New Immunomodulating Polyhydroxypregnane Glycosides from the Roots of Cynanchum otophyllum C.K.Schneid.

Article abstract of DOI:10.1002/cbdv.201900062

Seven new polyhydroxypregnane glycosides, named cynotophyllosides P–V, together with three known analogs were isolated from the roots of Cynanchum otophyllum C.K.Schneid. Their structures were elucidated by a variety of spectroscopic techniques, as well as acid-catalyzed hydrolysis. All isolates were tested for their immunological activities in vitro against Con A- and LPS-induced proliferation of mice splenocytes. Immunoenhancing (for 1, 9) and immunosuppressive (for 2) activities were observed. Furthermore, cynotophylloside R (3) showed immunomodulatory as it enhanced the proliferation of splenocytes in low concentration and suppressed immune cells in concentration more than 1.0 μg/ml.

Full text of DOI:10.1002/cbdv.201900062

Title: New immunomodulating polyhydroxypregnane glycosides from
the roots of Cynanchum otophyllum
Authors: Zha-Jun Zhan, Shu-Min Bao, Yue Zhang, Fei-Jun Qiu, Wei-
Guang Shan, and Liefeng Ma
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201900062
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900062

Chemistry & Biodiversity
10.1002/cbdv.201900062
New immunomodulating polyhydroxypregnane glycosides from the
roots of Cynanchum otophyllum
a)
a)
a)
b)
a)
a, b)
By Zha-Jun Zhan , Shu-Min Bao , Yue Zhang , Fei-Jun Qiu , Wei-Guang Shan , Lie-Feng Ma*
a) College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, P. R. China
Phone: +86-571-88871075; E-mail: maliefeng@zjut.edu.cn)
(
b) Zhejiang Xinguang Pharmaceutical Limited Liability Company, Shaoxing, 312400, P. R. China
1
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Chemistry & Biodiversity
10.1002/cbdv.201900062
Abstract
Seven new polyhydroxypregnane glycosides, named cynotophyllosides P‒V, together with
three known analogs were isolated from the roots of Cynanchum otophyllum Schneid. Their
structures were elucidated by a variety of spectroscopic techniques, as well as acid-catalyzed
hydrolysis. All isolates were tested for their immunological activities in vitro against Con A-
and LPS-induced proliferation of mice splenocytes. Immunoenhancing (1, 9) and
immunosuppressive (2) activities were observed. Furthermore, compound 3 showed
immunomodulatory as it enhanced the proliferation of splenocytes in low concentration and
suppressed immune cells in concentration more than 1.0 g/ml.
Keywords
Cynanchum otophyllum; polyhydroxypregnane glycoside; immunomodulating activity;
cynotophyllosides P‒V
Introduction
Genus Cynanchum, belonging to the family of Asclepiadaceae, consisted more than 200
species distributed worldwide. In China, a total of 53 species and 12 varieties are native to the
southwest region. Different parts of plants of this genus are used to treat common and chronic
diseases as herbal medicine by local people [1-3]. Cynanchum otophyllum Schneid is a
perennial herbaceous vine, its roots have been employed as traditional medicine for the
treatment of snake bites, bruises, rheumatoid arthritis and eczema [4]. Phytochemical
2
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Chemistry & Biodiversity
10.1002/cbdv.201900062
researches revealed this specie was a natural source of C21 steroidal glycosides, some of which
were reportedly possess neuroprotective activity [5], antidepressant activity [6], cytotoxicity
[
7, 8], MDR-reversing [9], anti-aging [10] and anti-epileptic activity [11]. In our continuous
effort to search for novel and biologically active natural products, seven new
polyhydroxypregnane glycosides 1‒7, named cynotophyllosides P‒V, together with three
known compounds (8‒10), were obtained from the roots of C. otophyllum. The structures were
identified by spectroscopic analysis, chemical methods, and comparison of observed and
reported spectroscopic data. These compounds were assessed for their immunological activities
against Con A- and LPS- induced proliferation of mice splenocytes in vitro. Herein, their
isolation, structure elucidation, and bioactivity evaluation are reported.
Results and discussion
The roots of C. otophyllum were extracted with 95% EtOH. The crude extract was
suspended in H
2
O and partitioned with CHCl . Then the organic fraction was subjected to
3
extensive column chromatography and semi-preparative HPLC to obtain ten steroidal
gylcosides 1‒10. Seven (1‒7) of these compounds were undescribed previously, they all
showed positive Lieberman-Burchard and Keller-Kiliani reactions, indicating the presence of
steroids and 2-deoxysugars moieties [12].
The presence of the monosaccharides in the hydrolysates of each compound was
13
determined by comparison of its C NMR spectroscopic data with whose reported data and
confirmed by co-TLC with authentic sugars, and the optical rotation was compared between
the 2,6-dideoxysugars in the new compounds with those reported in the literature (see
3
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Chemistry & Biodiversity
10.1002/cbdv.201900062
Experimental Part). Three kinds of 2,6-dideoxysugars were revealed in these new compounds,
they were digitoxopyranoside, diginopyranoside and cymaropranoside. Furthermore, a survey
of glycosides from the Asclepiadaceae family reveals that all the -linked 2,6-dideoxysugars
have the D configuration, whereas the -linked sugars are mostly L-sugars [13, 14]. To the 2,6-
1
3
dideoxysugars obtained in this study, most significant differences in the C NMR data between
D- and L- sugars are the shift of C-2, in the L-formed 2-deoxysugar it is ca. 32.0 ppm or less,
while that of C-2 in the D-formed sugar appears above 34.0 ppm [6, 15].
Compound 1 was obtained as white amorphous powder. Its molecular formula was
‒
1
established as C42
H
68
O
15, based on the negative HRESIMS (m/z 811.4488 [M ‒ H] ). The H
1.25 (s, H-18), 1.16 (s, H-
1.22 (d, J = 6.5 Hz, H-6′′), 1.25 (d, J =
.8 Hz, H-6′′′) and 1.30 (d, J = 6.2 Hz, H-6′′′′), three methoxyls attributed to the sugar moiety
3.43 (s), 3.41 (s) and 3.42 (s), and a trisubstituted olefinic proton at  5.37 (brs, H-6).
7.7 (C-18), 18.6 (C-
9), 28.2 (C-21), 17.5 (C-6′′), 18.0 (C-6′′′) and 18.3 (C-6′′′′), a pair of double bond carbon
signals at  140.4 (C-5), 117.9 (C-6), and a carbonyl group signal at  213.7 (C-20). The 1D
NMR spectroscopic signals arising from the aglycone of 1 were similar to those of
deacetylmetaplexigenin [16]. Meanwhile the NOE correlations of H -18/H -19 suggested they
were co-facial and were assigned to be -orientation, the cross peaks of H-3/H-1, H -19/H-
 indicated a -oriented 3-OH, the H-9 and H-12 were both assigned as  dur to the correlation
NMR spectrum of 1 showed signals of three tertiary methyls at 
H
1
6
9) and 2.34 (s, H-21), three secondary methyls at 
H
at 
H
H
1
3
The C NMR and DEPT spectra showed six methyl carbon signals at 
C
1
C
C
3
3
3
1
between H-12/H-9/H-1. These signals were consistent with literature [16], taken biosynthesis
involved in the genus of this plant into consideration, the relative configuration was assigned
4
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Chemistry & Biodiversity
10.1002/cbdv.201900062
as depicted. The glycosidation shift of C-3 from 
C
71.6 in deacetylmetaplexigenin to  77.8
C
confirmed that the sugar moiety located at C-3.
1
The H NMR spectrum of 1 exhibited three sugar anomeric protons at 
H
4.92 (d, J = 8.2
Hz, H-1′′), 4.98 (d, J = 3.4 Hz, H-1′′′) and 4.96 (d, J = 8.6 Hz, H-1′′′′), which correlated with
carbons at  95.7 (C-1′′), 100.8 (C-1′′′) and 99.1 (C-1′′′′) in the HSQC spectrum. This indicated
C
1
1
the sugar moiety was consisted with three 2-deoxysugar units. Detailed analysis of the H- H
COSY, HSQC, and HMBC spectra led to the assignment of the proton and carbon signals of
sugar units in 1. The existence of one -cymaropranoside (
C
34.3 C-2′′), one -
cymaropranoside ( 32.4 C-2′′′′) and one -diginopyranoside (
C
C
31.6 C-2′′′) unit was
confirmed by co-TLC of the acid hydrolysate with the standard sugars and comparison of its
spectroscopic data with those in the literatures [11, 17]. The sugar sequence of 1 was revealed
by the HMBC correlations of H-1′′′ of -L-diginopyranoside to C-4′′ of -D-cymaropranoside,
H-1′′′′ of -L-cymaropranoside to C-4′′′ of -L-diginopyranoside suggested the connection of
the three sugars via two (1 → 4) linkages as shown in Figure 2. The saccharide was connected
to C-3 of the aglycone, due to the glycosidation shift of C-3 and the HMBC correlation between
H-1′′ and C-3. Thus, the structure of compound 1 was established as deacetylmetaplexigenin
3
-O--L-cymaropranosyl-(1 → 4)--L-diginopyranosyl-(1 → 4)--D-cymaropranoside, and
named cynotophyllosides P.
Compound 2 was suggested to have the molecular formula C44
70
H O16, based on the m/z
–
8
54.4622 ([M ‒ H] ) in the HRESIMS, which was 42 amu higher than that of 1. Mild acid
hydrolysis of 2 afforded the same sugar compositions as compound 1. In the NMR spectra of
1
and 2 (Table 1 and 2), the structure of 2 corresponded to 1 except an additional acetyl group
5
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Chemistry & Biodiversity
10.1002/cbdv.201900062
at 
H
1.95 (s, H
3
-2′), 
C
20.7 (C-2′) and 169.9 (C-1′). The downfield shifts of H-8 from 
H
3.68
to 4.51 and C-8 from 
C
69.4 to 72.5 indicating the attachment of acetyl group to C-8, this was
further confirmed by the correlation from H-12 to C-1′ in HMBC experiment, hence the
aglycone was determined to be metaplexigenin [18]. The structure of 2 was revealed as
metaplexigenin 3-O--L-cymaropranosyl-(1 → 4)--L-diginopyranosyl-(1 → 4)--D-
cymaropranoside, and named cynotophylloside Q.
Cynotophylloside R (3) was obtained as a white amorphous powder. Based on the positive
HRESIMS data, the molecular formula of 3 was determined to be C51
74
H O16 (m/z 965.4876 [M
+
+
Na] ). In the 1D NMR data, signals at 
C
165.7 (C-1′), 117.6 (C-2′), 145.4 (C-3′), 134.2 (C-
4
′), 128.2 (C-5′), 128.9 (C-6′), 130.4 (C-7′) , 128.9 (C-8′), 128.2 (C-9′) demonstrated the
existence of the cinnamyl unit. Cinnamylation of the hydroxyl group at C-12 was confirmed
from the downfield shifts of their corresponding protons and carbons, as well as the HMBC
experiment which showed correlation from H-12 of the aglycone moiety to the carbonyl carbon
of the cinnamyl group. This indicated the aglycone was kidjoranin [19]. Mild acid hydrolysis
of 3 afforded the same sugar residues as those of 1, meanwhile they shared the same NMR data
of saccharide with each other. Thus, the structure of 3 was determined as kidjoranin 3-O--L-
cymaropranosyl-(1 → 4)--L-diginopyranosyl-(1 → 4)--D-cymaropranoside.
Compound 4 had the molecular formula of C43
68
H O16, as the negative HRESIMS showed
–
a deprotonated ion at m/z 839.4401 ([M ‒ H] ). Molecular weight of 4 was 14 amu less than
1
3
that of cynotophyllosides Q (2), meanwhile, comparison of the C NMR data of 4 with those
of 2 suggested they possessed same aglycone. This indicated the difference was in the
saccharide moiety. The anomeric carbons at  95.9 (C-1′′), 97.7 (C-1′′′) and 94.3 (C-1′′′′), and
C
6
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Chemistry & Biodiversity
10.1002/cbdv.201900062
the corresponding proton signals at 
H
4.93 (d, J = 9.7 Hz), 4.90 (d, J = 2.6 Hz) and 4.78 (d, J
=
9.6 Hz) showed correlations in the HSQC experiment. These coupling constants indicated -
linkages for two sugar units, and an -linkage for the other one [8]. The carbon shifts of each
sugar unit were assigned by HMBC and HSQC experiments. Comparison of the spectral data,
and co-TLC of acid hydrolysate of 4 with those of analogs, indicated the existence of one -D-
digitoxopyranoside, one -L-cymaropyranoside and one -D-cymaropyranoside. The
sequence of those sugar units of 4 was inferred by the long-rang correlations in HMBC analysis
from H-1′′′′ of -D-cymaropyranoside (
H
4.78) to C-4 of -L-cymaropyranoside (
C
76.5),
80.0).
and H-1′′′ of -L-cymaropyranoside (
H
4.90) to C-4 of -D-digitoxopyranoside (
C
Consequently, 4 was established to be metaplexigenin 3-O--D-cymaropranosyl-(1 → 4)--L-
cymaropyranosyl-(1 → 4)--D-digitoxopyranoside.
The molecular formulas of 5 and 6 were determined as C41
H
68
O15 and C40
66
H O15 by the
–
deprotonated molecular ion peaks at m/z 799.4465 and 785.4333 ([M ‒ H] ) in the HREIMS
respectively. Inspection of the NMR data revealed the saccharide moiety in 5 was identical
with those of 4, indicating they shared the same sugar sequence. Main differences were
observed in the aglycon, as the acetyl and carbonyl groups in 4 were not detected in 5. This
suggested the aglycone was replaced by sarcostin [15, 20]. It was further confirmed by TLC
analysis with authentic sample [12]. The NMR data suggested 6 shared the same aglycone with
1
5
. The H NMR spectrum of 6 showed three secondary methyl signals at 
H
1.48 (d, J = 5.6
3.47 (s), and three
5.52 (d, J = 8.0 Hz), 5.14 (d, J = 3.2 Hz) and 5.61 (dd, J = 9.6, 1.5 Hz).
Hz), 1.52 (d, J = 6.4 Hz) and 1.67 (d, J = 6.2 Hz), one methoxyl signal at 
H
anomeric signals 
H
That information proved that 6 had two sugar units with -linkages and the other 2,6-
7
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Chemistry & Biodiversity
10.1002/cbdv.201900062
dideoxysugar with -linkage. The carbon shifts of each sugar unit were assigned by HMBC
and HSQC experiments. Compared with the spectral data of analogs, it suggested the sugar
chain of 6 was formed from two D-digitoxopyranosides and one L-cymaropyranoside. The
sequence of three sugar units of 6 was illustrated by HMBC long correlations from H-1′′′′ at 
H
5
5
.61 of -D-digitoxopyranosyl to C-4′′′ at 
C
77.4 of -L-cymaropyranosyl, and H-1′′′ at 
H
.14 of -L-cymaropyranosyl correlated with C-4′′ at 
C
81.5 of -D-digitoxopyranosyl.
Therefore, the structures of 5 and 6 were established as sarcostin 3-O--D-cymaropranosyl-(1
4)--L-cymaropyranosyl-(1 → 4)--D-digitoxopyranoside and sarcostin 3-O--D-
→
digitoxopyranosyl-(1
respectively.
→
4)--L-cymaropyranosyl-(1
→
4)--D-digitoxopyranoside,
Compound 7 was isolated as colorless powder. According to the HRESIMS, its molecular
1
formula was determined to be C30
H
46
O
10. The H NMR showed one anomeric proton signal at

H
4.78 (d, J = 9.5 Hz), its coupling constant suggested a -linkage for the sugar unit. Acid
hydrolysis of 7 gave cymarose as residue. The aglycone showed resemblance to those of 2 and
. The HMBC correlation between  4.52 (q, J = 6.8 Hz, H-12) and  169.9 (C-1′) confirmed
4
H
C
the existence of metaplexigenin. Thus, compound 7 was established as metaplexigenin 3-O--
D-cymaropranoside, and given the name of cynotophylloside V.
The known compounds were identified as deacetylmetaplexigenim 3-O--D-
diginopyranosyl-(1
→
4)--L-digitoxopyranoside (8) [21], sarcostin 3-O--D-
cymaropyranosyl-(1 → 4)--L-digitoxopyranoside (9) [21] and 12--O-acetyl-20-O-(2-
methylbutyryl)-sarcostin (10) [22].
In Generally, all compounds could be further divided into two different groups depending
8
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Chemistry & Biodiversity
10.1002/cbdv.201900062
on the aglycone. Compounds 1‒4 and compounds 7‒8 shared same C21 polyhydroxypregnane
core with a carbonyl group in C-20 position. The aglycone of the other group, containing
compounds 5‒6 and 9‒10, was sarcostin with a hydroxyl group in C-20. Meanwhile,
compounds 1‒3 shared the same sugar sequence as -L-cymaropranosyl-(1 → 4)--L-
diginopyranosyl-(1 → 4)--D-cymaropranoside, and compounds 4‒5 shared another sugar
moiety. These compounds were tested for their immunomodulating activities.
The effect of 1‒10 on mitogen-stimulated mice splenocyte proliferation in vitro were
shown in Figure 3 and 4. Compounds 1 and 9 enhanced Con A- and LPS-induced mice
splenocyte proliferation in a dose-dependent manner. Compound 2 inhibited the proliferation
of the splenocyte significantly. Meanwhile, compound 3 displayed variable activity on both
Con A- and LPS-induced mice splenocytes proliferation as it could enhance the proliferation
in low concentration while inhibition was observed in high concentration remarkably.
Interestingly, compound 6 showed inhibition on the Con A-stimulated mice splenocyte
proliferation significantly, while enhancement was revealed in the LPS-induced group.
Comparing the bioactivities of compounds 1‒3 suggested the immunomodulating activities
were significantly affected by the substituents on the aglycon. Meanwhile, compounds 4 and
7
, possessing the aglycon of metaplexigenin with different sugar sequences, showed little
difference with each other on the bioactivity. This suggested that the different substitutions in
the aglycone showed more effect on the immunomodulating activities in the
polyhydroxypregnane glycosides consisted with a three-unit saccharide.
9
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Chemistry & Biodiversity
10.1002/cbdv.201900062
Conclusions
Isolation and structural elucidation of seven new polyhydroxypregnane glycosides
together with three known analogs from the root of Cynanchum otophyllum were demonstrated
in this study. As this species was used in traditional Chinese medicine to treat rheumatism,
these compounds were tested for their immunological activities in vitro against Con A- and
LPS-induced proliferation of mice splenocytes. Compounds 1 and 9 possessed
immunoenhancing activities, compound 2 was immunosuppressive. Compound 3 showed
variable activity, it could enhance the proliferation of splenocytes in low concentration and
suppress the immune cells in concentration more than 1.0 g/ml. These observations may
suggest that the pregnane glycosides from these species were responsible for the folk usage to
treat rheumatism as they possessed immunomodulatory activity.
Experimental
General
All solvents were of analytical reagent (AR) grade and used as received. Thin layer
chromatography (TLC) was performed on precoated silica gel GF254 plates (with glass support,
Qingdao Marine Chemical Inc.), detected under UV light (254 nm) and by spraying the plates
with 10% sulfuric acid ethanol solution followed by heating. Purification by column
chromatography was carried out using silica gel (200‒300 mesh, Qingdao Marine Chemical
Inc.), Sephadex LH-20 (Amersham Pharmacia Biotech), and reverse-phase SiO
2
(ODS-A 12
nm S-150; YMC Co. Ltd.) with indicated eluents Semi-preparative HPLC was done on an
1
0
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Chemistry & Biodiversity
10.1002/cbdv.201900062
Alltech model 1500 series (Alltech, IL, USA) with YMC-packed ODS-A column (5 um, 10
mm  250 mm, YMC Co. Ltd. Kyoto, Japan). Optical rotations were carried on Rudolph-
AutoPol (Rudolph Technologies, German). IR spectra were recorded on Thermo-Nicolet-6700
FT-IR microscope instrument (Thermo Fisher Scientific, MA, USA). The NMR spectra were
measured on a Bruker-AM-500 with Me Si as internal standard,  in ppm and J in Hz (Bruker
4
Daltonics, Bremen, Germany). ESIMS were detected on Agilent-6210-LC/TOF mass
spectrometer (Agilent Technologies, CA, USA)
Plant material
The plant used in this study was purchased from Yunnan Province, P.R. China, in July 2016,
and identified by Prof. Yong-Hong Zhang of the Fujian Medical University, P.R. China. A
voucher specimen (No. 20160810A) has been deposited in Pharmacognosy and Natural
Medicine Research Institute of Zhejiang University of Technology.
Extraction and isolation
The roots of C. otophyllum (3.2 kg) were pulverized ant extracted with 95% aq. EtOH (3

4 L, 7d each) at room temperature to give a crude extract (57 g). The crude extract was
subjected to CHCl /H O partition to give 39 g of the organic fraction. The CHCl soluble
fraction was subjected to CC (SiO (200−300 mesh), 8  70 cm, PE/EtOAc 10:1 to 0:1):
fractions A−J were obtained based on TLC profile. Fraction D (19.5 g) was then subjected to
CC (SiO (200−300 mesh), 3  70 cm, PE/acetone 10:1 to 2:1) to yield fractions D1–D5.
Fraction D3 was separated by CC (Sephadex LH-20; SiO (500 mesh), CHCl /MeOH 10:1)
3
2
3
2
2
2
3
1
1
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Chemistry & Biodiversity
10.1002/cbdv.201900062
and further purified by semi-preparative HPLC using MeOH/H
0 (t 17.1 min, 5.7 mg), 6 (t 20.3 min, 7.5 mg), and 7 (t
and 9 (39.7 mg) were isolated by CC (SiO (500 mesh), CHCl
in Fraction D4. Fraction D5 was further separated by CC (ODS-A; MeOH/H
gradiently) to obtain three subfractions D5A–D5C. Compound 4 (11.5 mg) and 1 (20.3 mg)
were obtained from fraction D5A(1.8 g) by CC (SiO (500 mesh), CHCl /MeOH 8:1) followed
by chromatographic column with Sephadex LH-20 gel (MeOH). Fraction D5B (2.2 g) was
separated firstly by CC (SiO (500 mesh), CHCl /MeOH 9:1) then semi-preparative HPLC
using MeOH/H O 50% to obtain compound 8 (t 21.5 min, 8.7 mg), 3 (t 23.7 min, 10.9 mg).
Compound 2 (10.4 mg) was obtained from Fraction D5C (0.3 g) by CC (Sephadex LH-20; SiO
500 mesh), CHCl /MeOH 8:1)
Cynotophylloside P (1) Colorless amorphous powder. []
2
O 55% to obtain compounds
1
R
R
R
24.8 min, 8.4 mg). Compound 5
/MeOH 15:1; Sephadex LH-20)
O 35% to 95%
2
3
2
2
3
2
3
2
R
R
2
(
3
2
5
D
= +15.6 (c = 0.32, CHCl
3
).
-1
1
13
IR(KBr): max 3510, 2934, 1703, 1645, 1372, 1266, 1165, 1087, 999 cm . H- and C- NMR
−
H O
15−; calc. 811.4485).
see Table 1. HRESIMS: m/z 811.4482 ([M ‒ H] , C42
67
2
5
Cynotophylloside Q (2) Colorless amorphous powder. []
D
= +36.0 (c = 0.22, CHCl ).
3
-1
1
13
IR(KBr): max 3455, 2931, 1711, 1635, 1367, 1238, 1163, 1090, 991 cm . H- and C- NMR
−
H O
16−; calc. 854.4625).
see Table 1. HR-ESI-MS: m/z 854.4622 ([M ‒ H] , C44
69
2
5
Cynotophylloside R (3) Colorless amorphous powder. []
D
= +16.0 (c = 0.16, CHCl ).
3
-1
1
13
IR(KBr): max 3470, 2932, 1710, 1637, 1449, 1350, 1277, 1165, 1084, 988 cm . H- and C-
+
74NaO16+; calc.
NMR see Table 1 and Table 2. HR-ESI-MS: m/z 965.4876 ([M + Na] , C51
H
9
65.4869).
2
0
Cynotophylloside S (4) Colorless amorphous powder. []
D
= ‒64.0 (c = 0.10, CHCl ).
3
1
2
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Chemistry & Biodiversity
10.1002/cbdv.201900062
-1
1
13
IR(KBr): max 3430, 2931, 1710, 1643, 1446, 1374, 1239, 1162, 1082, 995 cm . H- and C-
−
H O
16−; calc.
NMR see Table 1 and Table 2. HR-ESI-MS: m/z 839.4401 ([M ‒ H] , C43
67
8
39.4435).
2
5
Cynotophylloside T (5) Colorless amorphous powder. []
D
= ‒37.3 (c = 0.22, CHCl ).
3
-1
1
13
IR(KBr): max 3432, 2928, 1708, 1635, 1450, 1372, 1243, 1157, 1081, 996 cm . H- and C-
−
H O
15−; calc.
NMR see Table 1 and Table 2. HR-ESI-MS: m/z 799.4465 ([M ‒ H] , C41
67
7
79.4485)
Cynotophylloside U (6) Colorless amorphous powder. []
IR(KBr): max 3364, 2922, 1705, 1649, 1450, 1374, 1234, 1165, 1075, 997, 867 cm . H- and
2
5
D
= ‒40.0 (c = 0.22, C
5
5
H N).
-1 1
1
3
–
H O
15−; calc.
C- NMR see Table 1 and Table 2. HR-ESI-MS: m/z 785.4333 ([M ‒ H] , C40
65
7
85.4329).
2
5
Cynotophylloside V (7) Colorless amorphous powder. []
D
= +43.4 (c = 0.19, CHCl ).
3
IR(KBr): max 3579, 3482, 3406, 3283, 2977, 2932, 1743, 1612, 1454, 1388, 1357, 1240, 1097,
-1
1
13
1
030, 984 cm . H- and C- NMR see Table 1 and Table 2. HR-ESI-MS: m/z 656.3022 ([M ‒
–
H O
10−; calc. 565.3018).
H] , C30
45
Acid hydrolysis of compounds 1−7 and comparison with standard sugars
To a solution of each compound (2 mg) in MeOH (1 mL), 0.2 M H
The solution was kept at 60℃ for 2 h and then diluted with H O (2 mL). The solution was
neutralized with satd. aq. Ba(OH) and concentrated under vacuum. The residue (a mixture of
2
SO
4
(1 mL) was added.
2
2
aglycone and sugars) was subjected to a CC (Sephadex LH-20, MeOH) to give fractions of
aglycone and sugars. Constituents of each sugar fraction were identified by co-TLC with
1
3
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Chemistry & Biodiversity
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authentic sugars: cymarose (R
.46), and digitoxose (R (CHCl
compounds were determined by its optical rotation, as the D-form of the digitoxoses with []
f
(CHCl
3
/MeOH 8:1) 0.50), diginose (R
f
(CHCl /MeOH 8:1)
3
0
f
3
/MeOH 8:1) 0.40). 2,6-dideoxysugars obtained from the new
2
0
D
2
0
20
=
+48.4, the L-diginose with []
D
of ‒53.4, and the cymarose with []
D
= +50.2 and ‒53.4
for D- and L- respectively.
Biological assay
Male BALB/c mice (8‒10 weeks old) were purchased from Zhejiang Academy of Medical
Sciences (Zhejiang province, China). All mice were housed at a constant temperature (25  2

C) and relative humidity (50  5%) under a standard light-dark cycle. All biological
experiments were carried out according to the Guide for the Care and Use of Laboratory
Animals (Ministry of Science and Technology of China, 2006), and were approved by Animal
Care and Use Committee of Zhejiang University of Technology.
Single-cell suspensions were prepared as described in literatures [23, 24]. Splenocytes
7
were seeded into four wells of a 96-well flat-bottom microtiter plate at a cell density of 1  10
per mLin 100 L complete medium. Where 100 Lof 1‒10 (0.01‒10 g/mL), and Con A(final
concentration 5 g/mL), LPS (final concentration 10 g/mL) or medium were then added. The
plate was incubated at 37 C in a humidified atmosphere with 5% CO . After 44 h, 50 L of
2
MTT solution was added to each well and incubated for 4 h. The microtiter plates were
centrifuged (1400 rpm, 5 min), and the untransformed MTT was removed carefully by pipetting.
2
00 L of a DMSO working solution (192 L of DMSO with 8 L of HCl 1 mol/L) were added
to each well, and the absorbance (A) was evaluated after 15 min in an ELISA reader at 570 nm.
1
4
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Chemistry & Biodiversity
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Acknowledgment
We gratefully acknowledge the National Science Foundation (No. 81872777) for financial
support.
Author Contribution Statement
The authors declare that there are no conflicts of interest.
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1
8
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Chemistry & Biodiversity
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Tables
1
Table 1. H-NMR spectroscopic data for compounds 1‒7
1a
2b
3 a
4 a
5 a
6c
7 a
1
.85−1.93 (m)
1.84−1.89 (m)
1.07−1.12 (m)
1.88−1.93 (m)
1.63−1.69 (m)
3.54−3.58 (m)
2.36−2.39 (m)
2.25−2.31 (m)
-
1.76−1.82 (m)
1.11−1.18 (m)
1.87−1.93 (m)
1.50−1.61 (m)
3.53−3.60 (m)
2.34−2.41 (m)
2.30−2.34 (m)
-
1.82−1.90 (m)
1.05−1.22 (m)
1.85−1.93 (m)
1.55−1.64 (m)
3.50−3.60 (m)
2.39−2.46 (m)
2.28−2.35 (m)
-
1.82−1.88 (m)
1.02−1.10 (m)
1.88−1.96 (m)
1.63−1.69 (m)
3.52−3.58 (m)
2.35−2.42 (m)
2.28−2.34 (m)
-
2.01−2.09 (m)
1.16−1.30 (m)
2.16−2.23 (m)
1.86−1.92 (m)
3.90−3.96 (m)
2.61−2.68 (m)
2.48−2.61 (m)
-
1.83−1.87 (m)
1.08−1.14 (m)
1.89−1.97 (m)
1.58−1.63 (m)
3.53−3.57 (m)
2.35−2.42 (m)
2.27−2.34 (m)
-
1
1
.08 (dt, 13.0, 2.8)
1
1
.90−1.98 (m)
.53−1.68 (m)
2
3
4
3.54−3.61 (m)
2
2
.38−2.43 (m)
.26−2.39 (m)
-
5
6
5.37 (brs)
5.36 (brs)
5.38 (s)
5.35 (brs)
5.36 (brs)
5.47 (brs)
5.36 (brs)
2
2
.12−2.17 (m)
.13−2.19 (m)
-
2.49−2.62 (m)
2.42−2.47 (m)
-
7
2.14−2.23 (m)
2.17−2.22 (m)
2.15−1.22 (m)
2.06−2.15 (m)
1.17−2.23 (m)
8
9
-
-
-
-
-
1.46 (dd, 13.0, 3.1)
-
1.48−1.56 (m)
1.53−1.62 (m)
1.51−1.56 (m)
1.41 (dd, 11.1, 2.0)
1.68−1.76 (m)
-
1.51−1.56 (m)
10
-
-
-
-
-
1
1
.85−1.92 (m)
.52−1.68 (m)
1.97−2.05 (m)
1.60−1.68 (m)
3.56−3.62 (m)
-
2.53−2.61 (m)
2.04−2.12 (m)
3.96−4.03 (m)
-
1
1
1.72−1.84 (m)
1.82−1.94 (m)
1.68−1.80 (m)
1.76−1.84 (m)
1
1
1
2
3
4
3.68−3.73 (m)
4.51 (dd, 9.0, 6.0)
4.69 (t, 7.0)
4.51 (dd, 9.0, 6.9)
4.52 (dd, 9.0, 6.8)
-
-
-
-
-
-
-
-
-
-
-
-
1
1
.95−2.01 (m)
.90−1.97 (m)
2.38−2.42 (m)
1.74−1.80 (m)
1.98−2.02 (m)
1.90−1.96 (m)
1.81−1.87 (m)
1.73−1.77 (m)
2.02−2.11 (m)
2.00−2.09 (m)
1.93−1.99 (m)
1.88−1.96 (m)
15
1.97−2.06 (m)
1
9
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Chemistry & Biodiversity
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2
1
.72−2.78 (m)
.92−1.97 (m)
-
2.82−2.91 (m)
2.83−2.92 (m)
1.80−1.85 (m)
1.71−1.78 (m)
-
2.05−2.13 (m)
2.83−2.92 (m)
1
6
2.84−2.91 (m)
1.82−1.93 (m)
-
1.82−1.87 (m)
1.99−2.08 (m)
-
1.77−1.84 (m)
1
1
1
2
2
7
8
9
0
1
-
-
-
1.25 (s)
1.16 (s)
-
1.42 (s)
1.13 (s)
-
1.47 (s)
1.43 (s)
1.12 (s)
-
1.35 (s)
2.03 (s)
1.42 (s)
1.13 (s)
-
1.14 (s)
1.18 (s)
1.48 (s)
-
4.06 (q, 6.4)
1.19 (d, 6.4)
4.55−4.61 (m)
1.60 (d, 6.4)
2.34 (s)
2.24 (s)
-
2.21 (s)
2.25 (s)
-
2.24 (s)
-
1









-
2
1.95 (s)
6.31 (d, 16.2)
7.62 (d, 16.2)
-
1.95 (s)
1.95 (s)
3
4
5
6
7
8
9
7.42−7.56 (m)
7.26−7.41 (m)
7.26−7.41 (m)
7.26−7.41 (m)
7.42−7.56 (m)
D-Cym
D-Cym
D-Cym
D-Digit
D-Digit
D-Digit
D-Cym
1


4.92 (d, 8.2)
4.83(d, 9.5)
2.12−2.21 (m)
1.55−1.63 (m)
3.64−3.68 (m)
4.84 (d, 9.2)
2.18−2.24 (m)
1.53−1.62 (m)
3.68−3.73 (m)
4.93 (d, 9.7)
2.06−2.14 (m)
1.70−1.77 (m)
4.06−4.14 (m)
4.95(d, 8.8)
1.97−2.14 (m)
1.70−1.79 (m)
4.04−4.11 (m)
5.52 (d, 8.0)
2.43−2.51 (m)
2.08−2.14 (m)
4.58 (d, 2.9)
4.78 (d, 9.5)
2.16−2.23 (m)
1.56−1.62 (m)
3.63 (dd, 2.8, 5.8)
2.15−2.22 (m)
2
1.58−1.63 (m)
3
4
5
6
3.68−3.73 (m)
3.25 (dd, 3.1, 9.7) 3.26(dd, 2.7, 9.4) 3.27 (dd, 2.7, 9.4) 3.21 (dd, 3.0, 9.5) 3.24 (dd, 9.3, 2.8) 3.56 (dd, 9.3, 2.8) 3.22 (dt, 3.6, 10.4)
3.79−3.86 (m)
1.22 (d, 6.5)
3.43 (s)
3.79−3.86 (m)
1.25 (d, 6.0)
3.42(s)
3.78−3.88 (m)
1.24 (d, 6.5)
3.43 (s)
3.79 (dd, 9.5, 6.4) 3.78 (dd, 9.4, 6.3)
4.12−4.17 (m)
1.48 (d, 5.6)
3.63−3.69 (m)
1.28 (d, 6.2)
3.44 (s)
1.23 (d, 6.1)
1.22 (d, 5.6)
OMe
L-Dig
L-Dig
L-Dig
L-Cym
L-Cym
L-Cym
1
4.98 (d, 3.4)
4.98(d, 3.5)
4.89 (brs)
4.90 (d, 2.6)
4.90 (d, 2.3)
5.14 (t, 3.2)
2
0
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Chemistry & Biodiversity
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2
.04 (dt, 12.7, 2.9)
2.01−2.08 (m)
2.05−2.10 (m)
1.80−1.91 (m)
3.60−3.66 (m)
3.86 (brs)
2.28−2.32 (m)
1.72−1.76 (m)
3.68−3.75 (m)
3.54−3.62 (m)
4.06−4.14 (m)
1.23 (d, 6.3)
3.42 (s)
2.23−2.31 (m)
1.70−1.77 (m)
3.70−3.76 (m)
3.56−3.62 (m)
4.08−4.16 (m)
1.24 (d, 5.7)
3.43 (s)
2.38−2.49 (m)
2

1.80−1.84 (m)
1.80−1.88 (m)
3.60−3.64 (m)
3.86 (brs)
1.93−2.04 (m)
3.96−4.03 (m)
3.93−4.04 (m)
4.65−4.75 (m)
1.52 (d, 6.4)
3.47 (s)
3
4
5
6
3.58−3.65 (m)
3.87 (brs)
3.97 (q, 6.5)
1.25 (d, 6.8)
3.41 (s)
3.98 (q, 6.5)
1.30 (d, 6.0)
3.41(s)
3.98 (q, 6.5)
1.30 (d, 6.0)
3.42 (s)
OMe
L-Cym
L-Cym
L-Cym
D-Cym
D-Cym
D-Digit
1



4.69 (d, 8.6)
4.69 (d, 9.5)
2.34−2.41 (m)
1.88−1.94 (m)
3.64−3.67 (m)
3.28−3.32 (m)
3.54−3.58 (m)
1.22 (d, 6.5)
3.42 (s)
4.70 (t, 9.0)
2.35−2.42 (m)
1.72−1.81 (m)
3.51−3.58 (m)
3.22−3.27 (m)
3.51−3.58 (m)
1.23 (d, 6.5)
3.43 (s)
4.78 (d, 9.6)
2.22−2.29 (m)
1.57−1.65 (m)
3.60−3.68 (m)
3.18−3.27 (m)
3.51−3.56 (m)
1.27 (d, 6.2)
3.42 (s)
4.78 (d, 8.5)
2.26−2.29 (m)
1.56−1.64 (m)
3.63−3.67 (m)
3.18−3.24 (m)
3.53−3.59 (m)
1.27 (d, 6.2)
3.42 (s)
5.61 (dd, 9.5, 1.5)
2.44−2.55 (m)
2.07−2.15 (m)
4.50−4.58 (m)
3.71 (dd, 9.4, 3.0)
4.33−4.37 (m)
1.67 (d, 6.2)
2
.38−2.43 (m)
.71−1.76 (m
3.63−3.68 (m)
2
1
3
4
5
6
 3.31 (dd, 10.3, 3.5)


3.52−3.57 (m)
1.30 (d, 6.2)
3.42 (s)
OMe
a: measured at 400 MHz, in chloroform-d
b: measured at 500 MHz, in chloroform-d
c: measured at 400 MHz, in pyridine-d
1
1
5
2
1
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Chemistry & Biodiversity
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13
Table 2. C-NMR spectroscopic data for compounds 1‒7
1a
2b
3 a
4 a
5 a
6c
7 a
1
2
3
4
5
6
7
8
9
38.8
29.0
77.8
38.7
140.4
117.9
34.2
74.2
44.1
37.0
28.0
69.4
60.9
87.8
33.3
32.4
91.8
7.7
38.8
28.8
77.5
38.7
140.9
117.4
34.1
74.4
43.6
37.2
24.1
72.5
57.0
88.1
33.0
32.1
91.6
9.2
38.8
28.9
77.8
38.8
140.6
117.7
34.3
74.2
43.7
37.2
24.2
72.8
58.1
88.0
33.2
31.9
91.4
9.4
38.8
28.8
77.9
38.8
141.1
117.3
34.1
74.5
43.7
37.1
24.2
72.5
57.6
88.2
32.6
32.1
91.7
9.2
38.9
29.0
77.9
38.7
139.8
118.4
34.6
73.8
43.7
37.1
28.5
70.8
57.8
87.8
33.4
32.6
88.0
10.1
18.3
72.5
17.0
39.2
30.1
77.9
39.5
139.3
119.9
35.5
74.2
44.7
37.5
29.3
70.9
58.8
89.0
34.3
34.7
89.1
11.5
18.8
73.2
17.9
38.7
28.8
77.8
38.7
140.9
117.4
34.1
74.4
43.6
37.2
24.1
72.5
57.6
88.1
32.6
32.1
91.6
9.2
10
11
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
1
1
1
2
2
18.6
213.7
28.2
18.7
209.3
27.3
169.9
20.7
18.6
209.1
27.4
165.7
117.6
145.4
134.2
128.2
128.9
130.4
128.9
128.2
18.7
209.4
27.3
169.9
20.7
18.7
209.4
27.3
169.9
20.7
1
2
3
4
5
6
7
8
9
D-Cym D-Cym D-Cym D-Digit D-Digit D-Digit D-Cym
1
2
3
4
5
6






95.7
34.3
77.1
81.8
68.9
17.5
57.0
95.8
34.4
77.1
81.8
68.9
17.5
57.6
95.7
34.4
77.1
81.8
68.9
17.5
57.0
95.9
37.2
67.6
80.0
68.3
18.1
95.7
37.1
67.6
80.0
68.3
18.2
96.5
39.0
67.9
81.5
69.0
18.6
95.7
34.0
77.5
72.5
70.8
18.3
57.3
OMe
L-Dig
100.8
L-Dig
100.8
L-Dig L-Cym L-Cym L-Cym
100.8 97.7 97.6 98.5
1
2
2
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Chemistry & Biodiversity
10.1002/cbdv.201900062
2
3
4
5
6





31.6
73.9
74.2
66.8
18.0
55.6
31.6
73.9
74.2
66.8
18.0
55.6
31.6
74.0
74.3
66.8
18.0
55.6
31.7
72.6
76.5
64.7
17.9
56.9
31.7
72.6
76.4
64.7
17.9
57.1
32.7
73.7
77.4
68.3
18.4
57.3
OMe
L-Cym L-Cym L-Cym D-Cym D-Cym D-Digit
1
2
3
4
5
6






99.1
32.4
77.5
72.2
71.1
18.3
57.0
99.1
32.7
77.2
72.2
71.1
18.3
57.0
99.1
33.0
77.5
72.2
71.1
18.3
57.0
94.3
33.6
77.2
72.5
70.9
18.2
57.2
94.3
33.5
77.2
72.4
70.9
18.1
57.0
96.2
39.7
68.7
74.2
70.5
19.1
OMe
a: measured at 100 MHz, in chloroform-d
b: measured at 125 MHz, in chloroform-d
c: measured at 100 MHz, in pyridine-d
1
1
5
2
3
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Chemistry & Biodiversity
10.1002/cbdv.201900062
Figures
Figure 1. Chemical structures of compounds 1–10
Figure 2. Key 2D NMR correlations of compound 1
2
4
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Chemistry & Biodiversity
10.1002/cbdv.201900062
0
0
g/ml
.01 g/ml
2.5
0
.1 g/ml
g/ml
1
2.0
1
0 g/ml
1.5
1.0
0.5
1
2
3
4
5
6
7
8
9
10
Figure 3. Effect of compounds 1‒10 on Con A-stimulated splenocyte proliferation in vitro
2.0
1.5
1.0
0.5
0
0
g/ml
.01 g/ml
0
.1 g/ml
1
1
g/ml
0 g/ml
1
2
3
4
5
6
7
8
9
10
Figure 4. Effect of compounds 1‒10 on LPS-stimulated splenocyte proliferation in vitro
2
5
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