyranoses being shown by four anometic proton
signals at δH 4.77 (1H, d, J = 7.7 Hz, H-1′), 5.14 (1H, d, J = 9.5 Hz, H-1″), 5.51 (1H, d,
J = 9.7 Hz, H-1′′′) and 5.21 (1H, d, J = 3.2 Hz, H-1′′′) , which correlated to the correspond-
ing anomeric carbon signals at δC 102.8 (C-1′), 99.9 (C-1″), 99.2 (C-1′′′) and 101.5 (C-1′′′)
respectively, in the HSQC spectrum, and four methyls at δH 1.41 (3H, d, J = 6.3 Hz, H-6′),
1.31 (3H, d, J = 6.2 Hz, H-6″), 1.40 (3H, d, J = 6.2 Hz, H-6′′′) and 1.57 (3H, d, J = 6.2 Hz,
H-6′′′). In addition, three methoxyl signals at δH 3.93 (3H, s), 3.55(3H, s), 3.32 (3H, s) were
1
also observed in the H NMR spectrum, which were compatible with three methylated
6-deoxypyranoses. ese proton signals together with distinctive olefinic ptotons at δH
6.01(1H, d, J = 9.8 Hz, H-6) correlating to the carbon at δC 137.7 (C-6), showing HMBC
Figure 2. Key hMBc correlations of compounds 1 and 2.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
correlations (Figure 2) with the carbons at δC 42.2 (C-10), 73.1 (C-5), 76.2 (C-8) and 39.5
(C-4), the other olefinic proton at δH 6.64 (1H, d, J = 9.8 Hz, H-7) correlating to the carbon
at δC 133.4 (C-7), showing HMBC correlations with the carbons at δC 52.3 (C-9), 111.5
(C-14) and 76.2 (C-8), indicated the presence of a 5, 8, 14-trihydroxyl-14, 15-secopregn-
6-ene type skeleton aglycone moiety. Further, by consideration of the molecular formula
and 13C NMR spectroscopic data (Table 1), three hydroxyls were proposed to be linked
at C-5, C-8, and C-14, respectively. C14-OH was confirmed by the HMBC correlations
from 14-OH at δH 8.01 to the carbons at δC 58.6 (C-13), 76.2 (C-8), and 111.5 (C-14),
and based on the generally accepted β-orientation of Me-19 in 14, 15-secopregnane-type
skeleton, the β-orientation of 14-OH could be confirmed by the NOE correlation between
14-OH and H-19 (Figure 3). Because of the failure of detecting the labile protons from
hydroxyls, 8-OH was confirmed as α-orientation by comparing with stauntoside H [6]. But
the configuration of 5-OH was not able to be confirmed using this method. e linkage
positions and sequence of the four sugars were ascertained by HMBC correlations from
δH 5.21 (H-1′′′ of α-diginopyranose) to δC 82.6 (C-4′′′ of β-digitoxopyranose) , from δH
5.51 (H-1′′′ of β-digitoxopyranose) to δC 83.2 (C-4″ of β-cymaropyranose), from δH 5.14
(H-1″ of β-cymaropyranose) to δC 83.6 (C-4′ of β-thevetopyranose) and from δH 4.77 (H-1′
of β-thevetopyranose) to δC 75.4 (C-3). e splitting patterns of anomeric proton signals
indicated that 2 had three sugar units with β-linkages and one with α-linkage. e β-D-dig-
itoxopyranose, β-D-thevetopyranose, β-D-cymaropyranose and α-L-diginopyranose were
1
speculated by comparing the H and 13C NMR spectroscopic data of 2 with those in the
literature [6]. Acid hydrolysis of 2 afforded four sugars (digitoxose, cymarose, diginose
and thevetose). Identification of D-digitoxose, D-thevetose, D-cymarose and L-diginose
were performed by HPLC analysis with an optical rotation detector. us, the structure of
2 was determined to be 5, 8, 14-trihydroxyl-3-O-α-L- diginopyranosyl -(1→4)-β-D-digi-
toxopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-thevetopyranosyl-14, 16:15, 20: 18,
20-triepoxy-14, 15-secopregn-6-ene, and was given the trivial name limproside B.
Limprosides A and B were tested for their cytotoxicity against HL-60 and Caco-2 cell
lines using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide)
colorimetric assay. Unfortunately, the two compounds exhibited no cytotoxicity.
Figure 3. Key noesY correlations of compounds 1 and 2.

6
J.-C. LIU ET AL.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a JASCO DIP-370 digital polarimeter (JASCO interna-
tional Co., Ltd., Tokyo, Japan). IR spectra were obtained from a Bruker IFS-55 spectrom-
eter (Bruker Co., Karlsruhe, Germany). HR-ESI-MS were acquired using a Micro-mass
Autospec-Untima TOF mass spectrophotometer (Waters, Milford, U.S.A.). One- and
two-dimensional NMR spectra were recorded on a Bruker Avance-400/-600 spectrometer
(Bruker Co., Karlsruhe, Germany) with tetramethylsilane as an internal standard. CD spec-
tra were recorded with a Biologic MOS-450 spectrometer (Bio-Logic Co., Claix, France).
Sugars analytical HPLC was carried out on a Jasco PU-4180 pump and an OR-4090 detector
(JASCO international Co., Ltd., Tokyo, Japan). HPLC was performed with an Asahipak
NH2P-50 4E column (4.6 mm × 250 mm, 5 μm). Silica gel GF254 (10–40 μm) prepared for
TLC and silica gel (200–300 mesh) for column chromatography (CC) were obtained from
Qingdao Marine Chemical Factory (Qingdao, China). Sephadex LH-20 was obtained from
GE Healthcare (Little Chalfont, Buckinghamshire, U.K.). C18 reversed-phase (RP-18) silica
gel (50 μm, YMC Co. Ltd., Kyoto, Japan) was used for CC. RP-18 F254S (Merck, Darmstadt,
Germany) was used for reversed phase plates.
3.2. Plant materials
e dried roots (20 kg) of Cynanchum limprichtii Schltr. were bought from Anhui Economy
People Pharmaceutical Co., Ltd. A voucher specimen was identified by Prof. Jincai Lu of
Shenyang Pharmaceutical University and has been deposited in the School of Traditional
Chinese Materia Medica of Shenyang Pharmaceutical University (No. 2013052).
3.3. Extraction and isolation
e dried roots of C. limprichtii (20 kg) were extracted with 95% EtOH for three times (each
2 h) and the combined solution evaporated to dryness by a vacuum rotary evaporator to
afford a crude extract (3500 g). e crude extract was successively partitioned with petro-
leum ether, ethyl acetate, and n-butanol to yield three extracts. e ethyl acetate extract
(300 g) was subjected to silica gel CC using a gradient of CH2Cl2–MeOH (100:0–0:100, v/v)
to obtain five fractions (Fr.A-E) based on TLC analyses.
Fr.B (92 g) was separated by silica gel CC eluted with petroleum ether-CH3COCH3
(100:10–100:100, v/v) to afford nine fractions (Fr.B1-B9). Fr.B9 (38 g) was further separated
by silica gel CC using a gradient of petroleum ether-CH3COCH3 (100:15–100:100, v/v) to
obtain ten fractions (Fr.B9A-B9 J). Fr.B9F (10 g) yielded four fractions (Fr.B9F1-B9F4) by
silica gel CC eluted with CH2Cl2–MeOH (100:2–100:10, v/v). Fr.B9F1(5 g) further produced
nine fractions (Fr.B9F1A-B9F1I) by silica gel CC eluted with petroleum ether-CH3COCH3
(100:10–100:50, v/v). Fr.B9F1E (1 g) was subjected to Sephadex LH-20 gel CC eluted with
MeOH to afford five fractions (Fr.B9F1E1-B9F1E5). Fr.B9F1E2 (350 mg) was subjected to
RP-C18 CC by using MeOH-H2O (50:50–80:20, v/v) as the eluting solvent, and followed
by purification on the silica gel CC using CH2Cl2–CH3COCH3–MeOH (30:2:1, v/v) to
provide compound 1 (16 mg). Fr.B9H (12 g) was subjected to silica gel CC eluted with
petroleum ether-CH3COCH3 (100:20–100:40, v/v) to give nine fractions (Fr.B9H1-B9H9).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Fr.B9H7 (4 g) further produced six fractions (Fr.B9H7A-B9H7F) by silica gel CC eluted with
petroleum ether-EtOAc (1:3, 1:5, 1:7, v/v). Fr.B9H7F (1.8 g) was separated by silica gel CC
eluted with petroleum ether-CH3COCH3 (100:50–100:100, v/v) to afford eight fractions (Fr.
B9H7F1-B9H7F8). Fr.B9H7F7 (330 mg) was subjected to RP-C18 CC eluted with MeOH-
H2O (60:40–80:20, v/v) to give compound 2 (10 mg).
3.3.1. Limproside A (1)
White amorphous powder.[ꢀ]D20 −84.5 (c 0.2, MeOH). CD (MeOH)Δε249 nm + 2.85. IR (KBr)
νmax: 3439, 2933, 2659, 1696, 1655, 1632, 1403, 1384, 1271, 1062, 833 cm−1. 1H (pyridine-d5,
400 MHz) and 13C NMR (pyridine-d5, 100 MHz) spectral data see Table 1. HR-ESI-MS
(positive-ion mode) m/z: 801.3618 [M + Na]+ (calcd for C40H58O15Na, 801.3668).
3.3.2. Limproside B (2)
White amorphous powder. [ꢀ]D20 −43.3 (c 0.05, CH3COCH3). CD (CHCl3) Δε205 nm + 8.83,
Δε215 nm −3.00. IR (KBr) νmax: 3425, 2932, 2659, 1649, 1632, 1403, 1384, 1271, 1069, 832 cm−1.
1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz) spectral data see
Table 1. HR-ESI-MS (positive-ion mode) m/z: 995.4910 [M + Na]+ (calcd for C48H76O20Na,
995.4828).
3.4. Acid hydrolysis of compounds 1 and 2
Each solution of 1 and 2 (each 6 mg) in MeOH (5 ml) was added 0.1 N H2SO4 (5 ml) and
kept at 50 °C for 30 min. e solution was diluted with 10 ml of water and then concen-
trated to 10 ml. e solution was kept at 60 °C for another 30 min, and then neutralized
with aqueous saturated Ba(OH)2 and the precipitates were filtered off. e filtrate was
concentrated and analyzed by TLC with three solvent systems: solvent A, CHCl3–CH3OH
(9:1); solvent B, CH2Cl2–C2H5OH (9:1); and solvent C, PE-acetone (3:2). e Rf values of
cymarose, diginose, digitoxose, thevetose, and canaropyranose, in the order of 0.56, 0.40,
0.21, 0.59, and 0.31 with solvent A; 0.47, 0.28, 0.15, 0.53, and 0.27 with solvent B; and 0.28,
0.18, 0.10, 0.37, and 0.17 with solvent C, respectively [8–10].
3.5. Determination of absolute configurations of sugar moieties in 1 and 2
e determination of absolute configurations of the deoxysugars obtained from water layer
of the hydrolysis solution of the compounds by HPLC under the following conditions:
column, Asahipak NH2P-50 4E column; flow rate, 1.0 ml/min; solvent, 75% MeCN-H2O;
detection, OR-4090 detector, identification of D-cymarose, L-cymarose, D-digitoxose,
L-diginose, D-canaropyranose, and D-thevetose in each sugar fraction was carried out
by comparison of the retention times and polarities with those of authentic samples (the
standards of sugars were obtained by acid hydrolysis of known compounds with the same
method of those new compounds). D-cymarose (tR 8.76 min, positive polarity), L-cymarose
(tR 9.15 min, negative polarity), D-digitoxose (tR 11.5 min, positive polarity), L-diginose
(tR 7.21 min, negative polarity), D-canaropyranose (tR 14.9 min, positive polarity), and
D-thevetose (tR 6.02 min, positive polarity).

8
J.-C. LIU ET AL.
3.6. Cytotoxic assay
e MTT assay [11,12] was used to determine the cytotoxicity of each compound against
two cultured human cancer cell lines HL-60 and Caco-2. 5-Fluorouracil was used as a pos-
itive control. e cytotoxicity of 5-fluorouracil against the HL-60 and Caco-2 cell lines was
estimated by their IC50 values of 6.38 and 17.01 μM, respectively, for these two cell lines.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was financially supported by National Natural Science Foundation of China [grant number
31370375].
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